本文選題：艾滋病病毒1型 + 負調控因子�。� 參考：《湖北大學》2014年碩士論文

【摘要】：人類免疫缺陷病毒1型(human immunodeficiency virus1,HIV-1)是一種單股正鏈核糖核酸(RNA)逆轉錄病毒,其具有高復制率、高重組率、高突變率等特點。因為它有快速變異的基因組,所以它能夠逃脫宿主細胞的免疫壓力。艾滋病毒已經在世界范圍內泛濫幾十年,對于尋找治療艾滋病毒的新方法需要全人類共同努力。負調控因子(negative factor, NEF)是人免疫缺陷病毒附屬蛋白之一,其基因在HIV-1、HIV-2和猴免疫缺陷病毒中具有很高的保守性。國內外對Nef進行了大量的研究,發(fā)現(xiàn)其具有以下幾個主要功能：(1)介導CD4的下調；(2)介導CD28的下調；(3)介導MHC分子的下調；(4)干擾病毒粒子的復制能力和感染性；(5)介導細胞凋亡的抑制。因為對于其作用機制了解不夠透徹,通過酵母雙雜交系統(tǒng)篩選與Nef相互作用的蛋白質,我們能夠更好的對Nef調控HIV-1的機制以及AIDS疾病進程影響機制進行研究。 酵母雙雜交系統(tǒng)最主要特點是能夠快速檢測蛋白之間是否具有相互作用。本實驗首先以pNL4-3為模板擴增得到目的基因Nef,然后將目的基因Nef和載體pGBKT7進行雙酶切反應,使目的基因Nef克隆到載體pGBKT7上,構建成pGBKT7-Nef誘餌質粒。完成誘餌蛋白pGBKT7-Nef在AH109酵母菌株里進行自激活性和毒性檢測后,再進行酵母雙雜交實驗。將接種到AH109酵母菌株中表達的pGBKT7-Nef與人的B淋巴細胞文庫進行酵母雙雜交篩選。首先將酵母菌株AH109活化,然后將誘餌質粒pGBKT7-Nef轉入到酵母AH109中,然后與帶有誘餌質粒pGBKT7-Nef的AH109的單克隆與人類B淋巴胞文庫質粒共轉,然后運用X-a-gal分析(藍白斑的鑒定)的方進,將單克隆在涂有X-a-gal的四缺培養(yǎng)基上觀察其生長狀態(tài)以及顏色的變化。根據(jù)顯藍斑的編號,找出相對應的陽性克隆提取酵母質粒,對感受態(tài)細胞大腸桿菌DH5α轉化,然后提取質粒,送去測序。將測序結果在NCBI上進行nucleotide blast進行同源性分析,從而找到與Nef蛋白相互作用的蛋白。最終通過篩選和分析得到兩個可能會與Nef相互作用的蛋白,分別是纖維凝膠蛋白(ficolin)和波形蛋白(vimentin)。 通過酵母雙雜交系統(tǒng)篩選出與Nef存在相互作用的蛋白,但是這是在酵母細胞水平上獲得,但是動物細胞內的環(huán)境與酵母細胞內的環(huán)境會有一定的區(qū)別,可能還需要在動物細胞內進行相關實驗,比如免疫共沉淀、GST pull-down進一步確定蛋白相互作用的關系。如果證實了纖維凝膠蛋白、波形蛋白和Nef存在相互作用,將進一步對闡明Nef對于HIV-1調控的機制以及藥物預防提供了新的線索。
[Abstract]:Human immunodeficiency virus type 1 (immunodeficiency virus1) is a single strand positive strand RNA RNA (RNA) retrovirus with high replication rate, high recombination rate and high mutation rate. Because it has a rapidly mutated genome, it can escape the immune pressure of host cells. HIV has spread around the world for decades, and finding new ways to treat it requires a concerted effort from all of humanity. Negative factor (NEF) is one of the accessory proteins of human immunodeficiency virus. Its gene is highly conserved in HIV-1 HIV-2 and simian immunodeficiency virus. A large number of studies on Nef have been carried out at home and abroad. It has been found that Nef has the following main functions: 1) mediating the down-regulation of CD4 (2) / 3) (3) mediating the down-regulation of MHC (4) interfering with the replication of virus particles and inhibiting apoptosis by infective 5). Because of the lack of thorough understanding of its mechanism, we can better study the mechanism of Nef regulating HIV-1 and the influence mechanism of AIDS disease process through yeast two-hybrid system to screen proteins interacting with Nef. The main characteristic of yeast two-hybrid system is the ability to quickly detect the interaction between proteins. In this experiment, pNL4-3 was used as template to amplify the target gene Nef. then the target gene Nef and vector pGBKT7 were digested by double enzyme digestion, and the target gene Nef was cloned into the vector pGBKT7 and constructed as pGBKT7-Nef bait plasmid. After the bait protein pGBKT7-Nef was tested for self-activation and toxicity in AH109 yeast strain, yeast two-hybrid experiment was carried out. The pGBKT7-Nef expressed in AH109 yeast strain and human B lymphocyte library were screened by yeast two-hybrid. First, the yeast strain AH109 was activated, then the bait plasmid pGBKT7-Nef was transferred into yeast AH109, then co-transformed with the clone of AH109 with bait plasmid pGBKT7-Nef and human B lymphoid library plasmid, and then X-a-gal analysis (identification of blue and white spot) was used. The growth state and color change were observed on the four deficiency culture medium coated with X-a-gal. According to the number of locus coeruleus, the corresponding positive clones were found to extract yeast plasmid, then transformed into E. coli DH5 偽, then the plasmid was extracted and sequenced. The sequencing results were sequenced by nucleotide blast homology analysis on NCBI, and the proteins interacting with Nef protein were found. Finally, two proteins which may interact with Nef were obtained by screening and analysis, namely fibronectin and vimentin. Proteins that interact with Nef are screened by yeast two-hybrid systems, but this is obtained at the yeast cell level, but the environment in animal cells is somewhat different from that in yeast cells. It may also be necessary to conduct experiments in animal cells, such as immunoprecipitation pull-down to further determine the relationship between protein interactions. If the interaction between fibronectin, vimentin and Nef is confirmed, it will provide a new clue to elucidate the mechanism of Nef regulating HIV-1 and drug prevention.
【學位授予單位】：湖北大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R512.91

【參考文獻】

相關期刊論文 前3條

1 余勇,肖庚富,李敏,詹睿,張文濤;人類免疫缺陷病毒-1進入細胞的分子機制及相關藥物的研究[J];生物化學與生物物理進展;2003年01期

2 張宏偉;吳昊;;HIV Nef的結構特點及生物學作用[J];首都醫(yī)科大學學報;2011年02期

3 姚茜茜;呂鈞;葉榮;;人類免疫缺陷病毒1型gp41結構與功能的研究進展[J];微生物與感染;2011年04期

，

本文編號：1888007