Search

Cancer-associated gene fusions serve as a potential source of highly immunogenic neoantigens. In this study, we identified fusion proteins from fusion genes and extracted fusion peptides to accurately predict Breast cancer (BRCA) neo-antigen candidates by high-throughput artificial intelligence computation. Firstly, Deepsurv was used to evaluate the prognosis of patients, providing a landscape of prognostic fusion genes in BRCA. Next, AGFusion was utilized to generate full-length fusion protein sequences and annotate functional domains. Advanced neural networks and Transformer-based analyses were implemented to predict the binding of fusion peptides to 112 types of HLA, thereby forming a new immunotherapy candidates' library of BRCA neo-antigens (n = 7791, covering 88.41% of patients). Among them, 15 neo-antigens were validated and factually translated into mass spectrometry data of BRCA patients. Finally, AlphaFold2 was applied to predict the binding sites of these neo-antigens to MHC (HLA) molecules. Notably, we identified a prognostic neoantigen from the TBC1D4–COMMD6 fusion that significantly improves patient prognosis and extensively binds to 16 types of HLA alleles. These highly immunogenic and tumor-specific neoantigens offer emerging targets for personalized cancer immunotherapies and act as prospective predictors for tumor survival prognosis and responses to immune checkpoint therapies. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections, with no currently available small-molecule drugs that are both safe and effective. A major obstacle in antiviral drug development is the rapid emergence of drug-resistant viral strains. Targeting multiple viral compounds may help mitigate the development of resistance. Herein, we conducted a drug screening using the Antiviral Traditional Chinese Medicine Active Compound Library, aiming to identify compounds that simultaneously target the RSV fusion (F) protein, glycoprotein (G), and the host heparan sulfate proteoglycans (HSPGs). From this screening, 10 candidate compounds were identified for their ability to interact with all three targets. Among these 10 candidates, chebulagic acid (CHLA) and punicalagin (PUG) demonstrated the most potent inhibition of RSV replication. In vitro dose-response assays confirmed the antiviral efficacy of CHLA (IC50: 0.07864 µM) and PUG (IC50: 0.08065 µM). Further experiments revealed both CHLA and PUG disrupt RSV attachment and membrane fusion by targeting the RSV-F and G proteins, rather than HSPG. Notably, CHLA and PUG were found to bind to the CX3C motif of the RSV-G protein, with docking assays predicting their binding sites at cysteines 176 and 182. Additionally, CHLA enhanced the conformational stability of the RSV-F protein before fusion. In an in vivo study, both CHLA and PUG were shown to alleviate RSV-induced pulmonary pathology by reducing viral titers, mitigating lung injury, and suppressing the inflammatory responses in the lungs. Our findings suggest that CHLA and PUG hold potential as therapeutic agents for RSV infection. [ABSTRACT FROM AUTHOR]

The respiratory syncytial virus (RSV) fusion protein (F) facilitates virus-cell membrane fusion, which is critical for viral entry, and cell-cell fusion. In contrast to many type I fusion proteins, RSV F must be proteolytically cleaved at two distinct sites to be fusogenic. Cleavage at both sites results in the release of a 27 amino-acid fragment, termed Pep27. We examined proteolytic processing and the role of Pep27 for RSV F from both RSV A2 and RSV B9320 laboratory-adapted strains, allowing important comparisons between A and B clade F proteins. F from both clades was cleaved at both sites, and pulse-chase analysis indicated that cleavage at both sites occurs early after synthesis, most likely within the secretory pathway. Mutation of either site to alter the furin recognition motif blocked cell-cell fusion activity. To assess the role of Pep27 in F processing and expression, we deleted the Pep27 fragment, but preserved the cleavage sites. Deletion of Pep27 reduced F surface expression and cell-cell fusion. Two conserved N-linked glycosylation sites within Pep 27 are present in both the RSV A2 and RSV B9320 F. Randomization of the Pep27 sequence, while conserving the two N-liked glycosylation sites, did not significantly change surface expression, and only modestly reduced cell-cell fusion. However, the disruption of either Pep27 glycosylation site reduced cell-cell fusion. This work clarifies the timing of RSV F proteolytic cleavage and offers insight into the crucial role the N-linked glycosylation sites within Pep27 play in the biological function of F. [ABSTRACT FROM AUTHOR]

Coinfections with porcine circovirus types 2, 3, and 4 (PCV2, PCV3, and PCV4) are increasingly being detected in the swine industry. However, there is no commercially available vaccine which prevents coinfection with PCV2, PCV3, and PCV4. The development of a vaccine expressing capsid (Cap) fusion proteins of multiple PCVs represents a promising approach for broadly preventing infection with PCVs. In this study, we developed a PCV subunit vaccine candidate (Cap 2-3-4) by predicting, screening, and fusing antigenic epitopes of Cap proteins of PCV2, PCV3, and PCV4. Immunoprotection assays showed that the prokaryotic expression of Cap 2-3-4 could effectively induce high levels of PCV2, PCV3, and PCV4 Cap-specific antibodies and successfully neutralize both PCV2 and PCV3. Furthermore, Cap 2-3-4 demonstrated a potent ability to activate cellular immunity and thus prevent lung damage in mice. This study provides a new option for the development of broad vaccines against PCVs. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children, elderly and immunocompromised patients worldwide. The RSV fusion (F) protein, which has 5–6 N-glycosylation sites depending on the strain, is a major target for vaccine development. Two to three of these sites are located in the p27 peptide, which is considered absent in virions. Prior research from our group showed that removing the N-glycan at position 116 (N116) in p27 led to higher neutralizing antibody responses and better protection against RSV. In this study, the effect of single, double and triple N-glycan deletion mutations in F p27 was evaluated. Surprisingly, all mutants exhibited similar expressions and functionality to the wild-type F protein. All F p27 glycomutants induced neutralizing antibodies and lowered lung viral loads after an RSV challenge in a mouse model. Although N-glycans in p27 influence immune responses, their exact role in RSV biology remains unclear. Possibly, these glycans, which are mostly conserved, play a role in other aspects of virus replication and biology. [ABSTRACT FROM AUTHOR]

