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Peste des petits ruminants virus (PPRV), belonging to paramyxoviruses, has six structure proteins (such as matrix protein (M), nucleocapsid proteins (N), fusion protein (F) and hemagglutinin protein (H)) and could cause high morbidity and mortality in sheep and goats. Although a vaccine strain of PPRV has been rescued and co-expression of M and N could yield PPRV-like particles, the roles of structure proteins in virion assembly and release have not been investigated in detail. In this study, plasmids carrying PPRV cDNA sequences encoding the N, M, H, and F proteins were expressed in Vero cells. The co-expression of all four proteins resulted in the release of virus-like particles (VLPs) with similar release efficiency to that of authentic virions. Moreover, the co-expression of M together with F also resulted in efficient VLPs release. In the absence of M protein, the expression of no combination of the other proteins resulted in particle release. In summary, a VLPs production system for PPRV has been established and M protein is necessary for promoting the assembly and release of VLPs, of which the predominant protein is M protein. Further study will be focused on the immunogenicity of the VLPs., (Copyright © 2017. Published by Elsevier Ltd.)

Eimeria stiedai (E. stiedai) is a coccidian that infects the liver of the domestic rabbit and may cause severe hepatic coccidiosis. Virus-like particles in E. stiedai were discovered by Revets et al. However, the complete genome sequence of the E. stiedai virus has yet to be determined. A novel virus was isolated from E. stiedai in the present study. The complete genome sequence of the E. stiedai virus was 6219 bp in length and contained two open reading frames (ORFs) with a tetranucleotide overlap (AUGA). ORF1 (2400 bp) encoded a putative coat protein of 799 amino acids (86.471 kDa) that exhibited a high level of amino acid sequence similarity to that of Eimeria tenella (E. tenella) RNA virus 1 (EtRV1; 43 % identity, NC_026140), whereas ORF2 (3303 bp) encoded a putative RNA-dependent RNA polymerase (RdRp) of 1100 amino acids (118.850 kDa) that exhibited a high level of amino acid sequence similarity to that of the E. tenella RNA virus 1 (EtRV1; 51 % identity, NC_026140). Phylogenetic analysis revealed that the E. stiedai virus was a new member of the family Totiviridae. The sequence data provided sufficient information for classification of eimeriaviruses.

The tapeworm Taenia solium is an important human zoonotic parasite that causes great economic loss and also endangers public health. At present, an effective vaccine that will prevent infection and chemotherapy without any side effect remains to be developed. In this study, codon usage patterns in the T. solium genome were examined through 8,484 protein-coding genes. Neutrality analysis showed that T. solium had a narrow GC distribution, and a significant correlation was observed between GC12 and GC3. Examination of an NC (ENC vs GC3s)-plot showed a few genes on or close to the expected curve, but the majority of points with low-ENC (the effective number of codons) values were detected below the expected curve, suggesting that mutational bias plays a major role in shaping codon usage. The Parity Rule 2 plot (PR2) analysis showed that GC and AT were not used proportionally. We also identified 26 optimal codons in the T. solium genome, all of which ended with either a G or C residue. These optimal codons in the T. solium genome are likely consistent with tRNAs that are highly expressed in the cell, suggesting that mutational and translational selection forces are probably driving factors of codon usage bias in the T. solium genome.

The nematode-trapping fungi possess a unique capability of predating and invading nematodes. As a representative nematode-trapping fungus, Arthrobotrys oligospora has been widely used to study the interactions between nematode-trapping fungi and their hosts. Serine proteinase is one of the important virulence factors during process of invasion of the nematode-trapping fungi into nematodes. In this study, using reverse transcription polymerase chain reaction, we amplified the gene sequence of serine proteinase 186 from A. oligospora, cloned it into pPIC9K vector and expressed it in the yeast Pichia pastoris. The expressed recombinant serine proteinase186 (reP186) was purified via Ni-affinity chromatography. The in vitro nematode-degrading activity of reP186 was analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis revealed that reP186 with molecular weight of 33 kDa was successfully obtained. ReP186 was capable of degrading a series of protein substrates including casein, gelatin, bovine serum albumin, denatured collagen and nematode cortical layer. The reP186 exhibited the maximal activity at pH 8.0 and 55 °C and was highly sensitive to the inhibitor, phenylmethanesulfonylfluoride. Treatment of Caenorhabditis elegans and Haemonchus contortus with reP186 for 12, 24 and 36 h, respectively, resulted in 62, 88 and 100 % of killing rates for C. elegans, and 52, 65 and 84 % of killing rates for H. contortus, respectively, indicating a relatively strong nematode-degrading bioactivity of reP186.

