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The NS1protein, a nonstructural protein of Tembusu virus, plays a key role in the pathogenesis of TMUV. To research host proteins that interact with NS1 protein, the cDNA library of duck embryo fibroblasts (DEF) was successfully constructed. The recombinant plasmid, pGBKT7-NS1, was transformed into the yeast Y2H to be cloned and tested for autoactivation and toxicity. The autoactivation and toxicity test of bait showed that the yeast two hybrid test could be carried out normally. A total of 7 clones from the library were got by Yeast Two-Hybrid System, and 4 proteins, including RPS7, MORC3, GABARAPL1 and MTSS1, may be interacted with DTMUV NS1 after sequencing and blast. Then we chose the host protein of RPS7 for GST pull down assay and the recombinant plasmid of pGEX-6p-1-NS1and pEGFP-RPS7 were constructed. Then the proteins of GST-NS1 and GFP-RPS7 were successfully expressed in vitro for GST pull down assay. The results showed that there was a real interaction between the two proteins when the protein of GST-NS1-GFP-RPS7 was obtained eventually. The Real-time RT-PCR was used to detect the expression level of RPS7, MDM2 and P53 mRNA after the recombinant plasmid of pEGFP-NS1 was expressed in 293 T cells. It is showed that the expression of NS1 protein causes the low expression of RPS7 and MDM2 mRNA and eventually causes the high expression of P53 mRNA. This research lays the foundation for clarifying the pathogenic mechanism of Tembusu virus and the function of NS1 protein in virus propagation process. [ABSTRACT FROM AUTHOR]

Thirty clinical isolates of H. parasuis from pig farms in eastern China were screened for antimicrobial susceptibility. A novel plasmid, designated pHPSGC, was extracted from one isolate with evidence of resistance (elevated minimum inhibitory concentration) to florfenicol. DNA sequencing demonstrated that pHPSGC (5297 base pairs) contains three open reading frames (ORFs), corresponding to the genes rep , floR and lysR . The rep gene of pHPSGC shared 99% sequence identity with the rep gene of pHPS1019. In addition, the region containing floR and lysR in pHPSGC shared 99% similarity with the corresponding region of pCCK381. pHPSGC may be derived from a recombination event between pHPS1019 and pCCK381. A florfenicol resistance gene in H. parasuis may have been transferred via recombination between different plasmids. [ABSTRACT FROM AUTHOR]

• Ts14–3–3.3 could be a potential antigen molecule to Taenia solium vaccine. • Recombinant plasmids pMZ-X3-Ts14–3–3.3 and pMZ-X3-sp-Ts14–3–3.3 can induce specific cellular and humoral immune responses in BALB/c mice with immunization. • Recombinant plasmid pMZ-X3-sp-Ts14–3–3.3 has a better immune effect. There is a lack of vaccine against human cysticercosis, thus making a huge population at the risk of infection. In this study, we chose a novel potential antigen molecule Taenia solium 14–3–3.3 (Ts14–3–3.3) and optimized it as sp-Ts14–3–3.3 (sp is immunoglobulin H chain V-region precursor, partial) in order to construct recombinant plasmids pMZ-X3-Ts14–3–3.3 and pMZ-X3-sp-Ts14–3–3.3. BALB/c mice were divided into four groups for immunization: pMZ-X3-Ts14–3–3.3, pMZ-X3-sp-Ts14–3–3.3, pMZ-X3 plasmid control group and PBS control group. Compared with two control groups, the proliferation level of splenic lymphocytes increased significantly in pMZ-X3-Ts14–3–3.3 and pMZ-X3-sp-Ts14–3–3.3 groups and reached the maximum in week 6. And the same case arose as cytokines associated with Th1 response, IFN-γ, and IL-2 while those with Th2 response, IL-4, IL-10 went up and reached the maximum in week 4. The levels of serum specific IgG, IgG1 and IgG2a rose and reached the maximum in week 6, 4 and 6, respectively. Meanwhile, the proportion of CD4+/CD8+ splenic T lymphocytes increased and reached the peak in week 6. The results indicated that the recombinant plasmids pMZ-X3-Ts14–3–3.3 and pMZ-X3-sp-Ts14–3–3.3 can induce specific cellular and humoral immune responses in BALB/c mice with immunization. Notably, the recombinant plasmid pMZ-X3-sp-Ts14–3–3.3 has a better immune effect, which proves that Ts14–3–3.3 enjoys a higher possibility as a potential antigen molecule to T. solium vaccine. [ABSTRACT FROM AUTHOR]

