Construction of recombinant CHO cell strain for high expression of HBsAg / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To overexpress hepatitis B virus S gene in CHO cells cultured in serum-free media.</p><p><b>METHOD</b>Plasmid was constructed by cloning of HBV S gene and then it was transfected into CHO cells. After cell screen, the positive clones were identified and isolated into a serum-free media followed by the serological and morphological characterization of the expression product.</p><p><b>RESULT</b>CHO cell strains which can express HBsAg efficiently and stably were obtained. Spherical and filamentous HBsAg could be detected under electronic microscope. The titer of the expression product was up to 1:5000.</p><p><b>CONCLUSION</b>Serum-free media cultured CHO cell strain for overexpression of HBsAg was successfully constructed and the expression product was high antigenic.</p>

Antigenic analysis of two chimeric hepatitis B core particles presenting the preS1 neutralizing epitopes / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct full-length hepatitis B core particles presenting preS1 aa 21-47 epitope and truncated core particles presenting preS1 aa 37-45 epitope on their surface and compare their antigenicity.</p><p><b>METHODS</b>PreS1 aa21-47 epitope and aa 37-45 epitope were inserted respectively into full-length hepatitis B core (aa 1-183) and truncated HBcAg (aa 1-144), between the 78th (Asp) and 79th (Pro). The genes synthesized after the codon optimization were ligated to the pET43. 1a vector with the same cohesive terminal (NdeI and XhoI) and expressed in the E. coli expression system. The morphology of the proteins of interest were observed by electron microscope and characterized by ELISA and Western Blotting.</p><p><b>RESULTS</b>The morphology of the virus-like particles were confirmed by electron microscope. H2 were solid particles with a diameter of (31.61 +/- 1.27) nm, while H3 were hollow particles with a diameter of (28.46 +/- 1.16) nm. Statistical analysis showed that H2 is larger than H3 in the diameter (P < 0.01). The antigenicity of the inserted epitopes and carrier protein were identified by ELISA and Western Blotting.</p><p><b>CONCLUSION</b>Chimeric hepatitis B core particles presenting the preS1 neutralizing epitopes on their surface have been expressed, purified and identified, which lays the foundation for its application in vaccine research.</p>

Detection of hepatitis A virus RNA with an improved loop-mediated isothermal amplification assay / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a novel improved loop-mediated isothermal amplification (LAMP) technique to detect hepatitis A virus (HAV).</p><p><b>METHODS</b>A novel improved LAMP assay based on the addition of an acceleration primer was developed for hepatitis A virus nucleotide acid detection.</p><p><b>RESULTS</b>Precision and reproducibility analysis proved its high stability and reliability. Comparison between the improved and conventional LAMP assays revealed that the former was more sensitive with a detection limit of 5 TCID50/ml. The novel detection method displayed 100% consistency with the TaqMan real-time PCR assay when applied to clinical specimens collected from patients with confirmed acute HAV infection.</p><p><b>CONCLUSION</b>This novel technique is widely applicable as a simple diagnostic tool in the clinical field as well as for the surveillance and investigation of the infectious disease in developing countries where HAV is endemic.</p>

Gene optimization, protein expression, purification and characterization of the HDV antigen for diagnosis / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare HDAg with biological activities as a candidate of diagnostic reagent.</p><p><b>METHODS</b>To synthesize HDV gene fragment after codon optimization. To construct a thio-fused recombinant plasmid based on M48 expression vector. To express in E. coli induced by IPTG. To purify the protein by affinity chromatography followed by characterization in ELISA:</p><p><b>RESULTS</b>Plasmid construction was verified by enzyme digestion. SDS-PAGE indicated the molecular weight of the protein was the same as we expectation. ELISA proved its affinity with HDV antibodies.</p><p><b>CONCLUSION</b>HDAg was obtained successfully and it will pave the road to the research of HDV diagnostic reagent.</p>

Effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering.</p><p><b>METHODS</b>Based on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized. Both the original gene and the optimized gene expressed in prokaryotic cells, SDS-PAGE was made to analyze the protein expression yield and to decide which protein expression style was more proportion than the other. Furthermore, two antigens were purified by chromatography in order to compare the purity by SDS-PAGE and Image Lab software.</p><p><b>RESULTS</b>SDS-PAGE indicated that the molecular weight of target proteins from two groups were the same as we expected. Gene optimization resulted in the higher yield and it could make the product more soluble. After chromatography, the purity of target protein from optimized gene was up to 96.3%, obviously purer than that from original gene.</p><p><b>CONCLUSION</b>Gene optimization could increase the protein expression yield and solubility of genetic engineering HDV small antigen. In addition, the product from the optimized gene group was easier to be purified for diagnosis usage.</p>

