The Study On Prophylactic Vaccine Of Human Papillomavirus 16L1

The infection of Human papillomavirus (HPV) belongs to the sex transmitted disease (STD), which threatens seriously human health. The molecular biology and epidemiology studies showed that the Human papillomavirus was the main cause of cervical cancer. As the advancement of molecular biology and of gene engineering technology, the scientists are trying to take these advantages to develop cervical cancer vaccines. Therefore, the social and economical significance of the present study is obvious.To date, most researches of the HPV vaccines are based on highly immunogenic VLPs from late proteins and subordinate late proteins produced in the late stage of infection. In this paper, we used both the eukaryotic Pichia pastoris yeast system and prokaryotic system to express of HPV16 L1 protein as a prophylactic vaccine. The main results are summarized as follows:1) About 30 complete sequences of HPV16 L1 genes were obtained from the Genebank and aligned with the software AlignX program to make sure that the one chosen for the target gene has the highest homology in the level of the amino acid sequences with others. To improve the expression efficiency of recombinant L1 protein in Pichia pastoris, we designed synthetic L1 genes based on Pichia codon usage and gave this sequence to Shanghai Sangon Biological Engineering Company which synthezed the whole gene into two fragments and cloned into pUC57 vector to generate pUC57(1-1048 and pUC57((986-1536), respectively. Both plasmids have a BglII site in the overlapping region. Then, these two fragments were digested with BglII, purified, ligated, and served as template for PCR amplification.2) The recombinant plasimd pAO819-16L1 was constructed by inserting PCR-amplified L1 gene into pAO819 vector and transformed into GS115 strain. The recombinant strains were selected on G418-containing plates. The expression of L1 protein was induced with the methyl alcohol and detected by Western Blot with specific antibody. A 55KD protein corresponding to L1 was observed.3) L1 gene was cloned into prokaryotic plasmid pET43.1a and transformed into the BL21 codonplus(DE3) and the BL21 codonplus(DE3)-RILP, respectively. The L1 protein was expressed in the both strains with inclusion bodies which could be denaturalized with 8M urea and purified with anion-exchange resin. The purity of the protein reached over 80%.4) The eukaryotic plasmid pDRVI1.0-16L1 was constructed and used to immunize the mice. After each immunization, we use perforation to boost the immune reaction. The antiserum of HPV16L1 which reacted with L1 protein by detection of Western Blot. The titer of the antiserum is relatively high, but the specificity is not high enough.