Minigenomes (MGs) are complementary DNAs of the synthetic analogs of genomic RNA. MGs are widely used to study the life cycle of the Paramyxoviridae family of viruses. MG-based studies have provided valuable insights into the mechanisms of viral replication and transcription in this family, including the roles of viral proteins, the location and boundaries of the cis-acting elements, the functional domains of trans-acting proteins, techniques for the measurement of neutralizing antibody, virus-host interactions, and the structure and function of viral RNA. This article provides a brief overview of the principle and application of MG technology in studies involving members of the Paramyxoviridae family. The advantages, potential limitations, and future scope of MG technology are also discussed., (Copyright © 2014. Published by Elsevier B.V.)

No endogenous insulin-like peptides in parasitic flatworms have been reported. Insulin receptors from flukes and tapeworms have been shown to interact directly with the host-derived insulin molecule, which suggests the exploitation of host-derived insulin. In this study, a strategy of genome-wide searches followed by comprehensive analyses of strictly conserved features of the insulin family was used to demonstrate the presence of putative insulin-like peptides in the genomes of six tapeworms and two flukes. In addition, whole insulin signaling pathways were annotated on a genome-wide scale. Two putative insulin-like peptide genes in each genome of tapeworms and one insulin-like peptide gene in each genome of flukes were identified. The comprehensive analyses revealed that all of these peptides showed the common features shared by other members of the insulin family, and the phylogenetic analysis implied a putative gene duplication event in the Cestoda during the evolution of insulin-like peptide genes. The quantitative expression analysis and immunolocalization results suggested a putative role of these peptides in reproduction. Entire sets of major components of the classic insulin signaling pathway were successfully identified, suggesting that this pathway in parasitic flatworms might also regulate many other important biological activities. We believe that the identification of the insulin-like peptides gives us a better understanding of the insulin signaling pathway in these parasites, as well as host-parasite interactions., (© 2013 FEBS.)

In this paper, we investigated the enhancement of adjuvant effects of porcine IL-2 (pIL-2) by packaging it into a solid lipid nanoparticle (SLN) delivery system. SLN-pIL-2 was prepared using hydrogenated castor oil and Polylactide-co-glycolide by double emulsion solvent evaporation methods (w/o/w). In animal trials, BALB/c mice were immunized with inactivated foot and mouth disease virus (FMDV) antigen combined with the SLN-pIL-2 adjuvant on days 0 and 14. Antibody titer, splenocyte proliferation, and secretion of IFN-γ and IL-4 cytokines were determined. Our results showed that SLN-pIL-2 could significantly enhance FMDV-specific antibody level compared with recombinant pIL-2 alone (p<0.05). In addition, SLN-pIL-2 significantly increased the proliferative responses of antigen-specific spleen cells. Furthermore, SLN-pIL-2 induced the secretion of IFN-γ at a level higher than that induced by recombinant pIL-2 alone. Our results indicate that packaging recombinant pIL-2 in SLNs can be an effective way of boosting the effectiveness of pIL-2 as an adjuvant to enhance immune responses of vaccines., (Copyright © 2013. Published by Elsevier Ltd.)