Construction and characterization of hepatitis B surface antigen "a" epitope virus-like particles / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct virus-like particles of hepatitis B core antigen, with HBsAg "a" epitope exposed on the surface.</p><p><b>METHODS</b>Hepatitis B surface antigen "a" epitope were inserted into the Hepatitis B core antigen, between the 78th (Asp) and the 79th (Pro) amino acids. The gene was synthesized after the codon optimized, then it was ligated to the express vector after been enzyme digest. The virus-like particles were observed by electron microscope and detected by ELISA after been expressed and purified. Immune the rabbits by the VLPs, then detect the antibody.</p><p><b>RESULT</b>The virus-like particles were confirmed by electron microscope. Its antigenicity and immunogenicity were identified by ELISA.</p><p><b>CONCLUSION</b>The prokaryotic express plasmid with the fusion gene has been constructed successfully. The virus-like particles have been expressed, purified and identified, which lays the foundation for its application in the further.</p>

Expression of hepatitis C virus subunit fusion protein and analysis of its immunogenicity / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Obtain the hepatitis C virus high purified subunit fusion protein and detect its immunogenicity.</p><p><b>METHODS</b>With the vector of pET-11d, fusion protein was expressed in Escherichia coli BL21 (DE3) after induced by IPTG. The protein was then purified by DEAE negative ion exchange chromatography and Ni2+ affinity chromatography. Western Blot analysis was used to detect the antigenicity of the fusion protein. At the same time, the sera were collected and prepared from the immunized experimental animals in order to investigate the immunogenicity of the protein by EIA.</p><p><b>RESULTS</b>High purified hepatitis C virus subunit fusion protein was obtained successfully. The EIA indicated that the fusion protein could elicit specific antibodies in the animals with very high titers.</p><p><b>CONCLUSION</b>The hepatitis C virus subunit fusion protein expressed in prokaryotic system was proved to have strong immunogenicity. It could provide some helpful and useful information to the hepatitis C virus prophylactic and therapeutic vaccine development.</p>

Preparation and identification of the monoclonal antibodies against VP1 capsid protein of Enterovius 71 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare monoclonal antibodies (McAbs) against VP1 capsid protein of Enterovirus 71.</p><p><b>METHODS</b>Two peptides, SP55 and SP70, containing amino acid 163-177 and 208-222 of VP1, were synthesized respectively. Immunized BALB/c mice with the synthetic peptides to establish the hybridoma cell strains secreting specific McAb to VP1. After the specific McAbs were prepared, identified and analyzed the titer by indirect ELISA assay. The positive clones were selected and their neutralization titer were determined by neutralization test.</p><p><b>RESULTS</b>Two high titered anti-VP1 antibodies secreted by the hybridoma cells showed good neutralization reaction with enterovirus 71 on RD cells, and the neutralization titer were 1:8 and 1:16 respectively.</p><p><b>CONCLUSION</b>Two high titered anti-VP1 antibodies, with good neutralization activity, secreted by the hybridoma cells, which lays the foundation for further study.</p>

Cloning of PD-1 gene and its prokaryotic expression in Escherichia coli / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To clone human PD-1 gene, construct a prokaryotic expression plasmid and express in E. coli.</p><p><b>METHODS</b>The human PD-1 cDNA was cloned by RT-PCR from the total RNA, which was extracted from peripheral blood lymphocyte cell of the patient with chronic hepatitis B. Recombinant PD-1 protein was been expressed and purified after the prokaryotic expression plasmid had been constructed. It was identified by SDS-PAGE, DNA sequencing and amino acid sequencing.</p><p><b>RESULTS</b>The PD-1 gene was cloned and confirmed by DNA sequencing. The recombinant protein was expressed in E. coli. The purified protein was obtained, then been confirmed by amino acid sequencing.</p><p><b>CONCLUSION</b>The human PD-1 gene was successfully cloned and expressed in E. coli, which lays the foundation for further study on the function and application of PD-1.</p>

Antigenic properties of mutant hepatitis B virus surface antigen / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the antigenic properties of mutant hepatitis B virus surface antigen, to understand the sensitivity of the commercially available HBsAg assays to the variants and to reduce the undetectability of the variants.</p><p><b>METHODS</b>Recombinant eukaryotic expression plasmids for HBsAg. The recombinant eukaryotic expression plasmids pSS1adr, pSS1adw2, pSS1adw2- 145Arg, pSS1adr-126 Asn and pSS1adr-126Ser were transfected into COS-7 cells. HBsAg in the supernatants of transfected cells was detected by using different commercial ELISA kits.</p><p><b>RESULTS</b>The absorbance value of pSS1adr-126 Asn and pSS1adr-126Ser plasmids were similar to that of the wild type HBsAg, the absorbance value of pSS1adw2-145Arg plasmids was lower than that of the wild type HBsAg.</p><p><b>CONCLUSION</b>It is estimated that the antigenicity of HBsAg mainly depended on the amino acid sequence of "a" antigen determinant and its conformation, so 145 amino acid substitutions led to the change of conformation and the antigenicity of variant HBsAg was lower than that of the wild type.</p>