Abstract: Eastern equine encephalitis virus (EEEV) poses a serious public health threat in many countries. Therefore, developing efficient vaccine against EEEV remains an important challenge in the field of disease control. To identify immunogenic proteins in EEEV, we constructed an expression vector containing the protein coding genes C, E3, E2, 6k, and E1 (pcDNA3.1-C-E). After verifying the target gene expression in 293 T cells, we immunized BALB/c mice with the pcDNA3.1-C-E vector as a DNA vaccine in conjunction with either CpG or poly (I:C) or a mixture of both adjuvants and monitored various aspects of the immune response. After two immunizations, the mice vaccinated with antigen plus mixed CpG/poly (I:C) adjuvant exhibited significantly stronger IFN-gamma responses and generated high-level CD4+ cell responses for the cytokines IL-2, IL-4, and IFN-γ and CD8+ T cell responses for the cytokines IL-2 and IFN-γ compared to the mice vaccinated with the corresponding antigen plus CpG or poly(I:C) alone. In addition, the higher antibody titers against EEEV effectively neutralized the EEEV pseudoviruses in the group immunized with antigen plus mixed CpG/poly (I:C) adjuvant after tertiary immunization. This study demonstrates that the pcDNA3.1-C-E plasmids in conjunction with mixed CpG/poly (I:C) adjuvant priming maximize the cellular immune response and specific antibody generation in mice. Moreover, this mixed adjuvant priming provides a promising strategy for enhancing the immune effectiveness of a DNA vaccine against EEEV. [Copyright &y& Elsevier]

Abstract: Karenia mikimotoi is a dinoflagellate that is widely distributed in China that has recently become increasingly involved in harmful algal blooms (HABs). Therefore, it is urgent and necessary to study this dinoflagellate in order to monitor and forecast HABs. In this study, we designed a set of primers and a probe specific to K. mikimotoi (within its internal transcribed spacer (ITS) regions) and developed a real-time quantitative PCR (qPCR) method for the purpose of identification and quantification. We successfully prepared a 10-fold series of recombinant plasmid solutions containing ITS regions of K. mikimotoi as standard samples; these were used to generate a standard curve. Additionally, we quantified the genomic DNA of K. mikimotoi cells and utilized this DNA to prepare another 10-fold standard sample series and standard curve. The mathematic relationship between the cell numbers and their corresponding plasmid copy numbers was also established. We used laboratory and field samples to test the effectiveness of the qPCR method. For the laboratory samples, greater than 102 cells of K. mikimotoi could be quantified precisely utilizing both centrifugation and filtration techniques. The qPCR method was efficient even at low cell density and could detect 5 cells of K. mikimotoi. Field samples were collected from the East China Sea during a spring cruise in 2011; K. mikimotoi was detected (147±20cellsL−1) in these samples while light microscopy failed to detect K. mikimotoi. The qPCR and conventional PCR amplification products (utilizing dinoflagellate-specific primers LH2 and Dlam) were sequenced, and two sequences of 111bp and 684bp were determined to be highly similar to K. mikimotoi. Therefore, we conclude that the microalgae detected by the qPCR method was K. mikimotoi. [Copyright &y& Elsevier]

Abstract: Study of the mechanism of transmission of hepatitis B virus is important for public health. An improved experimental model is described for studying vertical transmission of hepatitis B virus (HBV) via human spermatozoa. Recombinant plasmid pIRES2-EGFP-HBx which would express enhanced green fluorescent protein (EGFP) used as a marker for the expression of hepatitis B virus X (HBx) gene was constructed successfully and confirmed by PCR, EcoR I and Sal I digestion, and DNA sequencing. After exposure to the plasmid, human spermatozoa were used to fertilize zona-free hamster ova in vitro. Two-cell embryos were collected and classified into group A with green fluorescence and group B without green fluorescence under fluorescence microscope. The results showed that HBx DNA positive bands were detected in the embryos with green fluorescence (PCR and RT-PCR) and positive controls (PCR) indicating presence and expression of HBx gene. In contrast, HBx gene expression was not detected in the embryos without green fluorescence and negative controls (PCR and RT-PCR). This improved experimental model is more efficient, accurate and reliable for studying further perinatal transmission of HBV or other viruses causing chronic human disease possibly via the male germ line. [Copyright &y& Elsevier]

