HPV EXPRESSION AND ANTIGENICITY

人类乳头瘤病毒（HPV）的研究需要使用

The study of human papillomaviruses (HPV) requires the use of molecular cloning, DNA sequencing, and expression vectors to identify and characterize viral genes because of the inability to propagate HPVs in culture. As the basis for much of this study, HPV polypeptides (identified as open reading frames (ORFs) from DNA sequence analysis) will be expressed in bacterial vectors and used as a source to purify viral antigens. Initially, sequences of HPV 6 and 16 will be expressed in plasmid vectors containing the E. coli trp E gene and in the bacteriophage lambda gtll. The fusion proteins will be purified and inoculated into rabbits to produce polyclonal antibodies and into mice to produce monoclonal antibodies, and assayed for their ability to recognize type-common and type-specific epitopes. Two approaches will be used to monitor gene expression in clinical tissues. First, the cloned HPV ORFs will be used as probes to detect viral RNA both by in situ hybridization and using Northern blot analysis. Secondly, the antibodies will be used to detect viral proteins by immunohistochemical methods and using Western blots. To assay the humoral immune response to HPV infection, patient antisera will be reacted with the fusion proteins in immunoblot assays. Subsequently, defined antigens will be used in Elisas. As a measure of the cell mediated immune response, purified antigens will be used to stimulate lymphocyte proliferation in vitro. Because the transcriptional pattern of HPV is likely to be complex it is probable that spliced mRNAs will give rise to proteins consisting of more than one ORF. Mapping of RNA transcripts in human tissues will be extremely difficult if not impossible, therefore the genomes of HPV6 and 16 will be cloned into vectors containing transcriptional enhancers, strong promoters and the SV40 replication origin, and transfected into COS cells to produce HPV transcripts in abundance. cDNA clones will be constructed , mapped, and inserted into the protein expression vectors to produce HPV polypeptides composed of combinations of ORFs.