Lysosomal storage diseases (LSDs) are genetic disorders caused by mutations in lysosomal enzymes, lysosomal membrane proteins or genes related to intracellular transport that result in impaired lysosomal function. Currently, the primary treatment for several LSDs is enzyme replacement therapy (ERT), which involves intravenous administration of the deficient lysosomal enzymes to ameliorate symptoms. The efficacy of ERT largely depends on the mannose-6-phosphate (M6P) modification of the N-glycans associated with the enzyme, as M6P is a marker for the recognition and trafficking of lysosomal enzymes. In cells, N-glycan processing and M6P modification occur in the endoplasmic reticulum and Golgi apparatus. This is a complex process involving multiple enzymes. In the trans-Golgi network (TGN), M6P-modified enzymes are recognized by the cation-independent mannose-6-phosphate receptor (CIMPR) and transported to the lysosome to exert their activities. In this study, we used the 9th domain of CIMPR, which exhibits a high affinity for M6P binding, and fused it with the Fc domain of human immunoglobulin G1 (IgG1). The resulting fusion protein specifically binds to M6P-modified proteins. This provides a tool for the rapid detection and concentration of M6P-containing recombinant enzymes to assess the effectiveness of ERT. The advantages of this approach include its high specificity and sensitivity and may lead to the development of new treatments for LSDs. [ABSTRACT FROM AUTHOR]

Xylene and its derivatives are bulk raw materials in the chemical industry, and their oxidation products, including p‐toluic acid and terephthalic acid, are also crucial in the production of fine chemicals. Unspecific peroxygenases (UPOs) are heme‐thiolate enzymes that are capable of oxidizing diverse organic compounds. In this study, a UPO from Thielavia terrestris (TteUPO) showed the ability to oxidize p‐xylene to p‐toluic acid with >99% chemoselectivity. To address the sensitivity of TteUPO to H2O2 during the reaction, a fusion protein of TteUPO and formate oxidase from Aspergillus oryzae (AoFOx) was constructed for in situ H2O2 regeneration. Additionally, site‐directed saturation mutagenesis of TteUPO was performed, giving the mutant F63S/A155 V, which exhibited a 2.4‐fold increase in the specific activity toward p‐xylene compared with wild‐type TteUPO. The engineered fusion protein TteUPOF63S/A155V‐AoFOx achieved 3.8 mM p‐toluic acid under 8 mM p‐xylene loading, which is approximately 60‐fold higher than previously reported p‐toluic acid concentrations through bio‐oxidation. It also showed the capability to convert other monocyclic and polycyclic aromatic hydrocarbons, indicating its potential for the high‐value conversion of xylene and its derivatives. [ABSTRACT FROM AUTHOR]

Objective: Blood lipid levels play a critical role in the progression of atherosclerosis. However, even with adequate lipid reduction, significant residual cardiovascular risk remains. Therefore, it is necessary to seek novel therapeutic strategies for atherosclerosis that can not only lower lipid levels but also inhibit inflammation simultaneously. Methods: The fusion protein FD03-IL-1Ra was designed by linking the Angiopoietin-like 3 (ANGPTL3) nanobody and human interleukin-1 receptor antagonist (IL-1Ra) sequences to a mutated human immunoglobulin gamma 1 (IgG1) Fc. This construct was transfected into HEK293 cells for expression. The purity and thermal stability of the fusion protein were assessed using SDS-PAGE, SEC-HPLC, and differential scanning calorimetry. Binding affinities of the fusion protein to ANGPTL3 and IL-1 receptor were measured using Biacore T200. The biological activity of the fusion protein was validated through in vitro experiments. The therapeutic efficacy of the fusion protein was evaluated in an ApoE-/- mouse model of atherosclerosis, including serum lipid level determination, histological analysis of aorta and aortic sinus sections, and detection of inflammatory and oxidative stress markers. ImageJ software was utilized for quantitative image analysis. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc test. Results: The FD03-IL-1Ra fusion protein was successfully expressed, with no polymer formation detected, and it demonstrated good thermal and conformational stability. High affinity for both murine and human ANGPTL3 was exhibited by FD03-IL-1Ra, and it was able to antagonize hANGPTL3's inhibition of LPL activity. FD03-IL-1Ra also showed high affinity for both murine and human IL-1R, inhibiting IL-6 expression in A549 cells induced by IL-1β stimulation, as well as suppressing IL-1β-induced activity inhibition in A375.S2 cells. Our study revealed that the fusion protein effectively lowered serum lipid levels and alleviated inflammatory responses in mice. Furthermore, the fusion protein enhanced plaque stability by increasing collagen content within atherosclerotic plaques. Conclusions: These findings highlighted the potential of bifunctional interleukin-1 receptor antagonist and ANGPTL3 antibody fusion proteins for ameliorating the progression of atherosclerosis, presenting a promising novel therapeutic approach targeting both inflammation and lipid levels. [ABSTRACT FROM AUTHOR]

Cervical cancer is the fourth most common cause of cancer and the fourth most common cause of cancer deaths in women. Some reports have shown the effect of Streptococcus agalactiae proteins and capsule products against cancer cell lines. This study aimed to design and produce a fusion of recombinant protein (containing the capsules of Streptococcus agalactiaes with a linker and Antti cancer sequences) CpsA-CpsC-L-ACAN in the plasmid pET-22b (+) vector. Construct pET-22b (+) was designed by researchers, optimized with bioinformatics software, and synthesized by a Biometrics company. For the confirmation of recombinant protein, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were performed. Antibacterial activity was performed according to protocol CLSI2020. Based on bioinformatics analysis, the recombinant protein had good spatial structure and stability and half-life. Iterative Threading ASSEmbly Refinement (I-TASSER) results predicted a surfing topology with C-score values (−3.60). The results of Gastro-Oesophageal Reflux 4 (GOR4) analysis showed a little extended strand (30.63%), a random coil with a percentage (50.18%), and an alpha helix with a percentage (19.19%). The recombinant protein was confirmed by SDS-PAGE and western blotting with Anti-his tag. The purpose of peptide fusion design in this article is to help in the development of anti-cervical cancer medicine. Of course, this issue needs to be investigated in animal and human phases. In this article, only bioinformatics investigations and validation of the results in the laboratory have been discussed. [ABSTRACT FROM AUTHOR]

Copyright of Journal of China Medical University is the property of Journal of China Medical University Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

DNA Fragmentation Factor (DFF) is a heterodimer protein involved in DNA fragmentation during apoptosis, which acts as a trigger downstream of caspase-3 activation. DFF40 catalytically active homo-oligomers break down chromosomal DNA. Previous scientific investigations have revealed a link between DFF40 expression changes and various cancers. DFF40 deletion or down-regulation has been observed in some cancers. Consequently, therapeutic strategies involving the DFF40 molecule compensating led to an increased rate of cancer cell apoptosis. In this review article, we aimed to introduce cancers with low expression of this protein first. The second part of this paper focuses on studies that utilized exogenous DFF40 protein produced by recombinant DNA technology and surveyed during in vitro and in vivo tests. Finally, compensation for diminished expression of the mentioned protein via gene therapy-based techniques to make up for this apoptotic molecule's low expression is the topic of the last part of this review article. [ABSTRACT FROM AUTHOR]

