Xiaoguo Song

Double-antigen sandwich ELISA for the detection of anti-hepatitis C virus antibodies.

A double-antigen sandwich ELISA was developed a detection of HCV antibodies by a recombinant multi-epitope HCV antigen and a biotin-streptavidin amplification system. Three plasma specimens from 1708 individuals who were suspected previously to be HCV-positive using an HCV antibody diagnostic kit (Chuangxin, Xiamen, China) displayed negative results when using the ELISA. These results were validated by a recombinant immunoblotting assay (two were negative, and one was indeterminate). Among 889 blood specimens donated for clinical evaluation, 246 were positive and 630 were negative using the ELISA. The sensitivity and specificity of the ELISA were 98.7% and 100%, respectively. In 43 donors and 14 patients with chronic hepatitis C, the detectable rates for HCV IgM by both ELISA and the HCV anti-IgM detection reagents (Huimin, Shenyang, China) were 100%, and the detectable rate for HCV IgG using an indirect HCV-antibody detection kit (GWK, Beijing, China) was 98.3%. Thus, the double-antigen sandwich ELISA exhibits strong specificity and sensitivity and has been approved by the China State Food and Drug Administration (SFDA). The performance of the double-antigen sandwich ELISA was similar to the Ortho ELISA 3.0. It did not give false-negative results otherwise IgM was undetectable using an indirect HCV-antibody detection kit. This ELISA provides another method for the detection of HCV antibodies.

The use of a chimera HIV‐1/HIV‐2 envelope protein for immunodiagosis of HIV infection: Its expression and purification in E. coli by use of a translation initiation site within HIV‐1 env gene

A chimera HIV‐1/HIV‐2 envelope sequence composed of multiple conserved immunodominant epitopes of HIV‐1 envlope protein (HIV‐1 IIIB: env482‐518+env548‐675) and the HIV‐2 gp36 immunodominant epitope (env592‐603), was constructed and directly over‐expressed in E. coli by using a prokaryotic translation initiation sequence contained within the gene of HIV‐1 envelope. The recombinant product was purified and applied in antibody‐screening assay. The purified chimera antigen reacted with all the thirty‐eight HIV‐1 positive serum samples, the two HIV‐2 serum samples, and had no cross‐reaction with all the eighty‐eight normal healthy serum sample. The results indicated that this recombinant chimera HIV‐1/HIV‐2 envelope protein could be useful for diagnostic purposes of HIV infection.

Thioredoxin fusion/HIV-1 protease coexpression system for production of soluble human IL6 in E. coli cytoplasm

In this paper, thioredoxin (TRX) fusion expression system has been modified to produce soluble human IL6 (hIL6) without TRX moiety in E. coli cytoplasm. A novel TRX gene fusion vector was developed that contained at the 3′‐end of TRX gene a short DNA sequences encoding a linker peptide ‘‐GSGSGVSQNYPIVQHHHHHH‐’, serving not only as a specific HIV‐1 protease site but also providing six contiguous histidine (His) residues to foreign proteins. The cDNA for hIL6 was cloned into this vector resulting in plasmid pTRX@HISIL6. The cDNA for the HIV‐1 protease has been cloned into another compatible plasmid pHMM2, resulting in plasmid pHMM2‐PR. Both plasmids were transformed into E. coli strain GI724, and when induced for expression of both proteins, the correct processing of TRX@HISIL6 was obtained, producing hlL6 with His6‐tag at the N terminus named HISIL6. A fraction of HISIL6 was found in soluble form and could be purified to homogeneity by Ni‐NTA Superflow and ion‐exchange chromatography. The biological activity of purified HISIL6 was measured by MTT method in an IL‐6‐dependent cell line 7TD1 to be 2.1×108 unit/mg.

Cancerogenic effect of different fragments of the hepatitis C virus core protein.

The hepatitis C virus core protein plays an extremely important role in the hepatocarcinogenesis of hepatitis C virus. Little, however, is known about the oncogenic potency of fragments. Thus, the purpose of the present study is to investigate the cancerogenic effects of the different core protein fragments. Two series of recombinant plasmids containing hepatitis C virus core gene fragments encoding the different-length core protein were constructed using plasmid enhanced green fluorescent protein (pEGFP)-C1 and pcDNA3.1(+), respectively. Human hepatocyte L02 cells transiently transfected with pEGFP-C1-based plasmids were subjected to confocal laser scanning microscopy analysis to determine the localization of the different core protein fragments. The stably transfected L02 cells with the pcDNA3.1(+)-based core protein plasmids were used to investigate the ultrastructural effects of the core protein and the tumorigenicity of L02 cells expressing core protein fragments in athymic nude mice. The full-length core protein and Core130−191 were completely localized in the cytoplasm, while Core1−59 existed exclusively in the nucleus. On the other hand, Core50−140 and Core1−140 were observed in both the nucleus and the cytoplasm. Ultrastructural changes of L02 cells expressing the full-length core protein were comprehensive and included, for example, irregular nuclear, increased nuclear/cytoplasmic ratio and mitochondria swelling. The slight changes were observed in the cells expressing Core50−140 and Core130−191, whereas the ultrastructure of the cells expressing Core1−59 remained normal. All the L02 cells stably expressing different fragments of the core protein, with the exception of the C-terminal truncated fragment Core1−59, could induce the occurrence of tumor in the nude mice. The N-terminal fragment of the core protein, Core1−59, was not oncogenic, while the intermediate and posterior segments of the hepatitis C virus core protein had the cancerogenic potency. In view of the existence of many important immunogenic epitopes in it, the core protein anterior segment might be a safer candidate for the development of hepatitis C virus vaccine.

Construction, Expression, Purification and Biotin Labeling of a Single Recombinant Multi-Epitope Antigen for Double-Antigen Sandwich ELISA to Detect Hepatitis C Virus Antibody

Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.