HPV EXPRESSION AND ANTIGENICITY

人乳头瘤病毒（HPV）的研究需要使用 分子克隆、DNA测序和表达载体， 鉴定和表征病毒基因，因为无法 在培养物中繁殖HPV。 作为这项研究的基础， HPV多肽（鉴定为来自HPV106的开放阅读框（ORF））， DNA序列分析）将在细菌载体中表达 并用作纯化病毒抗原的来源。 起初， HPV 6和16的序列将在质粒载体中表达 含有E. colitrpE基因和噬菌体中 λ gtll. 将融合蛋白纯化并接种 在兔子体内产生多克隆抗体， 产生单克隆抗体，并测定其 识别类型共同和类型特异性表位。 两 方法将用于监测临床中的基因表达， 组织中 首先，克隆的HPV ORF将用作探针， 通过原位杂交和使用 北方印迹分析。 其次，抗体将用于 免疫组化检测病毒蛋白 Western印迹。 检测人乳头瘤病毒（HPV）的体液免疫应答 感染时，患者抗血清将与融合蛋白反应， 蛋白质免疫印迹分析。 随后，确定的抗原 将用于Elisas。 作为细胞介导的 免疫应答，纯化的抗原将用于刺激 体外淋巴细胞增殖。 因为HPV的转录模式很可能是 复杂的是，剪接的mRNA可能会引起 由一个以上ORF组成的蛋白质。 RNA作图 如果不这样做， 不可能，因此将克隆HPV6和16的基因组 导入含有转录增强子的载体， 启动子和SV40复制起点，并转染入 COS细胞大量产生HPV转录物。 cDNA 克隆将被构建、映射并插入到 产生HPV多肽的蛋白质表达载体 由ORF的组合组成。

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.