Expression of recombinant rubella virus E1 protein and initial application for detecting of antibody / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To apply recombinant rubella virus envelope protein-1 (E1) to detect human rubella virus IgG antibody.</p><p><b>METHODS</b>Rubella virus E1 protein was expressed in E. coli, purified E1 protein was used as the antigen for the detecting of anti rubella in human sera in the way of enzyme linked Immunosorbant assay (ELISA).</p><p><b>RESULTS</b>The antigenicity of the recombinant protein was checked by WHO rubella sera panel. We detected 200 sera samples, which came from Guangxi Guilin. 93% of these samples were positive.</p><p><b>CONCLUSION</b>The antigenicity of recombinant E1 is a satisfied candidate antigen for the detecting of human rubella virus antibody. The prevalence of anti rubella virus IgG in Guangxi is 93%. It is at the some level compared with other provinces in China.</p>

Expression of human IL-12 in mammalian cell and study on its biological activities / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To obtain recombinant human interleukin 12 by genetic engineering, and to explore possibility of its clinical application in treatment of tumor and chronic hepatitis.</p><p><b>METHODS</b>Bicistronic expression vector P35-IRES-P40 was constructed for the simultaneous translation of IL-12 p35 and p40 cDNA subunit through internal ribosomal entry sites (IRES). pCI-dhfr-P35-IRES-P40 vector was constructed for expression in CHO-DHFR- cells. Positively cloned cells were screened by means of ELISA. Pools of clones with increased expression of IL-12 could be generated by selection in methotrexate. To determine the biological activities of rhIL-12, PHA-activated lymphoblasts proliferation assay and IFN-gamma induction assay were used in this study.</p><p><b>RESULTS</b>Genetically engineered cells expressing hIl-12 were obtained and all the cell lines showed the stabile expression of rhIL-12 in high efficiency and good growth properties.</p><p><b>CONCLUSION</b>rhIL-12 have good biological activities, it can stimulate activation and proliferation of T cells and induce production of IFN-gamma.</p>

Influence of the mutants of hepatitis B surface antigen on the cell immunity / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the influence of the mutants of hepatitis B surface antigen on the cell immunity.</p><p><b>METHODS</b>The recombinant plasmids of NS2Swt, NS2S126, NS2S133, NS2S141 and NS2S145 were transfected into Chinese hamster ovary (CHO) cells and the expressed proteins were detected by means of ELISA. Following PHA-activated lymphoblasts proliferation assay and IFN-gamma, IL-2, IL-10 induction assay were done with these proteins.</p><p><b>RESULTS</b>It was identified that these proteins of HBsAg could stimulate human lymphoblasts proliferation. Besides, there were no significant difference between the mutants and the wild. It was deserved to point out that the HBsAg with T126S mutation could increase the expression of IFN-gamma in the culture medium while the HBsAg with M133T mutation induced more expression of IL-10.</p><p><b>CONCLUSION</b>The results suggested that the cellular immune response to mutants of HBV might not be strengthened or weakened. But it should not be ignored that HBV T126S or M133T mutation may assert a potential impact on the cell immunity.</p>

Antigenic analysis of the common recombinant mutant types of HBsAg / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To study the difference between the mutant types and the wild type of HBsAg on the binding efficiency with antibodies in order to find some methods to detect the mutants.</p><p><b>METHODS</b>The recombinant wild type of HBsAg and the mutants including 145 R, 133 T, 126 S, 141 E, 126 S+133 T, 126 S+133 T+145 R and 126 S+133 T+141 E were cloned, respectively. Then the expression vector containing the wild or mutant gene was transfected into COS7 cells, and the recombinant proteins were expressed. The proteins were detected with the routine HBsAg kits.</p><p><b>RESULTS</b>The binding efficiency with monoclonal antibodies of HBsAg expressed by 126 S was higher than that of the wild type, while the results of 145 R, 141 E, 126 S+133 T+145 R and 126 S+133 T+141 E were lower than that of the wild, and 133 T was similar to the wild. But most of the mutants had the same reactivity with the polyclonal antibody.</p><p><b>CONCLUSIONS</b>The effect of mutations on antibody binding differs depending on the amino acid involved and on the location within the "a" loop. So it is necessary to use polyclonal antibody or to find new kits to detect the mutants of HBsAg.</p>