Duck Tembusu virus (DTMUV) is a single-stranded positive-sense RNA virus that causes disease to emerge in duck flocks and results in huge economic losses to the duck industry. However, no vaccines and control measures are available in China to date. Development of reliable and fast detection methods is necessary to prevent and control this disease. Therefore, a one-step SYBR Green real-time reverse transcription polymerase chain reaction (RT-PCR) method is established here for DTMUV detection. The results show that the method can specifically detect DTMUV without cross-reactions with selected avian pathogens. The sensitivity of the assay was 1000 times greater than that of a conventional RT-PCR and able to test as few as 20 copies from RNA standard samples. The coefficients of variations of inter- and intra-assay values ranged from 0.09% to 0.36% and 0.1% to 0.23%, respectively. Testing 168 field samples and 96 experimentally infected samples by conventional RT-PCR and the one-step SYBR Green real-time RT-PCR, the positive rates were 35.1% and 73.8% from field samples and 30.2% and 64.6% from infected samples. The one-step SYBR Green real-time RT-PCR developed in this study was shown to be a sensitive, specific, high-throughput, cost-effective, and simple diagnostic tool for the rapid detection and epidemiological surveillance of the emerging DTMUV infection.

Duck Tembusu virus (DTMUV) is a newly emerging infectious agent in China. Since 2010, this presumably arthropod-borne virus has caused severe disease in duck flocks. A cell-adapted DTMUV, designated AH10, has been developed. For this particular virus, replication, pathogenicity, and antibody-dependent enhancement (ADE) infection mediated by a specific antibody was investigated in mammalian cells and mice. The virus propagated in Vero or Hepa1-6 cells with a stable titer, and induced a visible cytopathic effect. Expression of pro- and anti-inflammatory cytokine genes in Hepa1-6 cells was elevated during early infection stages. AH10 was able to replicate in the brain, spleen, liver and kidneys of mice, and resulted in a systemic infection by day 5 when administered intracerebrally. When naïve mice were first transfused intraperitoneally with virus-specific antisera, upon subsequent infection, ADE of virus symptoms was observed. Mice displayed clinical symptoms by day 3 post-infection; weight loss in the AH10-specific antibody-treated group was observed 2-3 days earlier than that in the control groups. These findings indicate that AH10 was able to replicate in the mouse model, and that anti-AH10 antibodies caused ADE. The molecular basis of pathogenicity and ADE in the mouse model should be investigated in the future., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Orf virus (ORFV) is an enveloped virus with a double-stranded DNA genome, causing a contagious pustular dermatitis, mainly in goats and sheep. In this study, two strains of ORFV were isolated from sheep and goat samples in Xinjiang and Shaanxi, China. The B2L and virus interferon resistance (VIR) genes of these two isolates were sequenced and analyzed after PCR amplification. Phylogenetic analysis showed that the two isolates clustered with other ORFV strains but were separated into different subgroups. The Xinjiang strain shared the highest homology with the Gansu strain, whereas the Shaanxi strain shared higher homology with the Taiwan and Hubei strains. This is the first report of the molecular characterization of ORFV in Northwest China, and it provides new information on the genotyping of the causative agents responsible for contagious ecthyma dermatitis outbreaks in China.

Rabbit cysticercosis, caused by the larval stage of Taenia pisiformis, is a serious parasitic disease of rabbits. It was reported that some cysteine peptidases have potential roles in the pathogenesis of various parasitic infections. To investigate the biochemical characteristics and roles in the pathogenesis/host-invasion of cysteine peptidases, a cDNA sequence encoding for a cathepsin L-like cysteine protease (TpCP) was cloned and identified from the T. pisiformis metacestodes. This sequence was 1220 bp in its length, which included a 1017 bp open reading frame encoding a 339 amino acid peptide. Multiple sequence alignments revealed a 28.9-88.5% similarity with cathepsin L-like cysteine proteases from other helminth parasites and mammals. The recombinant TpCP expressed in Escherichia coli did not show the proteolytic activity by zymography gel assay. However, the TpCP expressed in Pichia pastoris had typical biochemical activities that could hydrolyze rabbit immunoglobulin G, bovine serum albumin and fibronectin. Substrate studies indicated pronounced cleavage of Z-Phe-Arg-AMC. This activity was sensitive to cysteine protease inhibitor E-64 and immunohistochemistry results also indicated that TpCP was distributed as an intense positive reaction in the bladder wall. Our results gave us insights into future studies of TpCP's roles in the infection., (Copyright © 2013 Elsevier B.V. All rights reserved.)

