[Construction of recombinant CHO cell strain for high expression of HBsAg].

Objective: To overexpress hepatitis B virus S gene in CHO cells cultured in serum-free media.

Method: 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.

[Effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering].

Objective: To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering.

Methods: Based on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized.

[Gene optimization, protein expression, purification and characterization of the HDV antigen for diagnosis].

Objective: To prepare HDAg with biological activities as a candidate of diagnostic reagent.

Methods: To synthesize HDV gene fragment after codon optimization. To construct a thio-fused recombinant plasmid based on M48 expression vector.

[Expression of hepatitis C virus subunit fusion protein and analysis of its immunogenicity].

Objective: Obtain the hepatitis C virus high purified subunit fusion protein and detect its immunogenicity.

Methods: 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.

[Construction and characterization of hepatitis B surface antigen "a" epitope virus-like particles].

Objective: To construct virus-like particles of hepatitis B core antigen, with HBsAg "a" epitope exposed on the surface.

Methods: 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.

[Preparation and identification of the monoclonal antibodies against VP1 capsid protein of Enterovius 71].

Objective: To prepare monoclonal antibodies (McAbs) against VP1 capsid protein of Enterovirus 71.

Methods: 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.

[Expression of recombinant rubella virus E1 protein and initial application for detecting of antibody].

Objective: To apply recombinant rubella virus envelope protein-1 (E1) to detect human rubella virus IgG antibody.

Methods: 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).

[Antigenic properties of mutant hepatitis B virus surface antigen].

Objective: 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.

Methods: 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.

[Cloning of PD-1 gene and its prokaryotic expression in Escherichia coli].

Objective: To clone human PD-1 gene, construct a prokaryotic expression plasmid and express in E. coli.

Methods: 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.

[Expression of human IL-12 in mammalian cell and study on its biological activities].

Objective: To obtain recombinant human interleukin 12 by genetic engineering, and to explore possibility of its clinical application in treatment of tumor and chronic hepatitis.

Methods: 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.

[Antigenic analysis of the common recombinant mutant types of HBsAg].

Background: 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.

Methods: 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.