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

【摘要】： 一般來(lái)說(shuō),病毒與細(xì)胞膜上特異性受體結(jié)合是病毒感染細(xì)胞的起始環(huán)節(jié),對(duì)于病毒能否成功感染細(xì)胞具有重要意義。病毒受體具有特異性、高親和性、結(jié)合位點(diǎn)有限性及相關(guān)生物學(xué)功能等特征。識(shí)別和鑒定病毒特異性結(jié)合的細(xì)胞膜受體不僅有益于了解病毒的組織嗜性、致病機(jī)制和生活周期,而且有利于制定預(yù)防和治療措施。 漢坦病毒(Hantavirus,HV)感染是嚴(yán)重威脅人類生命健康的全球性公共衛(wèi)生問(wèn)題。盡管人們已經(jīng)對(duì)HV的基因組成、復(fù)制過(guò)程及生物學(xué)特征有相當(dāng)?shù)牧私?但對(duì)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(jié)合是治療HV感染的重要途徑之一。 目前關(guān)于HV受體的研究主要集中在細(xì)胞膜粘附蛋白β3整合素上,認(rèn)為β3整合素可能介導(dǎo)了HV入侵其宿主細(xì)胞,但是并沒(méi)有漢坦病毒包膜蛋白與β3整合素直接結(jié)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(jìn)一步揭示;同時(shí)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(jìn)一步的研究來(lái)證實(shí)。 本課題利用基因重組技術(shù)分別構(gòu)建了HV胞膜糖蛋白G2膜外區(qū)全長(zhǎng)及各功能片段的真核表達(dá)載體以及β3整合素膜外區(qū)各截短片段的真核表達(dá)載體,通過(guò)Western blot檢測(cè)表達(dá)的效果。結(jié)果顯示β3整合素膜外區(qū)各片段在細(xì)胞裂解上清中均獲得大量表達(dá),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(dá),其余片段均主要存在于某種細(xì)胞器中。將G2N81-140真核表達(dá)質(zhì)粒與β3整合素各片段真核表達(dá)質(zhì)粒分別成對(duì)共轉(zhuǎn)染HEK293細(xì)胞,免疫共沉淀驗(yàn)證兩者間的相互作用。結(jié)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(diǎn)位于G2 N端81-140片段與β3整合素27-133位氨基酸之間。構(gòu)建G2蛋白N端81-140片段的原核表達(dá)載體并通過(guò)SDS-PAGE檢測(cè)其在BL21表達(dá)菌中表達(dá)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(yàn)證它們之間的相互作用,結(jié)果也顯示兩者間存在著直接的相互作用。用生物素標(biāo)記HV易感細(xì)胞VeroE6及非允許細(xì)胞CHO細(xì)胞膜蛋白,將純化的原核表達(dá)G2N81-140片段作為探針與經(jīng)過(guò)GST蛋白預(yù)處理的含有生物素標(biāo)記細(xì)胞膜蛋白的VeroE6裂解液進(jìn)行孵育,同時(shí)以非允許細(xì)胞CHO和無(wú)關(guān)蛋白GST-TLM做為陰性對(duì)照,通過(guò)GST pull-down分離與G2蛋白相互作用的細(xì)胞膜蛋白。結(jié)果顯示一個(gè)約30KDa的細(xì)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(xì)胞膜候選受體或受體輔助分子之一。 本課題的研究結(jié)果為β3整合素作為HV特異性細(xì)胞膜受體提供了有力的證據(jù),同時(shí)也表明除β3整合素外HV入侵細(xì)胞還可能存在有其它候選受體或輔助受體分子,為進(jìn)一步研究HV入侵宿主細(xì)胞的機(jī)制奠定了基礎(chǔ)。
[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(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：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(xué);2008年

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本文編號(hào)：1565768