Hsp70 Fused with the Envelope Glycoprotein E0 of Classical Swine Fever Virus Enhances Immune Responses in Balb/c Mice / 病毒学报

Heat-shock protein (Hsp) 70 potentiates specific immune responses to some antigenic peptides fused to it. Here, the prokaryotic plasmids harboring the envelope glycoprotein E0 gene of classical swine fever virus (CSFV) and/or the Hsp70 gene of Haemophilus parasuis were constructed and expressed in Escherichia coli Rosseta 2(R2). The fusion proteins were then purified. Groups of Balb/c mice were immunized with these fusion proteins, respectively, and sera collected 7 days after the third immunization. Immune effects were determined via an enzyme-linked immunosorbent assay and flow cytometric analyses. E0-Hsp70 fusion protein and E0+Hsp70 mixture significantly improved the titer of E-specific antibody, levels of CD4+ and CD8+ T cells, and release of interferon-γ. These findings suggested that Hsp70 can significantly enhance the immune effects of the envelope glycoprotein E0 of CSFV, thereby laying the foundation of further application in pigs.

Expression and adjuvant effects of the fusion peptide TBP5 / 生物工程学报

Thymopentin (TP5) and bursopentin (BP5) are both immunopotentiators. To explore whether the TP5-BP5 fusion peptide (TBP5) has adjuvant activity or not, we cloned the TBP5 gene and confirmed that the TBP5 gene in a recombinant prokaryotic expression plasmid was successfully expressed in Escherichia coli BL21. TBP5 significantly promoted the proliferation of thymic and splenic lymphocytes of mice. The potential adjuvant activity of the TBP5 was examined in mice by coinjecting TBP5 and H9N2 avian influenza virus (AIV) inactivated vaccine. HI antibody titers, HA antibodies and cytokines levels (IL-4 and IFN-γ) were determined. We found that TBP5 markedly elevated serum HI titers and HA antibody levels, induced the secretion of both IL-4 and IFN-γ cytokines. Furthermore, virus challenge experiments confirmed that TBP5 contributed to inhibition replication of the virus [H9N2 AIV (A/chicken/Jiangsu/NJ07/05)] from mouse lungs. Altogether, these findings suggest that TBP5 may be an effective adjuvant for avian vaccine and that this study provides a reference for further research on new vaccine adjuvants.

Effect on Softening Liver and Reducing Enzyme Mixed Agent in Prevention of CCl4-induced Liver Damage Rats / 世界科学技术-中医药现代化

This study was aimed to observe the effect of Softening Liver and Reducing Enzyme Mixed Agent (SLREXA) in the prevention of acute liver injury rats induced by carbon tetrachloride (CCl4). A total of 60 male SD rats were randomly divided into 6 groups, which were the SLREXA low-, middle-, high-dose group, glucurolactone group, normal group and model group. Intraperitoneal injection of CCl4 was used to induce acute liver injury rat mod-el. Intragastric administration of SLREXA was given to each Chinese medicine group. Intragastric administration of distilled water was given to the normal group and the model group. Intragastric administration of glucurolactone aque-ous solution was given to the glucurolactone group. On the 12th day of the experiment, after 16-hour fasting, rats were killed. Pathological changes in liver tissues were examined. Blood serum was determined for alanine aminotrans-ferase (ALT) and aspartate aminotransferase (AST). The liver homogenate was determined for superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione (GSH), catalase (CAT) and malondialdehyde (MDA) in liver tis-sues of rats. RT-PCR was used to detect the expression level of mRNA in liver heme oxygenase-1 (HO-1). The re-sults showed that in the microscopic examination of liver tissues, compared with the model group, different doses of SLREXA can alleviate pathological damages of liver in varying degrees. Levels of blood serum ALT and AST content in different doses of SLREXA groups and glucurolactone group were significantly lower than those of the model group (P < 0.05 or P < 0.01). Compared with the model group, contents of GSH-Px, GSH, SOD, CAT in the liver ho-mogenate were significantly increased, and MDA content was decreased significantly (P< 0.05 or P< 0.01) in differ-ent doses of SLREXA groups and glucurolactone group; compared with the model group, the HO-1 mRNA relative expression quantity in the normal group and each treatment group increased obviously, with statistical significance (P< 0.05 or P< 0.01). It was concluded that SLREXA can prevent CCl4-induced liver injury rats with definite thera-peutic effect.