SigmaB factor is an important regulatory factor for stress response in Gram-positive bacteria such as Listeria monocytogenes (L. monocytogenes), Staphylococcus aureus and Bacillus subtilis. However, the activity of SigmaB factor is regulated by RsbV factor. Currently, the functional studies of RsbV factor are mostly focused on non-pathogenic B. subtilis, but the roles of RsbV factor in pathogenic L. monocytogenes during the regulation of environmental stress and virulence are still unclear. In the study, a ∆RsbV mutant of L. monocytogenes was constructed to explore the regulatory role of RsbV in environmental stress and virulence. The environmental stress experiments indicated that the growth and survival capability of ∆RsbV mutant obviously decreased in stress of low temperature, osmotic pressure, alcohol and acid, compared with EGD strain. The macrophage infection experiment indicated that ∆RsbV mutant had weaker survival capability than EGD strain, and the expression of PrfA, actA, PlcA and LLO was down-regulated in infected cells. Animal inoculation experiments indicated that RsbV deletion significantly reduced the pathogenicity of L. monocytogenes. Our data demonstrate that, in addition to regulating tolerance under environmental stress conditions, RsbV also contributes to regulation of L. monocytogenes virulence.

Objective: To study the effect of RsbV gene deletion on the virulence of Listeria monocytogenes., Methods: The fragment with RsbV deletion was generated by gene overlap extension PCR (SOE-PCR) and the mutant with RsbV deletion was obtained by homologous recombination based on the wild strain LM-XS5. The differences in virulence between the two strains were determined by LD50, bacterial counts in liver and spleen, and qRT-PCR experiments., Results: LD50 of the RsbV gene-deleted strain was 10(4) higher than that of the wild strain. The numbers of gene-deleted strain in the mouse's liver and spleen were significantly fewer than that of the wild strain (P < 0.05). Results of qRT-PCR show that four virulence factors' expression levels of the RsbV gene-deleted strain were significantly lower than that of the wild strain (P < 0.05). The RsbV gene-deleted strain induced a strong immune response in mice against the wild strain., Conclusion: RsbV regulates the expression of four virulence gene (inlA, LLO, PlcA and PrfA) of Listeria monocytogenes; The virulence of the RsbV gene-deleted strain is significantly reduced, but it still has good immunogenicity.

A tapeworm, Taenia solium, remains a great threat to human health, particularly in developing countries. The life cycle of T. solium is thought to be terminated via vaccination of intermediate hosts. In this study, we constructed a recombinant attenuated Salmonella typhimurium live vaccine strain χ4558 expressing a TSOL18 antigen. SDS-PAGE and Western blot confirmed the expression of the interest protein and its antigenic property. The recombinant strain stably propagated in vitro, of which the growth was not reversely influenced by TSOL18 protein expressed. It was also shown that mice survived 10(12) CFU of S. typhimurium χ4558, while all mice infected with 10(7) CFU of the wild-type died within five days. The mouse experiment indicated that vaccine strain χ4558 induced a high titer of specific antibody for a long time. In contrast to the controls, the vaccinated mice had an obvious augment of CD4(+) and CD8(+) T lymphocytes and the percentage of helper CD4(+)/CD8(+) T lymphocytes was significantly increased (p<0.01). After oral administration, S. typhimurium χ4558 was first colonized mainly in the Peyer's patches and then predominantly in the mesenteric lymph nodes and spleens in the vaccinated mice. In addition, the high levels of specific anti-TSOL18 antibodies were also observed in pigs administrated with S. typhimurium χ4558. Collectively, these results demonstrate the possibility of use of an attenuated S. typhimurium strain as a vector to deliver protective antigens of T. solium., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Listeria monocytogenes is an intracellular bacterium responsible for listeriosis in both humans and animals. Infected livestock is believed to be one source of this pathogen. Vaccination is an optimal approach to control the occurrence of this disease in livestock. However, inactivated vaccines have been reported to be insufficient to offer immune protection against L. monocytogenes. Here we evaluated the immune protection capacity of a combination of recombinant p60 and LLO. Mice immunized with p60 and LLO generated a high level of anti-L. monocytogenes antibodies. In addition, the elevated levels of IFN-γ and the decreased levels of IL-4 were also observed in these treated mice. Consistent with the colonization of L. monocytogenes post infection, all mice in the control group died within 5 days after infection of L. monocytogenes, while 40, 40, 80, and 100 % of animals immunized with inactivated L. monocytogenes vaccine (ILMV), LLO + ILMV, p60 + ILMV, and p60 + LLO + ILMV, respectively, survived for 2 weeks. Collectively, the results presented in this study demonstrate the capacity of a combination of LLO and p60 to elicit high protective immune responses against L. monocytogenes infection.