Abstract Cancer-associated gene fusions serve as a potential source of highly immunogenic neoantigens. In this study, we identified fusion proteins from fusion genes and extracted fusion peptides to accurately predict Breast cancer (BRCA) neo-antigen candidates by high-throughput artificial intelligence computation. Firstly, Deepsurv was used to evaluate the prognosis of patients, providing a landscape of prognostic fusion genes in BRCA. Next, AGFusion was utilized to generate full-length fusion protein sequences and annotate functional domains. Advanced neural networks and Transformer-based analyses were implemented to predict the binding of fusion peptides to 112 types of HLA, thereby forming a new immunotherapy candidates’ library of BRCA neo-antigens (n = 7791, covering 88.41% of patients). Among them, 15 neo-antigens were validated and factually translated into mass spectrometry data of BRCA patients. Finally, AlphaFold2 was applied to predict the binding sites of these neo-antigens to MHC (HLA) molecules. Notably, we identified a prognostic neoantigen from the TBC1D4–COMMD6 fusion that significantly improves patient prognosis and extensively binds to 16 types of HLA alleles. These highly immunogenic and tumor-specific neoantigens offer emerging targets for personalized cancer immunotherapies and act as prospective predictors for tumor survival prognosis and responses to immune checkpoint therapies.

PURPOSE: Evaluate the safety and efficacy of efbemalenograstim alfa for reducing the risk of febrile neutropenia in breast cancer patients undergoing myelosuppressive chemotherapy. METHODS: A phase III, randomized, double-blind, placebo-controlled study was conducted. A total of 122 subjects received up to 4 cycles of TA chemotherapy (75 mg/m2 docetaxel + 60 mg/m2 doxorubicin). Patients were randomized in a 2:1 ratio to subcutaneously inject a single 20 mg of efbemalenograstim alfa or placebo on day 2 of cycle 1, and all subjects received efbemalenograstim alfa on day 2 of cycles 2, 3, and 4. Duration of severe (grade 4) neutropenia (DSN), depth of neutrophil nadir, incidence of febrile neutropenia (FN), time to neutrophil recovery, and safety information were recorded. RESULTS: For the primary endpoint, the mean DSN in cycle 1 was 1.3 days and 3.9 days for efbemalenograstim alfa and placebo respectively (95% CI, 2.3, 3.4). As the lower bound of the 95% CI was > 0, superiority of efbemalenograstim alfa over placebo can be declared. In addition, the incidence of FN in Cycle 1 was lower in efbemalenograstim alfa group than in placebo group (4.8% vs. 25.6%; p = 0.0016). Patients in the efbemalenograstim alfa group required less intravenous antibiotics (3.6% vs. 17.9%; p = 0.0119). Most adverse events were consistent with those expected for breast cancer patient receiving TA chemotherapy. CONCLUSION: Efbemalenograstim alfa is effective and safe for significantly decreasing the duration of severe neutropenia and the incidence of febrile neutropenia in breast cancer patients who are receiving TA chemotherapy. TRIAL REGISTRATION: NCT02872103, August 19, 2016.

Avidin-biotin binding, the most robust non-covalent protein-ligand interaction occurring in nature, has wide-ranging applications in biotechnology. A frequent challenge in these applications is accurately determining the number of unoccupied biotin binding sites in avidin-containing fusion proteins. We delineate a novel assay protocol in miniaturized format to quantify available biotin binding sites based on the affinity of the anionic dye 4′-hydroxyazobenzene-2-carboxylic acid for biotin binding sites within avidin. We apply this assay as a quality control assay to evaluate the number of available biotin binding sites in different fusion protein production batches. This method offers a streamlined alternative to fluorescence-based assays commonly employed to assess biotin binding, is less time-consuming than other methods and is applicable to diverse fusion proteins.

Background: Variations in the fusion (F) protein of respiratory syncytial virus (RSV) with main antigenic sites I–V and Ø may affect the development of RSV vaccines and therapies. Methods: In the study, 30 respiratory specimens positive for RSV were randomly selected from children with acute lower respiratory infections (ALRI) in Beijing every year from 2012 to 2021 for F gene sequencing. Then, 300 F gene sequences and 508 uploaded to GenBank from China were subjected to phylogenetic analysis. Results: The results indicated the nucleotide identities were 95.4–100% among 446 sequences of RSV A, and 96.3–100% among 362 of RSV B. The most common variant loci were N80K (100.00%) and R213S (97.76%) for site Ø, and V384I/T (98.43%) for site I among sequences of RSV A, and M152I (100.00%), I185V (100.00%), and L172Q/H (94.48%) for site V, and R202Q (99.45%) for site Ø among sequences of RSV B. N276S appears in 95.29% sequences of RSV A, while S276N and N262 I/S appear in 1.38% and 0.55% sequences of RSV B, respectively. No variation was found in all sequences at the binding sites of 14N4 and motavizumab. Conclusions: There were cumulative variations of the RSV F gene, especially at some binding sites of antigenic sites.

Abstract The primary challenge to improving existing cancer treatment is to develop drugs that specifically target tumor cell. NGR peptide is tumor homing peptide that selectively target cancer cells while interleukin 15 is a pleiotropic cytokine with anticancer properties. This study computationally engineered a IL15-NGR fusion peptide by linking the homing peptide NGR with the targeting peptide IL-15. After evaluating and validating the chimeric peptide, we docked it to the IL-15Rα/IL-15Rβ/γc heterodimer receptor, examining the interactions and binding energy and lastly, molecular dynamics simulations were performed. The secondary and tertiary structures, along with physicochemical properties of the designed IL-15-NGR chimeric protein, were predicted using GOR IV, trRosetta and ProtParam online servers respectively. The quality and 3D structure validation were confirmed via ProSA-web and SAVES 6.0 analysis which predicted an ERRAT score of 96.72%, with 97.6% of residues in the Ramachandran plot, validating its structure. Finally, Docking, MD simulations and interaction analysis were performed using ClusPro 2.0 and GROMACS and PDBsum, which exhibited significant hydrogen bonding and salt bridges, confirming the formation of a stable docked complex. These results were further corroborated by simulation analysis, which demonstrated a stable and dynamic behavior of the docked complex in a biological environment. The predicted high expression value of fusion protein was 0.844 in E.coli using SOLUPROT tool. These findings suggest efficient expression of the IL15-NGR fusion protein if its gene is inserted into E. coli and indicates its potential as a safe and effective anticancer treatment, paving the way for targeted therapeutic interventions. Graphical abstract

