Ⅰ型人类免疫缺陷病毒包膜糖蛋白修饰体表达载体的构建及鉴定

目的构建V3环完全或部分缺失的I型人类免疫缺陷病毒(hauman immunodeficiency virus type I,HIV-1)ADA株包膜糖蛋白表达体系。方法分别设计包膜糖蛋白两端及V3环删除区域两端的引物,采用重叠延伸剪接法进行HIV-1 ADA株包膜糖蛋白V3环修饰体的构建。得到的PCR产物经EcoR I和Xho I双酶切,将酶切产物与pSM载体连接,转化大肠埃希菌后筛选阳性克隆,经PCR及基因序列测定进行鉴定。结果获得HIV-1 ADA株包膜糖蛋白V3环修饰体PCR产物,构建了其表达载体pSM-ADA△V3和pSM- ADAmV3,经转化和筛选获得了重组质粒,经PCR及基因测序鉴定显示重组质粒序列正确,为预期目的片段。结论成功构建了V3环修饰的HIV-1 ADA株包膜糖蛋白的表达载体。此结果为进一步构建伪病毒,观察V3环完全或部分缺失对病毒侵入靶细胞过程的影响,为开发阻止HIV-1进入靶细胞的药物或疫苗打下基础。

Construction and identification of expression vectors of human immunodeficiency virus type Ⅰ envelope with V3 loop modification

Objective To construct expression vectors bearing HIV-1 ADA envelope (env) with V3 loop modification. Methods Splicing by overlap extension (SOE) was used for modification of HIV-1 ADA V3 loop. Three pairs of primers were designed and overlap PCR was taken placed. Recombinant plasmid pSM-ADAAV3 and pSM-ADAmV3 were obtained via ligation of the plasmid pSM and the PCR product. The recombinant plasmids were identified with PCR amplification and sequencing. Results PCR products were obtained and correctly inserted into plasmid pSM. The recombinant plasmids were transformed into E.coli. Sequencing results had shown that the mutated env gene having correct nucleotide sequence was the expected fragment. Conclusions Expression vectors of HIV-1 ADA env with V3 loop modification were constructed successfully, which provides basis for constructing pseudovirus and observing the effects of partial or complete deletion of V3 loop on the entry of viruses into target cells. Also,these expression vectors have an important implication in developing vaccine and drug.