Betacoronavirus 1 (BCoV-1) is an important pathogen causing diarrhea in calves. In the current study, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of BCoV-1 was successfully developed. The primers were designed to target the highly conserved fragment of BCoV-1 nucleocapsid gene. The assay displayed high specificity detecting only BCoV-1 with no cross reaction with other viruses. When 418 clinical samples from 6 different geographical areas of Xinjiang province were tested by the RT-LAMP method, the results indicated that this test is a simple, rapid, accurate, and sensitive method for the detection of BCoV-1.

In the present study, we investigated the tissue distribution and expression of signaling lymphocyte activation molecule (SLAM) in 40 tissues and organs of goats by real-time RT-PCR, in order to determine the role of these receptors in tissue tropism. SLAM mRNA was detected in all the samples investigated. The expression of SLAM mRNA was detected at high levels in spleen, mesenteric lymph node, hilar lymph node, mandibular lymph node, superficial cervical lymph node, nasal mucosa, duodenum, heart, gallbladder, thymus and blood; this is similar to the tissue tropism of peste des petits ruminant virus. However, it was surprising that expression of SLAM was low in lungs, colon and rectum which are the major sites of replication of PPRV. In addition, very low levels were detected in larynx, tongue and esophagus, which suggest the possible presence of an alternative receptor for PPRV. This study provided the first data on caprine SLAM for use in further studies of the pathogenesis of PPRV in goats.

Molecularly imprinted polymers (MIPs) are tailor-made polymers with high selectivity for a given analyte, or group of structurally related compounds. The influence of the process parameters (the moles of functional monomer and cross-linker, the selection of functional monomer and solvent) on the preparation of oseltamivir (OS)-imprinted polymers was investigated. A mathematical method for uniform design to optimize these selected parameters and to increase the MIP selectivity for template molecules was applied. The optimal conditions to synthesize MIP were 0.69 mmol 30% acrylamide (AA) + 70% 4-Vinylpyridine (4-VP) and 5.0 mmol ethylene glycol dimethacrylate (EGDMA) copolymerized in 5 ml toluene in the presence of 0.1 mmol OS. MIP showed high affinity and selectivity for separation of the template molecule from other compounds. In the present study, we have established an effective LC-MS/MS method to identify and quantify OS with good sensitivity, accuracy and precision.

In this study, hollow mesoporous silica nanoparticles (HMSNs) were synthesized using the sol-gel/emulsion approach and its potential application in drug delivery was assessed. The HMSNs were characterized, by transmission electron microscopy (TEM), Scanning Electron Microscopy (SEM), nitrogen adsorption/desorption and Brunauer-Emmett-Teller (BET), to have a mesoporous layer on its surface, with an average pore diameter of about 2 nm and a surface area of 880 m2/g. Fluorescein isothiocyanate (FITC) loaded into these HMSNs was used as a model platform to assess its efficacy as a drug delivery tool. Its release kinetic study revealed a sequential release of FITC from the HMSNs for over a period of one week when soaked in inorganic solution, while a burst release kinetic of the dye was observed just within a few hours of soaking in organic solution. These FITC-loaded HMSNs was also found capable to be internalized by live human cervical cancer cells (HeLa), wherein it was quickly released into the cytoplasm within a short period of time after intracellular uptake. We envision that these HMSNs, with large pores and high efficacy to adsorb chemicals such as the fluorescent dye FITC, could serve as a delivery vehicle for controlled release of chemicals administered into live cells, opening potential to a diverse range of applications including drug storage and release as well as metabolic manipulation of cells.