Hippocampal neurons can be the first to be impaired with neurodegenerative disorders, including Alzheimer's disease (AD). Most drug candidates for causal therapy of AD cannot either enter the brain or accumulate around hippocampal neurons. Here, we genetically engineered insulin-fusion proteins, called hippocampal neuron-targeting (Ht) proteins, for targeting protein drugs to hippocampal neurons because insulin tends to accumulate in the neuronal cell layers of the hippocampus. In vitro examinations clarified that insulin and Ht proteins were internalized into the cultured hippocampal neurons through insulin receptor-mediated macropinocytosis. Cysteines were key determinants of the delivery of Ht proteins to hippocampal neurons, and insulin B chain mutant was most potent in delivering cargo proteins. In vivo accumulation of Ht proteins to hippocampal neuronal layers occurred after intracerebroventricular administration. Thus, hippocampal neuron-targeting technology can provide great help for developing protein drugs against neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Background: Nipah virus is a deadly infectious virus that was first isolated and identified from Malaysia. Since then, a number of Nipah virus outbreaks have been reported from Bangladesh and India. Transmission of the disease occurs through Pteropus genus fruit bats. The case fatality rate of this infection is very high when compared with other viral zoonoses. At present, there are no approved vaccines or drugs available to prevent or treat this infection. A number of studies are ongoing to develop an efficient vaccine candidate to combat this deadly virus. The majority of them concentrate on the structural and non-structural proteins, which are the main targets of neutralizing antibodies. Materials and Methods: Here, we analyzed the genome sequence identity of two Nipah virus strains, Indian and Malaysian, and also the amino acid identity between the two structural proteins (Attachment glycoprotein G and Fusion protein F) and one non-structural protein (W protein) of those two strains. Results and Discussion: It was found that there is a considerable amount of nucleotide sequence homology between the initial strain that originated from Malaysia and the strain that is now found in India. Furthermore, the Structural and Non-structural proteins of these two strains exhibit a high degree of similarity. Conclusion: Hence, a vaccine candidate designed using either NiV M or NiV B can be effectively used as a potent vaccine. [ABSTRACT FROM AUTHOR]

Cryo-electron tomography enables capture of 3D images of membrane fusion intermediate states with resolution of fusion proteins and membrane leaflet remodeling. Studies of class I, II, and III viral membrane fusion proteins are revealing general features as well as differences in stages of membrane reorganization, leading to complete fusion and pore formation. Viral fusion machinery is comprised of fusion proteins, viral membrane, and internal structural components such as matrix proteins that regulate fusion protein conformation and function. Tightly docked membrane contact zones with closely apposed proximal headgroup layers appear to be a near universal intermediate state along the fusion pathway. Computational modeling that builds upon observed structural information from cryo-electron tomography may offer a path towards a detailed mechanistic understanding of the forces and energetics that drive protein-mediated membrane fusion. Protein-mediated membrane fusion is the dynamic process where specialized protein machinery undergoes dramatic conformational changes that drive two membrane bilayers together, leading to lipid mixing and opening of a fusion pore between previously separate membrane-bound compartments. Membrane fusion is an essential stage of enveloped virus entry that results in viral genome delivery into host cells. Recent studies applying cryo-electron microscopy techniques in a time-resolved fashion provide unprecedented glimpses into the interaction of viral fusion proteins and membranes, revealing fusion intermediate states from the initiation of fusion to release of the viral genome. In combination with complementary structural, biophysical, and computation modeling approaches, these advances are shedding new light on the mechanics and dynamics of protein-mediated membrane fusion. [ABSTRACT FROM AUTHOR]

Fusing insect derived neurotoxic peptides with Galanthus nivalis agglutinin (GNA) has been shown to enhance the insecticidal activity of the neuropeptides, especially when administered orally. This study produced a recombinant scorpion insect specific neurotoxin BjαIT, GNA, and a fusion protein BjαIT/GNA using Pichia pastoris as an expression host. Recombinant rBjαIT/GNA was found to be easily degraded during expression in yeast which and produced a main protein product with a molecular weight of approximately 14 kDa. Cytotoxicity results showed that rBjαIT, rGNA, and rBjαIT/GNA had no toxicity to mammalian NIH/3T3 cells. Adding rBjαIT or rBjαIT/GNA at a concentration as low as 1 ng/mL to insect cell culture medium inhibited the proliferation of insect Sf9 cells, with rBjαIT exhibiting stronger cytotoxicity, while 20 ng/mL rGNA did not inhibit the proliferation of Sf9 cells. Silkworm larval injection results showed that rBjαIT/GNA was the most toxic of the three proteins, followed by rBjαIT, and rGNA. When rBjαIT/GNA was injected at a concentration of 0.129 nmol/g body weight 46.7% of silkworm died within 48 h. Feeding newly hatched silkworms with rBjαIT/GNA at a leaf surface concentration of 40 µg/cm2 resulted in 76.7% mortality within 24 h. However, rBjαIT/GNA showed almost no oral insecticidal activity against second instar silkworms. The results indicated that rBjαIT/GNA has stronger injection insecticidal activity and feeding insecticidal activity than rBjαIT and rGNA individually, making it more suitable for biological control. [ABSTRACT FROM AUTHOR]

The search for novel therapeutic agents to treat breast cancer has compelled the development of fusion proteins that synergize the functional benefits of different bioactive peptides. Leptulipin, derived from scorpion venom, exhibits antitumor properties. On the other hand, p28, a peptide from the bacterial protein azurin, enhances cell penetration. The current study investigated the design and computational evaluation of a Leptulipin-p28 fusion protein for breast cancer treatment. The amino acid sequences of Leptulipin and p28 were joined via a rigid linker to maintain structural and functional integrity. Secondary and tertiary structure predictions were performed using online servers of GOR-IV and I-TASSER. Physicochemical properties and solubility were analyzed using ProtParam and Protein-Sol. Validation and quality assessment of the fusion protein were confirmed through Rampage and ERRAT2. Finally, the fusion protein was docked with 2 receptors (VEGFR and Cadherin) and docked complexes were simulated on GROMACS. The Leptulipin-p28 fusion protein exhibited a stable structure exhibiting a high quality score of 92 on ERRAT and Ramachandran plot analysis highlighting 76.3% of residues in the favorable region. Docking studies with VEGFR and Cadherin receptors followed by 100 ns simulations on GROMACS showed stable complex formation. Molecular dynamics simulations confirmed the stability and robust interaction of the fusion protein-receptor complexes over time. The computational analysis indicates that the Leptulipin-p28 fusion protein holds promise as a multitarget therapeutic agent in breast cancer. The current findings warrant further investigation through in vitro and in vivo studies to validate the current outcomes. [ABSTRACT FROM AUTHOR]

