用酵母双杂交筛选与HIV Tat相互作用的宿主因子  

Identification of Host Factors Interacting with HIV Tat Protein by Yeast Two-Hybrid System

作　　者：路艳芳 刘为勇[1] 乔龙[2] 汪峰[1] 侯红艳[1] 孙自镛[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院检验科,武汉430030 [2]华中科技大学同济医学院附属同济医院肿瘤生物医学中心,武汉430030

出　　处：《华中科技大学学报（医学版）》2016年第4期398-401,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家科技重大专项项目(No.2012ZX10004207-004)

摘　　要：目的通过酵母双杂交试验筛选与HIV Tat蛋白相互作用的宿主因子。方法对构建的pGBKT7-tat进行自激活和半乳糖苷试验。运用酵母双杂交实验以HIV Tat作为诱饵,与人均一化基因库进行初筛。对筛选出来的阳性克隆进行测序分析。结果构建的pGBKT7-tat无自激活特性和细胞毒性,然后进行酵母双杂交试验。结果发现在DDO/X/A平板上出现47个阳性的蓝色斑点,在QDO/X/A平板上进行进一步筛选,对再次出现的8个蓝色斑点进行测序。在GenBank中对相互作用的人体基因进行BLAST分析,根据基因注释推测其在病毒与机体相互作用过程中可能发挥的作用。结果测出5个不同的基因,这5个不同的基因分别为羧肽酶N、酪氨酸磷酸酶受体M、ATP1B1、锌指蛋白MYM2和锌指蛋白MYM5。结论本研究新筛选出羧肽酶、酪氨酸磷酸酶受体和ATP1B1病程相关蛋白及两个锌指蛋白等5种功能蛋白与HIV Tat相互作用,为进一步研究HIV Tat蛋白功能提供依据。Objective To identify host factors that interact with HIV Tat protein by using the yeast two-hybrid system.Methods The self-activation and galactose glucoside experiments were conducted in the constructed pGBKT7-tat vector.Then,the yeast two-hybrid assay was employed to identify human proteins that could interact with Tat protein of HIV by screening the homogenized cDNA library.The positive clones were thereafter sequenced.Results The constructed pGBKT7-tat vector was found no self-activation and cell toxicity.The yeast two-hybrid analysis showed that there were 47 positive blue dots on the DDO/X/A plate.Further screening of the 47 positive blue dots on QDO/X/A plate showed eight blue dots,which were later sequenced.Blast analysis was performed on these gene in GenBank.Five different genes were found based on the gene annotation of which played an important role in the interaction of the virus and the host.They were PTPRM,CPN2,ATP1B1,ZMYM5,and ZMYM2,respectively.Conclusion Carboxypeptidase,tyrosine phosphatase receptor protein,ATP1B1course-related protein,and two zinc finger proteins that can interact with HIV Tat were found in the study,which provides the basis for further research on the function of HIV Tat.

关 键 词：酵母双杂交 HIV TAT 蛋白质相互作用 天然免疫 病毒复制 

分 类 号：R373.51[医药卫生—病原生物学]