HLA-A33分子的体外表达及鉴定

发布时间：2018-01-03 00:26

本文关键词：HLA-A33分子的体外表达及鉴定　出处：《山东农业大学》2011年硕士论文　论文类型：学位论文

【摘要】：人类白细胞抗原(HLA)是一种重要的遗传物质,是目前所知人体最复杂的多态系统。在我国人群中A33表位占A位点表位的21%,是临床中较常见的表位。根据近些年相关报道,HLA-A33表位在多种疾病的病程进展过程中起着重要作用。在我国南方以及Korea统计的结果,HLA-A33与HBV感染后慢性化有较明显的相关性;Luan-Yin Chang博士等的研究表明,HLA-A33型为EV71易感基因型;洪军等人的研究表明,HLA-A33与小儿急性淋巴细胞白血病有遗传相关性(曹海霞等2007)。本研究通过构建HLA-A33表达载体,经重组载体的鉴定、载体的转化及转染、目的蛋白的原核表达及表达水平的检测,初步研究HLA-A33分子与EV71病毒分子之间的相互作用,为研究HLA-A33在相关疾病中的致病作用和免疫机制提供理论基础和实验依据,本研究氛围两部分内容。第一部分:建立稳定表达HLA-A33分子的细胞系及其功能的相关研究。从GenBank查阅人类HLA-A33的基因序列,结合真核表达的特点,优化所得序列的密码子,并且添加必要的限制性酶切位点Nhe I、Xho I及Flag标签蛋白。通过四质粒慢病毒系统将目的基因片段整合到宿主细胞的基因组DNA上。构建的稳定细胞系通过puro筛选可稳定传代的细胞,通过IF,WB,PCR等方法鉴定细胞系。用EV71病毒感染所获得的稳定细胞系,尝试通过CoIP寻找HLA-A33与EV71之间存在相互作用的蛋白质。第二部分:HLA-A33基因在原核细胞中表达及检测。根据原核表达的特点重新优化密码子,选择pET28a作为表达载体,在序列两端添加Nco I和BamH I限制性酶切位点,通过T4连接酶将载体和序列连接,化学转化法转入大肠杆菌TOP10感受态细胞,得到大量克隆并用PCR和测序鉴定目的基因的插入情况。将重组质粒重新转化入表达菌柱BL21(DE3),通过IPTG诱导表达,SDS-PAGE凝胶电泳,考马斯亮蓝成像来检测HLA-A33蛋白在原核细胞内表达情况。原核表达的蛋白添加了BirA酶底物肽和链亲和素标签Streptavidin,为后续构建HLA-A33分子的tetramer四聚体提供实验基础。本实验取得了如下成果:用常用的分子克隆技术成功构建了HLA-A33的原核表达载体,实现HLA-A33蛋白在原核细胞内的高效表达,并对表达蛋白进行了检测分析;利用慢病毒系统转染法获得了HLA-A33稳定细胞系并利用稳定细胞系初步进行EV71与HLA-A33相互作用蛋白的分析。实验的成果对于HLA-A33的后续研究具有重要的理论意义并提供坚实的实验基础。
[Abstract]:Human leukocyte antigen ( HLA ) is an important genetic material , which is the most complex multi - state system known to the human body . According to the recent years , HLA - A33 epitopes play an important role in the course of disease progression . According to the recent years , HLA - A33 has a significant correlation with chronic disease after HBV infection . The study of Luan - Yin Chang et al . shows that HLA - A33 is a susceptible genotype of EV71 . Studies from Hongjun et al . show that HLA - A33 is genetically related to childhood acute lymphoblastic leukemia ( Cao Haixia et al 2007 ) . In this study , the interaction between HLA - A33 molecule and EV71 virus molecule was preliminarily studied by constructing HLA - A33 expression vector , identification of recombinant vector , transformation and transfection of vector , prokaryotic expression and expression level of target protein , and provided a theoretical basis and experimental basis for studying the pathogenic role and immune mechanism of HLA - A33 in related diseases . A stable cell line was constructed by puro screening for stably passaged cells , and the stable cell line obtained by using EV71 virus infection was used to search for the interaction between HLA - A33 and EV71 by CoIP . In the second part , the expression and detection of HLA - A33 gene in prokaryotic cells were studied . According to the characteristics of prokaryotic expression , the codons were re - optimized and pET28a was chosen as expression vector . The expression of HLA - A33 protein in prokaryotic cells was detected by means of T4 ligase . The expression of HLA - A33 protein in prokaryotic cells was detected by IPTG - induced expression , SDS - PAGE gel electrophoresis . In this experiment , the prokaryotic expression vector of HLA - A33 was successfully constructed by using conventional molecular cloning technology . The expression of HLA - A33 protein in prokaryotic cells was carried out . The HLA - A33 stable cell line was obtained by the transfection of lentivirus system and the interaction protein of EV71 and HLA - A33 was obtained by using stable cell line . The results of experiment were of great theoretical significance and provide a solid experimental basis for the subsequent study of HLA - A33 .

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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