Background: Variations in the fusion (F) protein of respiratory syncytial virus (RSV) with main antigenic sites I–V and Ø may affect the development of RSV vaccines and therapies. Methods: In the study, 30 respiratory specimens positive for RSV were randomly selected from children with acute lower respiratory infections (ALRI) in Beijing every year from 2012 to 2021 for F gene sequencing. Then, 300 F gene sequences and 508 uploaded to GenBank from China were subjected to phylogenetic analysis. Results: The results indicated the nucleotide identities were 95.4–100% among 446 sequences of RSV A, and 96.3–100% among 362 of RSV B. The most common variant loci were N80K (100.00%) and R213S (97.76%) for site Ø, and V384I/T (98.43%) for site I among sequences of RSV A, and M152I (100.00%), I185V (100.00%), and L172Q/H (94.48%) for site V, and R202Q (99.45%) for site Ø among sequences of RSV B. N276S appears in 95.29% sequences of RSV A, while S276N and N262 I/S appear in 1.38% and 0.55% sequences of RSV B, respectively. No variation was found in all sequences at the binding sites of 14N4 and motavizumab. Conclusions: There were cumulative variations of the RSV F gene, especially at some binding sites of antigenic sites. [ABSTRACT FROM AUTHOR]

The development of immune cell engagers (ICEs) can be limited by logistical and functional restrictions associated with fusion protein designs, thus limiting immune cell recruitment to solid tumors. Herein, a high affinity superantigen‐based multivalent ICE is developed for simultaneous activation and recruitment of NK and T cells for tumor treatment. Yeast library‐based directed evolution is adopted to identify superantigen variants possessing enhanced binding affinity to immunoreceptors expressed on human T cells and NK cells. High‐affinity superantigens exhibiting improved immune‐stimulatory activities are then incorporated into a superantigen‐based tri‐functional yeast‐display‐enhanced multivalent immune cell engager (STYMIE), which is functionalized with a nanobody, a Neo‐2/15 cytokine, and an Fc domain for tumor targeting, immune stimulation, and prolonged circulation, respectively. Intravenous administration of STYMIE enhances NK and T cell recruitment into solid tumors, leading to enhanced inhibition in multiple tumor models. The study offers design principles for multifunctional ICEs. [ABSTRACT FROM AUTHOR]

Introducing genetic material into hard-to-transfect mammalian cell lines and primary cells is often best achieved through retroviral infection. An ideal retroviral vector should offer a compact, selectable, and screenable marker while maximizing transgene delivery capacity. However, a previously published retroviral vector featuring an EGFP/Puromycin fusion protein failed to meet these criteria in our experiments. We encountered issues such as low infection efficiency, weak EGFP fluorescence, and selection against infected cells. To address these shortcomings, we developed a novel retroviral vector based on the Moloney murine leukemia virus. This vector includes a compact bifunctional EGFP and Puromycin resistance cassette connected by a 2A peptide. Our extensively tested vector demonstrated superior EGFP expression, efficient Puromycin selection, and no growth penalty in infected cells compared with the earlier design. These benefits were consistent across multiple mammalian cell types, underscoring the versatility of our vector. In summary, our enhanced retroviral vector offers a robust solution for efficient infection, reliable detection, and effective selection in mammalian cells. Its improved performance and compact design make it an ideal choice for a wide range of applications involving precise genetic manipulation and characterization in cell-based studies. [ABSTRACT FROM AUTHOR]

Chimeric RNAs, distinct from DNA gene fusions, have emerged as promising therapeutic targets with diverse functions in cancer treatment. However, the functional significance and therapeutic potential of most chimeric RNAs remain unclear. Here we identify a novel fusion transcript of solute carrier family 2‐member 11 (SLC2A11) and macrophage migration inhibitory factor (MIF). In this study, we investigated the upregulation of SLC2A11–MIF in The Cancer Genome Atlas cohort and a cohort of patients from Sun Yat‐Sen Memorial Hospital. Subsequently, functional investigations demonstrated that SLC2A11–MIF enhanced the proliferation, antiapoptotic effects, and metastasis of bladder cancer cells in vitro and in vivo. Mechanistically, the fusion protein encoded by SLC2A11–MIF interacted with polypyrimidine tract binding protein 1 (PTBP1) and regulated the mRNA half‐lives of Polo Like Kinase 1, Roundabout guidance receptor 1, and phosphoinositide‐3‐kinase regulatory subunit 3 in BCa cells. Moreover, PTBP1 knockdown abolished the enhanced impact of SLC2A11–MIF on biological function and mRNA stability. Furthermore, the expression of SLC2A11–MIF mRNA is regulated by CCCTC‐binding factor and stabilized through RNA N4‐acetylcytidine modification facilitated by N‐acetyltransferase 10. Overall, our findings revealed a significant fusion protein orchestrated by the SLC2A11–MIF–PTBP1 axis that governs mRNA stability during the multistep progression of bladder cancer. [ABSTRACT FROM AUTHOR]

The primary challenge to improving existing cancer treatment is to develop drugs that specifically target tumor cell. NGR peptide is tumor homing peptide that selectively target cancer cells while interleukin 15 is a pleiotropic cytokine with anticancer properties. This study computationally engineered a IL15-NGR fusion peptide by linking the homing peptide NGR with the targeting peptide IL-15. After evaluating and validating the chimeric peptide, we docked it to the IL-15Rα/IL-15Rβ/γc heterodimer receptor, examining the interactions and binding energy and lastly, molecular dynamics simulations were performed. The secondary and tertiary structures, along with physicochemical properties of the designed IL-15-NGR chimeric protein, were predicted using GOR IV, trRosetta and ProtParam online servers respectively. The quality and 3D structure validation were confirmed via ProSA-web and SAVES 6.0 analysis which predicted an ERRAT score of 96.72%, with 97.6% of residues in the Ramachandran plot, validating its structure. Finally, Docking, MD simulations and interaction analysis were performed using ClusPro 2.0 and GROMACS and PDBsum, which exhibited significant hydrogen bonding and salt bridges, confirming the formation of a stable docked complex. These results were further corroborated by simulation analysis, which demonstrated a stable and dynamic behavior of the docked complex in a biological environment. The predicted high expression value of fusion protein was 0.844 in E.coli using SOLUPROT tool. These findings suggest efficient expression of the IL15-NGR fusion protein if its gene is inserted into E. coli and indicates its potential as a safe and effective anticancer treatment, paving the way for targeted therapeutic interventions. [ABSTRACT FROM AUTHOR]

