Prokaryotic soluble expression of protein D of Haemophilus influenzae type b / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To express the recombinant D protein in prokaryotic expression system solubly and make preparation for producing D-carrier conjugate vaccine next step.</p><p><b>METHODS</b>The hpd gene fragment removed of signal peptide from genomic DNA of Hib CMCC was inserted into pET43. 1a. The recombinant plasmid was transformed to competent E. coli BL21 (DE3) for expression under induction of IPTG. The expressed recombination protein was precipitated with ammonium sulfate, purified by DEAE anion exchange column chromatography and identified for reactogenicity by Western Blot.</p><p><b>RESULTS</b>The expressed recombination protein, in a soluble form, constained about 50% of total somatic protein and showed specific reaction with the HIB antisera after preliminary purification.</p><p><b>CONCLUSION</b>The D protein recombined expression plasmid was constructed successfully and expressed D protein in prokaryotic cells in a solube form.</p>

Preparation of vero cell-adapted influenza H5N1 virus strain by genetic reassortment / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare Vero cell-adapted influenza H5N1 virus strain by Genetic Reassortment and produce influenza H5N1 vaccine using Vero cell as a substrate.</p><p><b>METHODS</b>Embryonated specific pathogen-free (SPF) hen's eggs and Vero cells were co-infected with Vero cell-adapted influenza virus A/Yunnan/1/2005 Va(H3N2) and A/Anhui/1/2005 (H5N1) via reverse genetics. The reassortant was screened with goat antibody against strain A/Yunnan/1/2005 Va(H3N2) and identified for subtype by hemagglutination-inhibition (HI) assays and gene analysis of HA and NA.</p><p><b>RESULTS</b>A Vero cell-adapted influenza H5N1 virus strain was obtained, and there was no significant difference in serum antibody titers of monovalent inactivated vaccine reassorted before and after (F = 0.857, P > 0.05).</p><p><b>CONCLUSION</b>The Vero cell-adapted influenza virus of epidemic strain may be reassortment between Vero cell-adapted and epidemic strains.</p>

Effective inactivatian test of inactivated hepatitis A vaccine using integrated cell culture/strand-specific reverse transcriptase-polymerase chain reaction / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish an quick, sensitive and specific assay for effective inactivatian test of inactivated hepatitis A vaccine.</p><p><b>METHODS</b>effective inactivatian test of inactivated hepatitis A vaccine were carried out using integrated cell culture/strand-specific RT-PCR (ICC/strand-specific RT-PCR) assay compared with traditional ELISA and nest RT-PCR assay.</p><p><b>RESULTS</b>all the samples were infectious negative detecting by both ICC/ strand-specific RT-PCR and ELISA assay,while some samples appeared false positive detecting by nest RT-PCR.</p><p><b>CONCLUSION</b>ICC/strand-specific RT-PCR assay is a novel, rapid, sensitive and reliable method for effective inactivatian test of inactivated hepatitis A vaccine. Shorting detection period largely, this assay may be used as an alternative method for routine inactivated hepatitis A vaccines test.</p>

Research on an improved Lowry method to determine content of protein in Sabin IPV / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a method for the content determination of protein in Sabin IPV.</p><p><b>METHODS</b>Using lowry method combined with being precipitated by trichloroacetic acid to determine the content of protein in Sabin IPV. Changing different conditions to optimize the experiment to establish a improved lowry method. And the sample recovery test was also conducted.</p><p><b>RESULTS</b>The method can exclude the interference of free aminoacid, phenols and some other additives. The calibration curve was in good linearity of protein within the range of 2.5 microg/ml-40 Microg/ml, r = 0.9998. Under the best conditions, the mean recovery was 95.32%, the CV in a batch and between batches were both < 10%.</p><p><b>CONCLUSION</b>The method can be used to determine the micro content of protein in vaccines.</p>