Abstract: Construction, electrochemically biosensing and discrimination of recombinant pEThIL-2 plasmid, with 5839bp size, on the basis of interleukine-2 (IL-2) DNA insert are described. Plasmid pEThIL-2 was constructed by PCR amplification of IL-2 encoding DNA and subcloning into pET21a(+) vector using BamHI and SacI sites. The recombinant pEThIL-2 plasmid was detected with a label-free DNA hybridization biosensor using a non-inosine substituted probe. The proposed sensor was made up by immobilization of a 20-mer antisense single strand oligonucleotide (chIL-2) related to the human interleukine-2 gene on the pencil graphite electrode (PGE) as a probe and then the sensing of recombinant pEThIL-2 plasmid was conducted by anodic differential pulse voltammetry (ADPV) based on guanine oxidation signal. Selectivity of the detection was assessed with pET21a(+) non-complementary plasmid, with 5443bp size, lacking IL-2 encoding DNA. Different factors such as electrode activation conditions and washing strategy were tested in order to eliminate the nonspecific adsorption of pET21a(+). We have found that the PGE activation for 300s produces a condition in which desorption of nonspecifically adsorbed plasmids from the electrode surface can be achieved by 300s washing of the electrode in 20mM Tris–HCl buffer solution (pH 7.0) containing 20mM NaCl. Diagnostic performance of the biosensor is described and the detection limit is found to be 10.31pg/μL. [Copyright &y& Elsevier]

Segregational stability of the recombinant plasmid pKA18 bearing the pga gene encoding penicillin G acylase (PGA) and the production stability for PGA was studied in Escherichia coli strain W and its descendants. The hosts differ in the number of indigenous plasmids (pRK1–3) and the specific activity of PGA. The production of PGA by the recombinant strain RE3(pKA18) is the highest among the hosts and the increase of activity is proportional to the increase of copy number (CN) of the plasmid pKA18. The plasmid is segregationally stable in spite of the presence of the indigenous plasmid pRK2 that belong to the same ColE1 plasmid family. Both plasmids (total sum of CN equals 271) are maintained without selection pressure during 102 generations. Subculture-based selection experiments taking advantage of a very high segregational and production stability of plasmid pKA18 in the host RE3 were used to study the optimization of the host–plasmid interaction. The clones with increased synthesis of the enzyme average value of 833 U/g DM were isolated: the expression of PGA per plasmid molecule increased in these strains from 3 to 4 U. Once this expression is reached, the population of segregationally stable, high-producing strains becomes heterogeneous: segregationally stable, low-producing clones and segregationally unstable, high-producing clones appear in population. [Copyright &y& Elsevier]

We exploited the humoral immunoreaction of mice induced by TCR DNA vaccine of β chain of T cell lymphoma. The plasmids of pcDNA3.1/TCR V β 8 was constructed. The BALB/c mice were randomly divided into four groups which were pcDNA3.1, pcDNA3.1/TCR V β 8, pcDNA3.1/TCR V β 8 + CpG + liposome and phosphorothioate CpG groups with six mice in each group. Vaccines were injected in bilateral musculus quadriceps femoris of mice in the 0, second, and fourth week, respectively. The antibody formation was tested by indirect immuofluorescence in the 0, second, fourth, sixth and eighth weeks, respectively, before and after immunization. Production of antibody against TCR V β 8 antigen was observed in the groups of pcDNA3.1/TCR V β 8 and pcDNA3.1/TCR V β 8 + CpG + liposome. The antibody titer began to rise in the fourth week and attain the maximal value in the sixth week. The antibody titer in the group of pcDNA3.1/TCR V β 8 + CpG + liposome was higher than that in the group of pcDNA3.1/TCR V β 8 in the fourth and eighth weeks (both P<0.01); the antibody titer in the group of pcDNA3.1/TCR V β 8 + CpG + liposome was markedly higher than that in the group of pcDNA3.1/TCR V β 8 in the sixth week (P<0.001). The result indicate that TCR V β 8 antigen can induce formation of special antibody in mice. CpG and liposome can improve the humoral immunoreaction induced by TCR V β 8 gene vaccine. [Copyright &y& Elsevier]