HxTx-Hv1h, a neurotoxic peptide derived from spider venom, has been developed for use in commercial biopesticide formulations. Cell Penetrating Peptides (CPPs) are short peptides that facilitate the translocation of various biomolecules across cellular membranes. Here, we evaluated the aphidicidal efficacy of a conjugated peptide, HxTx-Hv1h/CPP-1838, created by fusing HxTx-Hv1h with CPP-1838. Additionally, we aimed to establish a robust recombinant expression system for HxTx-Hv1h/CPP-1838. We successfully achieved the secretory production of HxTx-Hv1h, its fusion with Galanthus nivalis agglutinin (GNA) forming HxTx-Hv1h/GNA and HxTx-Hv1h/CPP-1838 in yeast. Purified HxTx-Hv1h exhibited contact toxicity against Megoura crassicauda, with a 48 h median lethal concentration (LC50) of 860.5 μg/mL. Fusion with GNA or CPP-1838 significantly enhanced its aphidicidal potency, reducing the LC50 to 683.5 μg/mL and 465.2 μg/mL, respectively. The aphidicidal efficacy was further improved with the addition of surfactant, decreasing the LC50 of HxTx-Hv1h/CPP-1838 to 66.7 μg/mL—over four times lower compared to HxTx-Hv1h alone. Furthermore, we engineered HxTx-Hv1h/CPP-1838 multi-copy expression vectors utilizing the BglBrick assembly method and achieved high-level recombinant production in laboratory-scale fermentation. This study is the first to document a CPP fusion strategy that enhances the transdermal aphidicidal activity of a natural toxin like HxTx-Hv1h and opens up the possibility of exploring the recombinant production of HxTx-Hv1h/CPP-1838 for potential applications. [ABSTRACT FROM AUTHOR]

The coronavirus disease 2019 (COVID-19) pandemic, which caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), lead to a crisis with devastating disasters to global public economy and health. Several studies suggest that the SARS-CoV-2 nucleocapsid protein (N protein) is one of uppermost structural constituents of SARS-CoV-2 and is relatively conserved which could become a specific diagnostic marker. In this study, eight single domain antibodies recognized the N protein specifically which were named pN01–pN08 were screened using human phage display library. According to multiple sequence alignment and molecular docking analyses, the interaction mechanism between antibody and N protein was predicted. ELISA results indicated pN01–pN08 with high affinity to protein N. To improve their efficacy, two fusion proteins were prepared and their affinity was tested. These finding showed that fusion proteins had higher affinity than single domain antibodies and will be used as diagnosis for the pandemic of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Recombinant human growth hormone (rhGH) and GH receptor antagonists (GHAs) are used clinically to treat a range of disorders associated with GH deficiency or hypersecretion, respectively. However, these biotherapeutics can be difficult and expensive to manufacture with multiple challenges from recombinant protein generation through to the development of long-acting formulations required to improve the circulating half-life of the drug. In this review, we summarize methodologies and approaches used for making and purifying recombinant GH and GHA proteins, and strategies to improve pharmacokinetic and pharmacodynamic properties, including PEGylation and fusion proteins. Therapeutics that are in clinical use or are currently under development are also discussed.

ABSTRACT Background TNF‐related apoptosis‐inducing ligand (TRAIL) belongs to the tumor necrosis factor superfamily. TRAIL selectively induces apoptosis in tumor cells while sparing normal cells, which makes it an attractive candidate for cancer therapy. Recombinant soluble TRAIL and agonistic antibodies against TRAIL receptors have demonstrated safety and tolerability in clinical trials. However, they have failed to exhibit expected clinical efficacy. Consequently, extensive research has focused on optimizing TRAIL‐based therapies, with one of the most common approaches being the construction of TRAIL fusion proteins. Methods An extensive literature search was conducted to identify studies published over the past three decades related to TRAIL fusion proteins. These various TRAIL fusion strategies were categorized based on their effects achieved. Results The main fusion strategies for TRAIL include: 1. Construction of stable TRAIL trimers; 2. Enhancing the polymerization capacity of soluble TRAIL; 3. Increasing the accumulation of TRAIL at tumor sites by fusing with antibody fragments or peptides; 4. Decorating immune cells with TRAIL; 5. Prolonging the half‐life of TRAIL in vivo; 6. Sensitizing cancer cells to overcome resistance to TRAIL treatment. Conclusion This work focuses on the progress in recombinant TRAIL fusion proteins and aims to provide more rational and effective fusion strategies to enhance the efficacy of recombinant soluble TRAIL, facilitating its translation from bench to bedside as an effective anti‐cancer therapeutic.

ABSTRACTHuman metapneumovirus (HMPV) is one of the main pathogens causing severe respiratory infections in children, as a common cause of immunodeficiency-related deaths in children and elderly individuals, the prevalence of HMPV has been showing an increasing trend during the last years. However, no vaccines or effective treatment plans are available currently. In this present, based on candidate proteins highly associated with viral virulence and has promising protective potential, we screened for immunodominant cytotoxic T cells, helper T cells, and Linear B-cell epitopes from the most promising candidate Fusion protein, together with G, SH, M, and M2. All epitopes were predicted to have strong antigenicity by Vaxijen and pose no potential toxicity, allergenicity, or hormonology to human proteins by Toxinpred, Allerpred, and Blast analysis, meanwhile, high conservancy is demanded to cover different subtypes. adjuvants β-defensin II and Pam2Cys was attached with EAAAK linkers to improve vaccine’s efficiency. Then, calculation of physicochemical properties proved the protein vaccine as a product can stably exist in the human body. Besides, we assessed the docking between the vaccine and immune receptors to evaluate its ability to stimulate immune responses, and the dynamic simulation further confirmed that the vaccine can tightly bind with immune receptors, which approved that the construction has the potential to induce strong humoral and cellular immune response. Finally, the vaccine was constructed into a multi-epitope mRNA vaccine, the immune simulations suggest that this is a vaccine candidate for controlling HMPV infection.

