HIV-1流行株去糖基化修饰gp41突变体的构建及其表达和纯化  

Construction,expression and purification of HIV-1 gp41-deglycosylation mutants

作　　者：邵继平 符健 SHAO Ji-Ping;FU Jian(Hainan Key Laboratory of Preclinical Pharmacology&Toxicology Research for Drugs,Hainan Medical University,Haikou 571199,China)

机构地区：[1]海南医学院海南省药物临床前药理毒理学研究重点实验室,海口571199

出　　处：《中国免疫学杂志》2019年第5期579-583,共5页Chinese Journal of Immunology

基　　金：海南省科技厅社会发展项目(ZDYF2016126)

摘　　要：目的:构建HIV-1ⅢB标准株、中国大陆地区主要流行株CRF-BC和海南省流行株CRF-AE野生型和突变型gp41表达载体,获得gp41重组蛋白。方法:利用基因定点突变的方法,将HIV-1ⅢB、CRF-BC和CRF-AE流行株野生型gp41基因中特定的N611Q糖基化位点天冬酰胺Asn突变为谷氨酰胺Gln,使该位点去糖基化。利用基因工程技术构建野生型pc DNA3. 1-gp41和突变型pc DNA3. 1-Mgp41表达载体。取测序正确的重组质粒转染293 T细胞,重组蛋白经Ni-NTA亲和层析柱纯化后采用免疫杂交方法进行分析。结果:测序结果显示野生型gp41的N611糖基化位点均由天冬酰胺(Asn)的编码基因(AAU)突变为谷氨酰胺(Gln)的编码基因(GAA),成功构建了野生型和突变型gp41表达载体。琼脂糖凝胶电泳分析显示野生型和突变型HIV-1 gp41基因大小约为700 bp,Western blot免疫杂交结果显示在26 k D处出现目的条带,与预期一致。结论:成功构建了HIV-1流行株去糖基化修饰gp41突变体,为研究去糖基化修饰gp41蛋白与疾病发生发展的分子机制奠定基础。Objective:To construct expression vectors of gp41 wild type and mutants with deletion of the glycosylation sites of HIV-1ⅢB,HIV-1 CRF-BC and HIV-1 CRF-AE strain and obtain the recombinant proteins of gp41.Methods:N611 glycosylation sites on gp41 wild type of HIV-1ⅢB,HIV-1 CRF-BC and HIV-1 CRF-AE strain were simultaneously mutated according to the substitution sites of asparagine with glutamine using site-directed mutagenesis.Recombinant pcDNA3.1-gp41 and pcDNA3.1-Mgp41 expression vectors were constructed using gene engineering technique.The positive plasmids were transfected into 293 T cells,the recombinant protein were purified with Ni-NTA affinity chromatography resin and their sizes were analyzed using SDS-PAGE.Results:DNA sequencing indicated that the asparagines(N)residues at the N611 glycosylation sites on HIV-1 gp41 wild type were replaced by glutamine(Q).Wild type and mutants of HIV-1 gp41 expression vectors were successfully constructed.The size of HIV-1 gp41 gene were about 700 bp,the size of His-tag fusion protein were approximately 26 kD,as expectation.Conclusion:Deletion of N-linked glycosylation site mutants of HIV-1 gp41 were successfully constructed and laid the foundation for investigate the mechanism of deglycosylation of HIV-1 gp41 and development of AIDs in hainan province.

关 键 词：HIV-1 病毒 包膜糖蛋白gp41 去糖基化突变体 表达和纯化 

分 类 号：R373.32[医药卫生—病原生物学]