β3整合素作为汉坦病毒受体的鉴定以及其它候选受体的筛选

发布时间：2018-03-04 13:09

本文选题：汉坦病毒受体　切入点：胞膜糖蛋白G2　出处：《重庆医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 一般来说,病毒与细胞膜上特异性受体结合是病毒感染细胞的起始环节,对于病毒能否成功感染细胞具有重要意义。病毒受体具有特异性、高亲和性、结合位点有限性及相关生物学功能等特征。识别和鉴定病毒特异性结合的细胞膜受体不仅有益于了解病毒的组织嗜性、致病机制和生活周期,而且有利于制定预防和治疗措施。汉坦病毒(Hantavirus,HV)感染是严重威胁人类生命健康的全球性公共卫生问题。尽管人们已经对HV的基因组成、复制过程及生物学特征有相当的了解,但对病毒感染的起始环节及受体的研究还不清楚),而阻断HV与其受体的结合是治疗HV感染的重要途径之一。目前关于HV受体的研究主要集中在细胞膜粘附蛋白β3整合素上,认为β3整合素可能介导了HV入侵其宿主细胞,但是并没有汉坦病毒包膜蛋白与β3整合素直接结合的证据,故β3整合素作为汉坦病毒的特异性受体还有待进一步揭示;同时也有一些研究提示HV可能还存在有其它的候选受体或辅助受体分子,但需进一步的研究来证实。本课题利用基因重组技术分别构建了HV胞膜糖蛋白G2膜外区全长及各功能片段的真核表达载体以及β3整合素膜外区各截短片段的真核表达载体,通过Western blot检测表达的效果。结果显示β3整合素膜外区各片段在细胞裂解上清中均获得大量表达,而HV胞膜蛋白G2仅有膜外区N端81-140位(G2N81-140)氨基酸片段在裂解上清中有大量表达,其余片段均主要存在于某种细胞器中。将G2N81-140真核表达质粒与β3整合素各片段真核表达质粒分别成对共转染HEK293细胞,免疫共沉淀验证两者间的相互作用。结果表明G2与β3整合素可能存在着直接的相互作用,且作用位点位于G2 N端81-140片段与β3整合素27-133位氨基酸之间。构建G2蛋白N端81-140片段的原核表达载体并通过SDS-PAGE检测其在BL21表达菌中表达及纯化的效果。将纯化后的片段与β3整合素27-133位氨基酸片段孵育,GST pull-down验证它们之间的相互作用,结果也显示两者间存在着直接的相互作用。用生物素标记HV易感细胞VeroE6及非允许细胞CHO细胞膜蛋白,将纯化的原核表达G2N81-140片段作为探针与经过GST蛋白预处理的含有生物素标记细胞膜蛋白的VeroE6裂解液进行孵育,同时以非允许细胞CHO和无关蛋白GST-TLM做为阴性对照,通过GST pull-down分离与G2蛋白相互作用的细胞膜蛋白。结果显示一个约30KDa的细胞膜蛋白与G2N81-140氨基酸片段之间存在着特异性相互作用,表明该30KDa蛋白可能是HV细胞膜候选受体或受体辅助分子之一。本课题的研究结果为β3整合素作为HV特异性细胞膜受体提供了有力的证据,同时也表明除β3整合素外HV入侵细胞还可能存在有其它候选受体或辅助受体分子,为进一步研究HV入侵宿主细胞的机制奠定了基础。
[Abstract]:Generally speaking, the binding of virus to specific receptors on cell membrane is the initial link of virus infection cells, and has important significance for the success of virus infection cells. Virus receptors have specificity and high affinity. The identification and identification of viral specific binding cell membrane receptors is not only helpful to understand the tissue tropism, pathogenesis and life cycle of the virus, but also conducive to the formulation of preventive and therapeutic measures. Hantavirus HVinfection is a global public health problem that poses a serious threat to human life and health, although the genetic composition, replication process and biological characteristics of HV are well understood. However, the initiation of virus infection and the study of its receptor are not clear, and blocking the binding of HV and its receptor is one of the important ways to treat HV infection. At present, the research on HV receptor is mainly focused on the 尾 3 integrin of cell membrane adhesion protein. It is believed that 尾 3 integrin may mediate HV invasion into its host cells, but there is no evidence of direct binding of Hantavirus envelope protein to 尾 3 integrin. Therefore, 尾 3 integrin as a specific receptor of Hantavirus remains to be further explored, and some studies suggest that there may be other candidate receptors or coreceptor molecules in HV, but further research is needed to confirm it. In this study, the eukaryotic expression vectors of the full length and functional segments of HV membrane glycoprotein G2 and the eukaryotic expression vectors of each truncated segment of 尾 3 integrin extracellular region were constructed by gene recombination technique. The expression of 尾 3 integrin extracellular region was detected by Western blot. The results showed that the extracellular regions of 尾 3 integrin were highly expressed in the supernatant of cell lysis, while the HV membrane protein G2 had only a large number of amino acid fragments expressed in the supernatant of the N terminal 81-140 of the extracellular region of G2N81-140. The other fragments were mainly found in some organelle. The eukaryotic expression plasmids of G2N81-140 and 尾 3 integrin were co-transfected into HEK293 cells respectively. The immunoprecipitation was used to verify the interaction between the two. The results showed that there might be direct interaction between G2 and 尾 3 integrin. The interaction site is between 81-140 fragment of G2 N terminal and the amino acid of 尾 3 integrin 27-133. The prokaryotic expression vector of N-terminal 81-140 fragment of G2 protein was constructed and its expression and purification effect in BL21 expression strain was detected by SDS-PAGE. GST pull-down was incubated with 尾 3 integrin 27-133 amino acid fragment to verify their interaction. The results also showed that there was direct interaction between them. Biotin was used to label VeroE6 and CHO membrane proteins in HV susceptible cells. The purified prokaryotic expression G2N81-140 fragment was incubated with the VeroE6 cleavage solution containing biotin labeled cell membrane protein pretreated with GST protein, and CHO and unrelated protein GST-TLM were used as negative control. Membrane proteins interacting with G2 protein were isolated by GST pull-down. The results showed that there was a specific interaction between a 30 KDa membrane protein and a G2N81-140 amino acid fragment. These results suggest that the 30KDa protein may be a candidate receptor or adjunctor for HV cell membrane. The results of this study provide strong evidence for 尾 3 integrin as HV specific cell membrane receptor, and also suggest that there may be other candidate receptors or coreceptor molecules in HV invading cells in addition to 尾 3 integrin. It lays a foundation for further study on the mechanism of HV invading host cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373;R392

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