Neurodegenerative diseases such as Alzheimer’s disease (AD) pose substantial challenges to patients and health-care systems, particularly in countries with aging populations. Immunotherapies, including the marketed antibodies lecanemab (Leqembi®) and donanemab (KisunlaTM), offer promise but face hurdles due to limited delivery across the blood–brain barrier (BBB). This limitation necessitates high doses, resulting in increased costs and a higher risk of side effects. This study explores transferrin receptor (TfR)-binding camelid single-domain antibodies (VHHs) for facilitated brain delivery. We developed and evaluated fusion proteins (FPs) combining VHHs with human IgG Fc domains or single-chain variable fragments (scFvs) of the anti-amyloid-beta (Aβ) antibody 3D6. In vitro assessments showed varying affinities of the FPs for TfR. In vivo evaluations indicated that specific VHH-Fc and VHH-scFv fusions reached significant brain concentrations, emphasizing the importance of optimal TfR binding affinities. The VHH-scFv fusions were further investigated in mouse models with Aβ pathology, showing higher retention compared to wild-type mice without Aβ pathology. Our findings suggest that these novel VHH-based FPs hold potential for therapeutic and diagnostic applications in AD, providing a strategy to overcome BBB limitations and enhance brain targeting of antibody-based treatments. Furthermore, our results suggest that a given bispecific TfR-binding fusion format has a window of “optimal” affinity where parenchymal delivery is adequate, while blood pharmacokinetics aligns with the desired application of the fusion protein.

ABSTRACT Human parainfluenza virus 3 (HPIV3) infection is driven by the coordinated action of viral surface glycoproteins hemagglutinin-neuraminidase (HN) and fusion protein (F). Receptor-engaged HN activates F to insert into the target cell membrane and drive virion-cell membrane fusion. For F to mediate entry, its precursor (F0) must first be cleaved by host proteases. F0 cleavage has been thought to be executed during viral glycoprotein transit through the trans-Golgi network by the ubiquitously expressed furin because F0 proteins of laboratory-adapted viruses contain a furin recognition dibasic cleavage motif RXKR around residue 108. Here, we show that the F proteins of field strains have a different cleavage motif from laboratory-adapted strains and are cleaved by unidentified proteases expressed in only a narrow subset of cell types. We demonstrate that extracellular serine protease inhibitors block HPIV3 F0 cleavage for field strains, suggesting F0 cleavage occurs at the cell surface facilitated by transmembrane proteases. Candidate proteases that may process HPIV3 F in vivo were identified by a genome-wide CRISPRa screen in HEK293/dCas9-VP64 + MPH cells. The lung-expressed extracellular serine proteases TMPRSS2 and TMPRSS13 are both sufficient to cleave HPIV3 F and enable infectious virus release by otherwise non-permissive cells. Our findings support an alternative mechanism of F activation in vivo, reliant on extracellular membrane-bound serine proteases expressed in a narrow subset of cells. The proportion of HPIV3 F proteins cleaved and infectious virus release is determined by host cell expression of requisite proteases, allowing just-in-time activation of F and positioning F cleavage as another key regulator of HPIV3 spread.IMPORTANCEEnveloped viruses cause a wide range of diseases in humans. At the first step of infection, these viruses must fuse their envelope with a cell membrane to initiate infection. This fusion is mediated by viral proteins that require a critical activating cleavage event. It was previously thought that for parainfluenza virus 3, an important cause of respiratory disease and a representative of a group of important pathogens, this cleavage event was mediated by furin in the cell secretory pathways prior to formation of the virions. We show that this is only true for laboratory strain viruses, and that clinical viruses that infect humans utilize extracellular proteases that are only made by a small subset of cells. These results highlight the importance of studying authentic clinical viruses that infect human tissues for understanding natural infection.

Heparinases, including heparinases I–III (HepI, HepII, and HepIII, respectively), are important tools for producing low‐molecular‐weight heparin, an improved anticoagulant. The poor thermostability of heparinases significantly hinders their industrial and laboratory applications. To improve the thermostability of heparinases, we applied a rigid linker (EAAAK)5 (R) and a flexible linker (GGGGS)5 (F) to fuse maltose‐binding protein (MBP) and HepI, HepII, and HepIII from Pedobacter heparinus, replacing the original linker from the plasmid pMAL‐c2X. Compared with their parental fusion protein, MBP‐fused HepIs, HepIIs, and HepIIIs with linkers (EAAAK)5 or (GGGGS)5 all displayed enhanced thermostability (half‐lives at 30°C: 242%–464%). MBP‐fused HepIs and HepIIs exhibited higher specific activity (127%–324%), whereas MBP‐fused HepIIIs displayed activity similar to that of their parental fusion protein. Kinetics analysis revealed that MBP‐fused HepIIs showed a significantly decreased affinity toward heparin with increased Km values (397%–480%) after the linker replacement, whereas the substrate affinity did not change significantly for MBP‐fused HepIs and HepIIIs. Furthermore, it preliminarily appeared that the depolymerization mechanism of these fusion proteins may not change after linker replacement. These findings suggest the superior enzymatic properties of MBP‐fused heparinases with suitable linker designs and their potential for the bioproduction of low‐molecular‐weight heparin. [ABSTRACT FROM AUTHOR]

In previous studies, we developed a novel fusion protein named "melittin‐MIL‐2" which exhibited more anti‐tumor activity. However, it remains unclear whether melittin‐MIL‐2 possesses antitumor immune effect on lung adenocarcinoma. In this study, the immune effect and mechanism of melittin‐MIL‐2 inhibiting the growth and invasion of lung adenocarcinoma will be investigated, in order to provide novel perspectives for the immunotherapy of lung cancer. The results indicated that melittin‐MIL‐2 promoted T cell proliferation, enhanced NK cell cytotoxicity, and boosted IFN‐γ secretion in PBMCs. After melittin‐MIL‐2 stimulation, perforin expression and LAK/NK‐like killing activities of human PBMCs and NK cells were significantly enhanced. Melittin‐MIL‐2 is capable of hampering the development and proliferation of lung adenocarcinoma cell A549. ICAM‐1 and Fas expression in A549 cells exposed to melittin‐MIL‐2 rose significantly. The expression levels of TLR8 and VEGF in A549 cells decreased significantly after melittin‐MIL‐2 stimulation. In vivo, melittin‐MIL‐2 substantially impeded the growth of lung adenocarcinoma and formed an immune‐stimulating microenvironment locally in tumor tissues. In conclusion, the novel fusion protein melittin‐MIL‐2 exhibits strong anti‐tumor immune effect in lung adenocarcinoma cell A549 via activating the LFA‐1/ICAM‐1 and Fas/FasL pathways to enhance cytolytic activity, upregulating the secretion of IFN‐γ and perforin, and boosting LAK/NK‐like killing activities. Immuno‐effector cells and their secreted cytokines can form immune stimulation microenvironment locally in lung adenocarcinoma Lewis mice tissue. [ABSTRACT FROM AUTHOR]