A lateral flow immunoassay (LFI) was developed to identify and diagnose foot-and-mouth disease virus (FMDV) serotypes O, A and Asia 1. Antibodies obtained from rabbits and guinea pigs immunized with cell-culture-adapted virus strains (O/CHA/99, A/GS/LX/66, Asia 1/CHN/05) and suckling-mouse adapted virus strains (O/AV99(L), A/AV88(L), Asia 1/YNBS/58) were used as capture antibodies. The diagnostic kit included three immunochromatographic strips of types O, A and Asia 1, and the type-specific results were confirmed by color on the test lines of the three strips. The LFI was evaluated using epithelial and vesicular samples (n=396) prepared from current and historical field samples (provide by the National Foot-and-Mouth Disease Reference Laboratory of China at Lanzhou Veterinary Research Institute). Negative samples (n=95) were collected from healthy animals. The diagnostic sensitivity of the LFI for FMDV serotypes O, A and Asia 1 was 88.3% compared to 89.7% obtained by the reference method of indirect-sandwich ELISA. The sensitivity of the LFI for FMDV type Asia 1 was higher at 92.1% compared to 90.5% for the ELISA. The specificity of the LFI was 97.1% compared with 97.4%., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In the present study, we report the sequences and characterization of the porcine integrin cDNAs encoding alphav, beta3, beta6 and beta8 subunits and compare them to those of other species. The coding sequences for the porcine alphav, beta3, beta6 and beta8 subunits were found to be 3141, 2289, 2367 and 2304 nucleotides in length, encoding 1046, 762, 788 and 767-amino-acid-residue protein, respectively. The porcine integrin alphav, beta3, beta6 and beta8 subunit shares common structural and functional elements with their counterparts from the other species. Phylogenetic trees showed that the porcine alphav, beta3, beta6 and beta8 were clustered into the Artiodactyla group, together with those of camels, sheep, and cattle, that are susceptible to FMDV infection. Real-time RT-PCR was used to investigate expression of the integrins alphavbeta3, alphavbeta6 and alphavbeta8 in different tissues of pigs in order to determine the role of these receptors in tissue tropism. Expression analysis showed that alphavbeta6 and alphavbeta8 mRNA expression were detected at high levels in tissues known to support replication of FMDV. Tissue distribution pattern of alphavbeta3 mRNA seems to be unrelated to the known tissue tropism of FMDV. This study provided the first data of porcine integrins for the further studies of the FMDV pathogenesis in pigs., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of classical swine fever virus (CSFV) and a set of primers designed based on the E2 gene reference sequences of the CSFV. The assay was optimized to amplify CSFV RNA by incubation at 63 degrees C for 50 min. The RT-LAMP amplification products had a ladder-like appearance when electrophoresed on an agarose gel. The RT-LAMP assay showed higher sensitivity than the conventional RT-PCR, and no cross-reactivity appeared with other related porcine viruses including porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus (PRRSV). The positive predictive value (PPV) of CSFV RT-LAMP for 227 field tissue samples was 94.7%, the negative predictive value (NPV) was 30.8% and showed better for the conventional RT-PCR method. Thus, the RT-LAMP assay is extremely rapid, sensitive, and specific and has potential usefulness for rapid laboratory diagnosis and pen-side detection for CSFV detection in pigs., ((c) 2009. Published by Elsevier B.V. All rights reserved.)

