应用酵母双杂交系统筛选与Nef相互作用的蛋白

发布时间：2018-05-14 13:18

本文选题：艾滋病病毒1型 + 负调控因子　；参考：《湖北大学》2014年硕士论文

【摘要】：人类免疫缺陷病毒1型(human immunodeficiency virus1,HIV-1)是一种单股正链核糖核酸(RNA)逆转录病毒,其具有高复制率、高重组率、高突变率等特点。因为它有快速变异的基因组,所以它能够逃脱宿主细胞的免疫压力。艾滋病毒已经在世界范围内泛滥几十年,对于寻找治疗艾滋病毒的新方法需要全人类共同努力。负调控因子(negative factor, NEF)是人免疫缺陷病毒附属蛋白之一,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。国内外对Nef进行了大量的研究,发现其具有以下几个主要功能：(1)介导CD4的下调；(2)介导CD28的下调；(3)介导MHC分子的下调；(4)干扰病毒粒子的复制能力和感染性；(5)介导细胞凋亡的抑制。因为对于其作用机制了解不够透彻,通过酵母双杂交系统筛选与Nef相互作用的蛋白质,我们能够更好的对Nef调控HIV-1的机制以及AIDS疾病进程影响机制进行研究。酵母双杂交系统最主要特点是能够快速检测蛋白之间是否具有相互作用。本实验首先以pNL4-3为模板扩增得到目的基因Nef,然后将目的基因Nef和载体pGBKT7进行双酶切反应,使目的基因Nef克隆到载体pGBKT7上,构建成pGBKT7-Nef诱饵质粒。完成诱饵蛋白pGBKT7-Nef在AH109酵母菌株里进行自激活性和毒性检测后,再进行酵母双杂交实验。将接种到AH109酵母菌株中表达的pGBKT7-Nef与人的B淋巴细胞文库进行酵母双杂交筛选。首先将酵母菌株AH109活化,然后将诱饵质粒pGBKT7-Nef转入到酵母AH109中,然后与带有诱饵质粒pGBKT7-Nef的AH109的单克隆与人类B淋巴胞文库质粒共转,然后运用X-a-gal分析(蓝白斑的鉴定)的方进,将单克隆在涂有X-a-gal的四缺培养基上观察其生长状态以及颜色的变化。根据显蓝斑的编号,找出相对应的阳性克隆提取酵母质粒,对感受态细胞大肠杆菌DH5α转化,然后提取质粒,送去测序。将测序结果在NCBI上进行nucleotide blast进行同源性分析,从而找到与Nef蛋白相互作用的蛋白。最终通过筛选和分析得到两个可能会与Nef相互作用的蛋白,分别是纤维凝胶蛋白(ficolin)和波形蛋白(vimentin)。通过酵母双杂交系统筛选出与Nef存在相互作用的蛋白,但是这是在酵母细胞水平上获得,但是动物细胞内的环境与酵母细胞内的环境会有一定的区别,可能还需要在动物细胞内进行相关实验,比如免疫共沉淀、GST pull-down进一步确定蛋白相互作用的关系。如果证实了纤维凝胶蛋白、波形蛋白和Nef存在相互作用,将进一步对阐明Nef对于HIV-1调控的机制以及药物预防提供了新的线索。
[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 伪, 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.
【学位授予单位】：湖北大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.91

【参考文献】