Although functional studies on carbohydrate-binding module (CBM) have been carried out extensively, the role of tandem CBMs in the enzyme containing multiple catalytic domains (CDs) is unclear. Here, we identified a multidomain enzyme (Lc25986) with a novel modular structure from lignocellulolytic bacterial consortium. It consists of a mannanase domain, two CBM65 domains (LcCBM65-1/LcCBM65-2), and an esterase domain. To investigate CBM function and domain interactions, full-length Lc25986 and its variants were constructed and used for enzymatic activity, binding, and bioinformatic analyses. The results showed that LcCBM65-1 and LcCBM65-2 both bind mannan and xyloglucan but not cellulose or β-1,3-1,4-glucan, which differs from the ligand specificity of reported CBM65s. Compared to LcCBM65-2, LcCBM65-1 showed a stronger ligand affinity and a preference for acetylation sites. Both CBM65s stimulated the enzymatic activities of their respective neighboring CDs against acetylated mannan, but did not contribute to the activities of the distal CDs. The time course of mannan hydrolysis indicated that the full-length Lc25986 was more effective in the complete degradation of mixed acetyl/non-acetyl substrates than the mixture of single-CD mutants. When acting on complex substrates, LcCBM65-1 not only improved the enzymatic activity of the mannanase domain, but also directed the esterase domain to the acetylated polysaccharides. LcCBM65-2 adopted a low affinity to reduce interference with the catalysis of the mannanase domain. These results demonstrate the importance of CBMs for the synergism between the two CDs of a multidomain enzyme and suggest that they contribute to the adequate degradation of complex substrates such as plant cell walls. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) remains the primary cause of lower respiratory tract infections, particularly in infants and the elderly. In this study, we employed reverse genetics to generate a chimeric influenza virus expressing neuraminidase‐3F protein conjugate with three repeats of the RSV F protein protective epitope inserted into the NA gene of A/California/7/2009 ca (CA/AA ca), resulting in rFlu/RSV/NA‐3F (hereafter, rFRN3). The expression of NA‐3F protein was confirmed by Western blotting. The morphology and temperature‐sensitive phenotype of rFRN3 were similar to CA/AA ca. Its immunogenicity and protective efficiency were evaluated in BALB/c mice and cotton rats. Intranasal administration of rFRN3 elicited robust humoral, cellular, and to some extent, mucosal immune responses. Compared to controls, rFRN3 protected animals from RSV infection, attenuated lung injury, and reduced viral titers in the nose and lungs post‐RSV challenge. These results demonstrate that rFRN3 can trigger RSV‐specific immune responses and thus exhibits potent protective efficacy. The "dual vaccine" approach of a cold‐adapted influenza vector RSV vaccine will improve the prophylaxis of influenza and RSV infection. rFRN3 thus warrants further clinical investigations as a candidate RSV vaccine. [ABSTRACT FROM AUTHOR]

Rearranged during transfection (RET) rearrangement oncoprotein-mediated Ras/MAPK signaling cascade is constitutively activated in cancers. Here, we demonstrate a unique signal niche. The niche is a ternary complex based on the chimeric RET liquid-liquid phase separation. The complex comprises the rearranged kinase (RET fusion); the adaptor (GRB2), and the effector (SHC1). Together, they orchestrate the Ras/MAPK signal cascade, which is dependent on tyrosine kinase. CCDC6-RET fusion undergoes LLPS requiring its kinase domain and its fusion partner. The CCDC6-RET fusion LLPS promotes the autophosphorylation of RET fusion, with enhanced kinase activity, which is necessary for the formation of the signaling niche. Within the signal niche, the interactions among the constituent components are reinforced, and the signal transduction efficiency is amplified. The specific RET fusion-related signal niche elucidates the mechanism of the constitutive activation of the Ras/MAPK signaling pathway. Beyond just focusing on RET fusion itself, exploration of the ternary complex potentially unveils a promising avenue for devising therapeutic strategies aimed at treating RET fusion-driven diseases. [ABSTRACT FROM AUTHOR]

Interleukin-35 (IL-35) is an important immunosuppressive cytokine that has been shown to play a role in the immune response of various diseases. In this study, we cloned the coding sequence of human IL-35 gene, constructed single subunit expression vectors pXC17.4-p35 and pcDNA3.1 (+)-EBI3, and co-transfected CHO-K1 cells to express IL-35 in vitro. No binding was found between subunits of p35 and EBI3. Knobs-into-Holes (KIH) can solve the problem of heavy chain mismatch of heterologous antibodies. Therefore, expression vectors pXC17. 4-p35-Fch and pcDNA3.1(+)-EBI3-Fck were constructed by fusing KIH structures on the basis of the original sequences to express the recombinant fusion protein of KIH-IL-35. The expression vectors of two subunits were exchanged at the same time to verify the influence of different vectors on the expression level of KIH-IL-35. The analysis of various protein detection methods showed that the correct expression rate of KIH-IL-35 structure was significantly improved. Affinity purification of KIH-IL-35 was performed after large amount of expression, and the binding activity of KIH-IL-35 to glycoprotein 130 (gp130) was detected by ELISA. The results showed that the binding of KIH-IL-35 to gp130 was concentration dependent. The indirect activity of KIH-IL-35 and M1 cells was detected by cell activity assay. Further results showed that the inhibition rate of M1 cells increased in a dose-dependent manner with the concentration of KIH-IL-35. In addition, a method for determining IL-35 activity by activated human peripheral blood mononuclear cells was successfully established. Activated PBMCs increased in a dose-dependent manner with KIH-IL-35 concentration. In summary, this study utilized the KIH-IL-35 model to enhance the expression of recombinant human IL-35 and validated its high activity in vitro, providing new ideas for the study of IL-35 and the recombinant expression of similar heterodimeric cytokines. [ABSTRACT FROM AUTHOR]