[Genetic analysis of NS1 fragment of human H5N1 influenza virus isolated in Anhui province and its expression in Escherichia coli].

NS1 gene was amplified from an H5N1 influenza virus, A/Anhui/01/2005 for cloning, sequence analysis and later expression in Escherichia coli. The result showed that NS1 of A/Anhui/01/2005 strain had the close phylogenetic relationship with that of H5N1 avian influenza strains recently isolated in Fujian and Hunan. Correspondingly, its amino acid sequence showed the highest homology with that of Fujian and Hunan strains. The amino acid position of 92 involved in virus virulence was Asp, contrast to Glu in A/HK/156/97. Five amino acid deletion from 80 to 84 was also found in A/Anhui/01/2005, which was considered as a contributor to virus resistance against cytokine, such as IFN, TNF, etc. A motif binding to CBSF, conveted to GFWEN, which was different from previous GLEWN found in other 19 strains. Besides, the PL motif of ESEV, binding to PDZ domain, is the same as previous high-mortality H5N1 isolates. Furthermore, this NS1 was efficiently expressed in Escherichia coli and the highly purified product demonstrated wonderful activity as confirmed by Western blot. As a result, the work paves the way for further understanding the role of NSI in human H5N1 infection and development of new antiviral drugs against influenza virus.

[Evaluation on the biosafety of classical swine fever DNA vaccine].

The biosafety of DNA vaccine is one of the key questions which should be solved before it is used in the clinical trail. In order to evaluate the biosafety of DNA vaccine, the CSFV DNA vaccine was used in the studying target, two main aspects of the vaccine were explored in the study. Firstly, the possibility of integration of two kinds of DNA vaccine plasmids into pig genome was analyzed by PCR technology after the different vaccines were injected through the intramuscular introduction. The results showed that both plasmids DNA were detected as the form were not integrated into pig genome, it can be detected 30 copies plasmid DNA in 1 microg total genomic DNA as the sensibility of PCR, indicated the safety of the DNA vaccine. Afterward the environmental fecal and soil samples in the experimental pens were picked up. Then the antibiotic resistant bacteria were isolated and its resistant genes were analyzed by PCR and gene sequencing. The results demonstrated that the transfer and spreading of two DNA vaccine plasmids studied into environmental bacteria from receptor pigs were not found. The results showed that the CSFV DNA vaccine is safe to both pigs and the surrounding environment.

[Subcloning of M1 gene fragment of H5N1 influenza virus and its expression in Escherichia coli].

OBJECTIVE: To generate the Escherichia col vector expressing human H5N1 influenza virus M1 protein. To provide useful tools for detection of human H5N1 influenza virus and study on biological function of M1 protein. METHODS: M1 gene fragment was amplified by PCR using the influenza virus gene segment 7 as template, and was subcloned into pQE80-L vector. The recombinant plasmid pQE80-L/M1 was transformed into Escherichia coil BL21 (DE3) strain. The expression of M1 was induced by isopropy-beta3-D-thiogalactopyranoside. We purified the recombinant M1 protein with polyhistidine tag with Ni2+ affinity chromatography. Mouse were immunized with the purified M1 protein for preparing antibodies against M1. RESULTS: The recombinant Ml protein was recognized by antiserum against H5N1 subtype influenza virus, elicit specific antibody in immunized animals. CONCLUSION: These confirmed that we successfully constructed the Escherichia coli vector expressing human H5N1 influenza virus M1 protein.

[Construction and immunogenicity study of DNA vaccine expressing human H5N1 influenza virus hemagglutinin].

Based on the first isolated human H5N1 influenza virus strain A/Anhui/1/2005 in China, HA and HA1 genes were amplified and cloned into the eukaryotic expression vector pStar. The recombinant plasmids pStar-HA and pStar-HA1 were transfected into COS7 cells. Western blot and IFA showed that the two recombinant DNA plasmids were successfully expressed in eukaryotic cells. BALB/c mice were immunized with the plasmids DNA by intramuscular injection. Anti-HA specific antibody in peripheral blood of immunized mice was tested by ELISA. The results showed that the recombinant plasmids successfully induced the anti-HA humoral immune response, and there was no significant difference between HA and HA1 as immunogen. This work provides basis for future development of novel avian flu vaccine.

[Expression of sTNFR-IgGFc fusion gene in endothelial cell and its application in gene therapy for rheumatoid arthritis].

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine, acting as a regulator of inflammation and immunity. TNFalpha plays a critical role in the pathogenesis of rheumatoid arthritis. Blocking of TNFa activity suppressed inflammatory tissue damage. In present study, the chimeric gene of soluble TNF receptor and IgG Fc fragment (sTNFR-IgG FC) was cloned into the mammalian cell expression vector pStar. When the plamid pStar/sTNFR-IgGFc-GFP was transfected into endothelial cells, a considerable expression of the sTNFR-IgG Fc fusion protein was detected. Moreover, the product in 100microL expression supernatant could completely antagonize the cytolytic effect of 1ng TNFa on L929 cells, even at 1/64 dilution. Then the plasmid was delivered into CIA-induced rheumatoid arthritis mice by tail vein injection. The expression of sTNFR-IgG Fc was detected in liver by RT-PCR. Animals in treatment group showed reduced symptoms of arthritis and more active. This treatment induced decrease of synovial incrassation and prevented the cartilage destruction of the mice RA model. These results show that tail vein injection is an effective way for gene therapy and sTNFR-IgGFc expression plasmid is potential for the treatment of rheumatoid arthritis.

[Screening for new binding proteins which interact with BM2 of influenza B virus with yeast two-hybrid system].

OBJECTIVE: To explore the role of BM2 protein in the life cycle of influenza B virus. METHODS: The authors screened human kidney MATCHMAKER cDNA library for new binding partners of BM2 of influenza B virus by using the yeast two hybrid system with truncated BM2 (26-109 aa) as the bait. RESULTS: Six positive plasmids encoding N-acetylneuraminate pyruvate lyase, angiopoietin 3, zinc finger protein 251, ribosomal protein S20, protein arginine N-methyltransferase 1 variant 1 (PRMT) and transcription factor-like 1 (TCFL1) were obtained. CONCLUSION: The results suggest that BM2 may play an important role in the life cycle of influenza B virus.