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  本文選題：漢坦病毒受體　切入點：胞膜糖蛋白G2　出處：《重慶醫(yī)科大學》2008年碩士論文　論文類型：學位論文

【摘要】： 一般來說,病毒與細胞膜上特異性受體結合是病毒感染細胞的起始環(huán)節(jié),對于病毒能否成功感染細胞具有重要意義。病毒受體具有特異性、高親和性、結合位點有限性及相關生物學功能等特征。識別和鑒定病毒特異性結合的細胞膜受體不僅有益于了解病毒的組織嗜性、致病機制和生活周期,而且有利于制定預防和治療措施。 漢坦病毒(Hantavirus,HV)感染是嚴重威脅人類生命健康的全球性公共衛(wèi)生問題。盡管人們已經(jīng)對HV的基因組成、復制過程及生物學特征有相當?shù)牧私?但對病毒感染的起始環(huán)節(jié)及受體的研究還不清楚),而阻斷HV與其受體的結合是治療HV感染的重要途徑之一。 目前關于HV受體的研究主要集中在細胞膜粘附蛋白β3整合素上,認為β3整合素可能介導了HV入侵其宿主細胞,但是并沒有漢坦病毒包膜蛋白與β3整合素直接結合的證據(jù),故β3整合素作為漢坦病毒的特異性受體還有待進一步揭示;同時也有一些研究提示HV可能還存在有其它的候選受體或輔助受體分子,但需進一步的研究來證實。 本課題利用基因重組技術分別構建了HV胞膜糖蛋白G2膜外區(qū)全長及各功能片段的真核表達載體以及β3整合素膜外區(qū)各截短片段的真核表達載體,通過Western blot檢測表達的效果。結果顯示β3整合素膜外區(qū)各片段在細胞裂解上清中均獲得大量表達,而HV胞膜蛋白G2僅有膜外區(qū)N端81-140位(G2N81-140)氨基酸片段在裂解上清中有大量表達,其余片段均主要存在于某種細胞器中。將G2N81-140真核表達質粒與β3整合素各片段真核表達質粒分別成對共轉染HEK293細胞,免疫共沉淀驗證兩者間的相互作用。結果表明G2與β3整合素可能存在著直接的相互作用,且作用位點位于G2 N端81-140片段與β3整合素27-133位氨基酸之間。構建G2蛋白N端81-140片段的原核表達載體并通過SDS-PAGE檢測其在BL21表達菌中表達及純化的效果。將純化后的片段與β3整合素27-133位氨基酸片段孵育,GST pull-down驗證它們之間的相互作用,結果也顯示兩者間存在著直接的相互作用。用生物素標記HV易感細胞VeroE6及非允許細胞CHO細胞膜蛋白,將純化的原核表達G2N81-140片段作為探針與經(jīng)過GST蛋白預處理的含有生物素標記細胞膜蛋白的VeroE6裂解液進行孵育,同時以非允許細胞CHO和無關蛋白GST-TLM做為陰性對照,通過GST pull-down分離與G2蛋白相互作用的細胞膜蛋白。結果顯示一個約30KDa的細胞膜蛋白與G2N81-140氨基酸片段之間存在著特異性相互作用,表明該30KDa蛋白可能是HV細胞膜候選受體或受體輔助分子之一。 本課題的研究結果為β3整合素作為HV特異性細胞膜受體提供了有力的證據(jù),同時也表明除β3整合素外HV入侵細胞還可能存在有其它候選受體或輔助受體分子,為進一步研究HV入侵宿主細胞的機制奠定了基礎。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R373;R392

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1 李青嶺;β3整合素作為漢坦病毒受體的鑒定以及其它候選受體的篩選[D];重慶醫(yī)科大學;2008年

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本文編號：1565768