A novel double expression shuttle vector to get marker-free recombinant modified vaccinia virus Ankara / 生物工程学报

<p><b>UNLABELLED</b>A novel double expression shuttle vector named pLR-gpt was constructed for marker-free recombinant modified vaccinia virus Ankara generation. A delectable Eco gpt marker was adopted with Cre/LoxP DNA recombination system and a BHK-21 cell line that can express Cre enzyme. Eco gpt gene controlled by P7.5 promoter from Vaccinia virus was cloned between two LoxP sites in the same direction. Additionally, two multiple cloning site under control of other two Vaccinia virus promoters were constructed outside LoxP sites. With this new transfer vector, Eco gpt marker in rMVA can be deleted on BHK-Cre with interaction between Cre enzyme and LoxP sequence. In order to verify the efficacy of this system, ORF5 and ORF6 gene of Porcine reproductive and respiratory syndrome virus (PRRSV) NJ-a strain were cloned into two multiple cloning sites of pLR-gpt to construct recombinant plasmid pLR-ORFS/ORF6. Homologous recombination between pLR-ORF5/ORF6 and wtMVA on BHK-21 cell was mediated by liposome by infecting cells with 0.01 MOI wtMVA two hours before transfection. After twelve cycles of selection, recombinant MVA with selecting marker Eco gpt was obtained and named as rMVAgpt-GP5/M. By infecting BHK-Cre, the Eco gpt marker in rMVAgpt-GP5/M was deleted and this rMVA was named as rMVA-GP5/M. Expression of GP5 and M protein was identified with Western blotting and IFA. Results from PCR and biological study for rMVA indicated that Eco gpt marker was completely deleted.</p><p><b>CONCLUSIONS</b>double expression transfer vector for marker-free recombinant Modified vaccinia virus Ankara generation was successfully constructed, and works well in MVA expression system.</p>

Influence of fusion protein of IBDV VP2 and chicken interleukin-2 on immune response in chicken / 生物工程学报

In order to research immunogenicity of the recombinant rVP2-IL-2 fusion protein, we obtained the rVP2-IL-2 fusion protein using Pichia pastoris expression system, and then evaluated its potential to induce immune responses in chicken. The effect was determined in the form of protective anti-IBDV VP2 titers, antibodies (IgG1 and IgG2a), lymphocyte proliferation, the levels of interferon-gamma and interleukin-4 cytokines, and challenge experiment. Antibody titers and proliferation lymphocyte level suggested that the fusion protein could elicit specific humoral immune and cellular immune responses, antibody sub-type results indicated that the rVP2-IL-2 fusion protein induced secretion both of IgG1 and IgG2a. The seem result elicited from cytokines ELISA test, secretion of both of Th1 (gamma-IFN) and Th2 (IL-4) were induced by the rVP2-IL-2 fusion protein. Challenge experiment result shown that chicken immunized the rVP2-IL-2 fusion protein obtained 85% protection. These results confirm that the fusion protein enhances the protection against IBDV through both humoral and cell-mediated immunity, and thus could serve as a candidate for the development of IBDV subunit vaccine.

Molecular Characterization of the Duck Enteritis Virus UL4 Gene / 中国病毒学·英文版

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious and fatal disease. In the present article, the DEV UL4 gene was cloned and sequenced from a vaccine virus. A degenerate oligonucleotide primer for the consensus site of herpesvirus UL3 gene and a specific primer located in UL5 were used in the polymerase chain reaction (PCR) to amplify a DNA product 2 086 bp in size. DNA sequence analysis revealed that a 714 bp open reading frame (ORF) of DEV encoding a 237 amino acid polypeptide is homologous to the family of herpesvirus UL4 proteins and therefore has been characterized as a DEV UL4 gene. Alignment of the DEV UL4 protein sequence with those of other alphaherpesviruses showed that 10 amino acid residues are completely conserved. Phylogenetic tree analysis showed that the seventeen alphaherpesviruses viruses analyzed were classified into four large groups, and the duck enteritis virus branched separately, closely related to the Mardiviruses group comprising Gallid herpesvirus 2 (GaHV-2), Gallid herpesvirus 3 (GaHV-3) and Meleagrid herpesvirus 1 (MeHV-1). The present study showed that the evolutionary relationship of the UL4 protein could be used for classification of alphaherpesviruses.