Effect of 2-phenoxyethanol on potency of Sabin inactivated poliomyelitis vaccine and its safety / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the effect of 2-phenoxyethanol on potency of Sabin inactivated poliomyelitis vaccine (IPV).</p><p><b>METHODS</b>Sabin IPV samples containing 5 mg or 7 mg 2-phenoxyethanol each dosage respectively were placed separately at 4 degrees C, 37 degrees C for 2 days and 7 days. D-antigen contents were tested with ELISA method. Then neutralizing antibodies in mice and guinea pigs were detected. The safety experiment was performed according to unusual toxicity test of China requirement for biological product.</p><p><b>RESULTS</b>After addition of 2-phenoxyethanol, the I, II, and III D-antigen contents of Sabin IPV did not change. The antibody levels in mice and guinea pigs were not different between experimental group and control group. Animals were safe during observation period.</p><p><b>CONCLUSION</b>2-Phenoxyethanol had no effect on potency and safety of Sabin IPV. It can be used as antiseptic for Sabin IPV.</p>

Construction of bicistronic vector and its application to combined DNA vaccine / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To study preparation of polyvalent DNA vaccine and the control of multiple gene expression.</p><p><b>METHODS</b>A bicistronic vector pcDNA3.0BA was constructed from pcDNA3.0. HCV PC154 gene and HBV preS2S gene were inserted into this vector to form bicistronic expression construct pcDNA3.0BAPC154S2S and monocistronic expression construct pcDNA3.0BAPC154 or pcDNA3.0BAS2S. These plasmids were transiently expressed in COS-7 cells and injected into muscles of BALB/c mice.</p><p><b>RESULTS</b>pcDNA3.0BA contains two cistronic units, which can co-express two kinds of genes, with the first immunogen gene and the second gene serving as additional immunogen or as modulator for the immune responses. HBV surface Ag and HCV core Ag were coexpressed in vitro. The antibody responses and lymphoproliferation to antigens were similar between bicistronic and monocistronic expression construct in mice.</p><p><b>CONCLUSION</b>pcDNA3.0BA is a novel vector, which can coexpress two proteins and elicit polyvalent immune responses.</p>

Expression and self-assembly of HCV structural proteins into virus-like particles and their immunogenicity / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The synthesis of virus-like particles (VLPs) provides an important tool to determine the structural requirements for viral particle assembly and virus-host interactions. Our purpose was to express simultaneously all three structural proteins of hepatitis C virus (HCV) in insect cells to investigate the proteins assembly into VLPs and the immunogenicity of these particles.</p><p><b>METHODS</b>HCV gene sequences encoding the structural proteins C, E1, and E2 were amplified with PCR, and recombinant baculoviruses were constructed using recombinant DNA techniques. The expression of HCV structural proteins in insect cells was analyzed by immunofluorescence and SDS-PAGE. The interaction of expressed structural proteins was investigated by immunoprecipitation and immunoblotting. The VLPs in the insect cells were visualized by electron microscopy (EM). VLPs were then purified by sucrose gradient centrifugation and used to immunize BALB/c mice. Antibodies against HCV were tested for in mouse serum samples by an ELISA assay.</p><p><b>RESULTS</b>The recombinant baculoviruses reBV/C and reBV/E1-E2 were constructed successfully. Insect cells co-infected with reBV/C and reBV/E1-E2 expressed HCV C, E1, and E2 proteins with the expected molecular weights of 20kD, 35kD, and 66kD, respectively. The results of immunoprecipitation and immunoblotting assays revealed the coimmunoprecipitation of C, E1, and E2 proteins, indicating association of the three structural proteins. Electron microscopy of insect cells co-infected with reBV/C and reBV/E1-E2 demonstrated spherical particles (40 to 60 nm in diameter) similar to the HCV virions from serum samples or hepatic tissue samples of HCV infected humans. The VLPs were partially purified. Antibodies to HCV were detectable in the serum of mice immunized with VLPs.</p><p><b>CONCLUSION</b>HCV structural proteins simultaneously expressed in insect cells can interact with each other and assemble into HCV-like particles, which are shown to be immunogenic in mice.</p>