Study On Character And Function Of HCV E1 And HCMV IE86 Proteins

This paper included two parts: (1) study on the expression of Hepatitis C Virus (HCV) El gene and the immunology character of El protein; (2) The Human Cytomegalovirus (HCMV) IE2 protein interacts with transcription activating protein.The full length of HCV El gene cDNA sequence of Wuhan strain was reported first time, and it was cloned respectively into pQE30, pBlueBacHisB and pcDNA3.1 (-). HCV El cDNA expression, antigenity and immunity of El protein from E. coli and eukaryote cells were overall analyzed. It is significant for further understanding the variation rule of El gene, the characteristics and functions of El protein. The studies on the expression of HCV El gene in E. coli and eukaryote cells, and the immunological properties of different forms of El protein and El DNA vaccine, will contribute greatly to the development of HCV diagnosis and vaccine.HCV El cDNA contained 576bp and encoded a protein of 192 amino acids. 74.48% nucleotide acids of El cDNA and 78. 13% amino acid of El protein are homologous between WH strain and Japan strain (HCV-J type). There were two preponderant epitopes, P1 (210-223aa) and P2 (315-327), which related to the immunity of El protein. 71.43% and 100% amino acids are homologous in P1 and in P2 respectively between the two strains. This indicated that P1 was a high variant, but P2 was high conservative.El fusion protein expressed in E. coli was non-glycosylated with the molecular weight of 23 kDa and formed inclusion body. HCV El inclusion body was used as antigen. El antibody could be detected in HCV positive human sera by ELISA. 48/90 of HCV positive sera could be detected by El antigen. The immunoreaction characteristic may correlate to a B-cell epitope in El protein at position 317-326aa, the RMAWDMMMNW amino acids domain. The reaction to the antibody was not related to protein glycosylation. The inclusion body of El protein was used as immunogen and could induce immune animals to produce IgG antibody specific to El. The mice IgG reached the highest value at the eighth week post immunization with the titer of 1 : 2400. The rabbits IgGreached the highest value at the eighth week post immunization with the titer of 1 : 5760. The antibody even could be detected in mucosa secretion. The protein induced mice CTL response at rather high level and DTH response. It also increase distinctly the ratio of CD8+T cells. It was presumed that the structure of the inclusion body was propitious to immunogen submitting. It showed that El protein had nice antigenity and immunogenicity.HCV El cDNA was expressed in sf9 cells by infection with Baculovirus. The products were mainly a protein with 40kDa and a series of protein expressed weakly with 29-40kDa. It was presumed that the difference in molecular weight resulted from glycosylation at different extents. El protein with 40kDa was used as antigen to detect HCV antibody in ELISA. 48/90 of HCV positive sera could be detected. The result was consistent with the inclusion body. But their reaction to antibody were different. The reaction produced by El glycoprotein was weaker than the El nonglycoprotein. HCV El glycoprotein with 40kDa as an immunogen could induce immune animals to produce specific immune response to HCV El. The mice IgG reached the highest titer at the sixth week post immunization. The titer was 1 I 2400. The rabbits IgG reached the highest value at the eighth week. The titer was 1 : 7680. HCV El glycoprotein induced mice slgG but not slgA response. The glycoprotein induced mice CTL response, distinct increase of CD8+T cells and DTH response. It indicated that HCV El glycoprotein could induce cell immunity response.HepG2 cells were transfected with pcE1 by lipofectamine. HCV El gene cDNA was experessed in HepG2 cells. The product was a single mature glycoprotein with 35kDa. The protein bound to HCV positive sera specifically. The mice thigh quadriceps cells incepted pcEl DNA and expressed as a glycoprotein that could be detected by affinity immunocytochemistry technology. Animals were immunized with bare pc...