Construction and biological characteristic for the recombinant modified vaccinia virus ankara co-expressing modified GP5 and M protein of porcine reproductive and respiratory syndrome virus / 生物工程学报

Modified ORF5 (MORF5) and ORF6 gene of PRRSV were cloned into two multiple cloning sites of MVA transfer vector pLR-gpt to construct the recombinant plasmid pLR-MORF5/ORF6. Homologous recombination between pLR-MORF5/ORF6 and the wtMVA on BHK-21 cell line was mediated with liposome by infecting the cell with 0.01 MOI wtMVA two hours before transfecting the recombinant plasmid into the cell. When the cytopathic effect (CPE) was obvious, virus was collected from the cell plate and the recombinant virus was selected with drug selecting medium (2% MXHAT). After 12 cycles of selection, rMVA with a selection marker Eco gpt was obtained and named as rMVAgpt-MGP5/M. By infecting BHK-Cre expressing Cre recombinant enzyme, the Eco gpt marker in rMVAgpt-MGP5/M was deleted and this rMVA was named as rMVA-MGP5/M. The insertion of MORF5 and ORF6 into the MVA genome was confirmed with PCR analysis and the expression of MGP5 and M protein was identified with Western blot and IFA. Through biological study on the recombinant MVA, no obvious difference was observed between rMVA-MGP5/M and the wtMVA regarding to the CPE and growth curve. The recombinant MVA constructed in this study could coexpress the modified GP5 and M protein and the expressed product had good immunocompetence. Furthermore, the insertion of the MORF5 and ORF6 into MVA genome had no obvious effect on the replication and biological characteristics of this virus.

Characterization of murine leukemia virus recombinants bearing PRRSV GP5 glycoproteins / 生物工程学报

The highly virulent PRRSV isolate strain HN-1/06 was cultivated on Marc-145. To study the viral entry mechanisms, the GP5 gene of PRRSV isolate was amplified by RT-PCR and cloned into pcDNA3.0 to generate the expressing plasmid pcDNA-GP5. pcDNA-GP5 was transfected into 293T by the calcium phosphate precipitation method. Analysis of flow cytometry confirmed that the GP5 proteins were expressed in surface of the 293T cells. Then 293T cells were transfected with pcDNA-GP5, pHIT60 and pHIT111 plasmids to generate pseudotyping virus. The pseudotyping virus supernatant was harvested 48 hours post-transfection and was detected by Western blotting and infection assay. Western blotting indicated that the GP5 glycoproteins were incorporated into the retroviral pseudotyped virus. Infection assay showed that the pseudotyped virus infected 293T and Mark-145 cell. The pseudotyped virus could be used to further study infectious mechanism of PRRSV.

Study on recombinant chicken ? interferon production in yeast Pichia pastoris and detection for its antiviral activity / 中国免疫学杂志

Objective:To obtain eukaryotic expressing protein of chicken interferon ? (ChIFN-?) and research its anti-virus activity.Methods: Chicken interferon ? mature protein gene was cloned and amplified by reverse transcripition-polymerase chain reaction(RT-PCR) from the total mRNA in the lymphocyte of chicken blood stimulated with ConA for 4～10 hours.The gene was inserted into the expression vector pPICZa-A,which had been cleaved by EcoR I and Xba I.The recombinant vector was linearized by Sac I and transferred into yeast Pichia pastoris,strain X33,anti-virus activity of the recombinant cytokine was detected by the classical experiment cell pathological effect inhibition assay.Results:The result showed that the preparation of recombinant interferon had higher anti-virus activity(10?48 U/ml).Conclusion: The recombinant chicken interferon ? with anti-virus bioactivity has been obtained.