Taenia solium is a cestode parasite that causes cysticercosis in humans and pigs. TSOL18 has been identified as a host-protective oncosphere antigen. To obtain mouse monoclonal antibodies (mAbs) against TSOL18 and to map its antigenic epitopes are potentials to develop a vaccine for the prevention of T. solium infection. In this study, mAbs were produced by the hybridoma technique using purified glycosylated TSOL18 produced in Pichia pastoris as the immunogen. mAb was used to define the B-cell epitopes of TSOL18 with phage-displayed random dodecapeptide library (Ph.D.-12), and some of the positive phage clones were sequenced and analyzed. The predominant mimotopes were ETTKLQRFQAML (L1) found in 83%, followed by DHTXF in 15% (L2: DHTLFAASHNHR, DHTLFSTGHSHG, and DHTFMQRYHTHQ). Comparison of the peptide sequences with native TSOL18 protein sequence using Clustal W software showed that they did not completely match, suggesting that the ETTKLQRFQAML and DHTXF sequences should be conformational epitopes. The sera of mice immunized with the selected phage clones obviously recognized the TSOL18 protein. Meanwhile, sera collected from TSOL18-vaccinated pigs reacted to both epitopes in enzyme-linked-immunosorbent serologic assay test. Our work demonstrated that the antigenic epitope could be mapped through screening the phage-displayed peptide libraries with mAbs and a mimotope of TSOL18, which could provide an alternative approach for the diagnosis and development of a vaccine for T. solium.

Integrins are heterodimeric adhesion receptors that participate in a variety of cell-cell and cell-extracellular matrix protein interactions. Many integrins recognize RGD sequences displayed on extracellular matrix proteins and the exposed loops of viral capsid proteins. Four members of the alphav integrin family of cellular receptors, alphavbeta3, alphavbeta6, alphavbeta1 and alphavbeta8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro, and integrins are believed to be the receptors used to target epithelial cells in the infected animals. To analyse the roles of the alphav integrins from a susceptible species as viral receptors, we have cloned Bactrian camel alphav, beta3 and beta6 integrin cDNAs and compared them to those of other species. The coding sequences for Bactrian camel integrin alphav, beta3 and beta6 were found to be 3165, 2289 and 2367 nucleotides in length, encoding 1054, 762 and 788 amino acids, respectively. The Bactrian camel alphav, beta3 and beta6 subunits share many structural features with homologues of other species, including the ligand binding domain and cysteine-rich region. Phylogenetic trees and similarity analyses showed the close relationships of integrin genes from Bactrian camels, pigs and cattle, which are each susceptible to FMDV infection, that were distinct from the orders Rodentia, Primates, Perissodactyla, Carnivora, Galliformes and Xenopus. We postulate that host tropism of FMDV may in part be related to the divergence in integrin subunits among different species.

Four members of the alphav integrin family of cellular receptors, alphavbeta1, alphavbeta3, alphavbeta6, and alphavbeta8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro, and integrins are believed to be the receptors used to target epithelial cells in the infected animals. To analyse roles of the alphav integrins from a susceptible species as viral receptors, we have cloned sheep alphav, beta1, and beta6 integrin cDNAs and compared them to those of other species. The coding sequences for sheep integrin alphav, beta1, and beta6 were found to be 3147, 2397, and 2364 nuclotides in length, encoding 1048, 798, and 787 amino acids, respectively. The sheep alphav, beta1, and beta6 subunits share many structural features including ligand binding domain and cysteine-rich region with homologues of other species. Phylogenetic trees and similarity analyses showed the close relationship of integrin genes among sheep, pigs, cattle and Bactrian camels that are susceptible to FMDV infection, which were distinct from the order Rodentia, Primates, Perissodactyla, Carnivora, Galliformes. We postulate that host tropism of FMDV may be related to divergence in integrin subunits among different species.

