HPV EXPRESSION AND ANTIGENICITY

人类乳头瘤病毒(HPV)的研究需要使用 分子克隆、DNA测序和表达载体 识别和表征病毒基因，因为无法 在培养中繁殖人乳头瘤病毒。作为这项研究的大部分基础， HPV多肽(鉴定为开放阅读框架(ORF)) DNA序列分析)将在细菌载体中表达 并用作提纯病毒抗原的来源。最初， HPV6和HPV16的序列将在质粒载体中表达 含有E.ColiTrp E基因的重组噬菌体 兰布达gtll。融合蛋白将被提纯并接种。 进入兔子以产生多克隆抗体，并进入小鼠以 产生单抗，并检测它们的能力 识别类型共同表位和类型特定表位。二 临床上将使用各种方法来监测基因的表达 纸巾。首先，克隆的HPVORF将作为探针用于 检测病毒RNA的原位杂交法和荧光检测法 Northern印迹分析。其次，抗体将被用来 用免疫组织化学方法检测病毒蛋白 西式印迹。检测人乳头瘤病毒的体液免疫应答 感染，患者的抗血清将与融合反应 免疫印迹分析中的蛋白质。随后，定义的抗原 将在ELISA中使用。作为细胞介导的一种衡量标准 免疫反应，纯化的抗原将被用来刺激 淋巴细胞体外增殖。 因为HPV的转录模式很可能是 复杂的剪接的mRNA很可能会引起 由不止一个开放阅读框架组成的蛋白质。RNA的作图 如果不是这样，在人体组织中的转录将是极其困难的 不可能，因此HPV6和16型的基因组将被克隆 转化为含有转录增强子的载体， 启动子和SV40复制起始点，并转化入 COS细胞产生大量的HPV转录本。Cdna 克隆将被构建、映射并插入到 用于生产HPV多肽的蛋白质表达载体 由ORF的组合组成的。

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.