To obtain the recombinant 18 kD protein with high purity and normal bioactivity of Cysticercus cellulosae (rCE18), E. coli cells with the rCE18 were disrupted ultra-sonically, and the inclusion bodies were washed with a solution containing 0.2% deoxycholic acid sodium (DOC)and 2% DOC, respectively. Then they were denatured with 0.9% sodium lauroyl sarcosine (SKL) followed by dialysis and gel filtration to refold and purify the target protein. At the same time, this method was compared with GST-FF affinity chromatography and recovering from SDS-PAGE gel. Biological activity of purified rCE18 was analyzed with indirect ELISA, and the purity of the products was identified using SDS-PAGE. The purity of refolded inclusion bodies exceeded 60% and the total recovery of activated protein rCE18 was about 41.3%. The specificity of rCE18 reached up to 97.2% using indirect ELISA. An effective way for purifying and refolding rCE18 expressed in E. coli as inclusion bodies was established, rCE18 with higher purity and activity was obtained, which has the potential for developing diagnosis methods of porcine cysticercosis.

Objective: The aim of the study is to establish in vitro cell line with stable and effective 3Dpol gene expression, so as to study the biological function of foot-and-mouth disease virus (FMDV ) 3Dpol and foot-and-mouth disease (FMD) gene engineering vaccine., Methods: FMDV 3D gene was amplified from pMD18-T-3D and inserted into pGEM-Teasy vector. By NotI/BamHI digestion, 3D gene with Not I/BamH I site was inserted into Not I/BamH I cloning site of the pBPSTR1 retroviral vector in order to obtain recombinant retroviral vector pBPSTR1-3D. The artifical retroviral viruses were obtained by both pBPSTR1-3D and pVSV-G envelope vector into the Gp2-293 package cells using Lipofectamine 2000. The BHK-21 cells were infected by artificial retroviral particles with 8 microg/mL Polybrene. The positive cell clones which genomes contained the 3Dpol gene were continually selected using puromycine and was regulated by tetracycline for 12 days .The single clone highly effective expressing 3D fusion protein was obtained by seeding the cells into 96-well plates with one cell per well., Results: By using retroviral gene transfer technology, the 3Dpol gene was integrated into the chromosome of BHK-21 cells, then under selection pressure, the cell lines stably expressing 3D were established. Finally, a cell line stably expressing the 3D fusion protein was established. The fusion protein was confirmed to be expressed correctly by Western-blot. The transfected genes in the cell line were consistently expressed during 35 passages of the host cells., Conclusion: Transgene cell strain stably carrying exogenous gene in subsequent passaging was successfully constructed. It provide a good experimental tool for the biological function of FMDV 3Dpol and FMD gene engineering vaccine research.

Objective: To purify and to detect reactivity of non-structural proteins 3A, 3B and 2C expressed in the Escherichia coli., Methods: FMDV NSP 3A, 3B and 2C containing the major B-cell antigenic sites were expressed in E. coli. We got renatured 2C protein by lysing of isolated inclusion body using high concentration of urea, and then diluted in a buffer system containing oxidized/reduced glutathione. Purified 3A, 3B and 2C were obtained by Ni-NTA His Bind Resin affinity chromatography. The reactivity of three NSPs with sera of different origin was measured using an indirect ELISA and Western-blot. The reactivity of three proteins was compared with 3ABC and 3D by detecting sera of clinically healthy sheep that were collected from epidemic region of Asia I FMD., Results: Proteins 3A and 3B were solubly expressed in bacteria, and 2C was expressed to form inclusion body. All three products could react specifically with sera from FMDV infected animal by western-blot and ELISA. The high coincident rates were observed between 3A, 3B, 2C and 3ABC., Conclusion: The results would provide useful materials for establishment of immunoelectro-transfer blot (EITB) diagnostic method, which could be used for differentiation of the FMDV infected animals from the vaccinated animals.

This paper presents a description of the rules of construction and function of genome as well as evolutional relationship between organisms, and opens out the essence of life through introducing the progress in genome of model organism. The other purpose of this review is to highlight the status and function of model organism in the research of comparing genomics so as to provide the model of cycle for researches into high creature life, especially human beings life.