人类免疫缺陷病毒1型病毒复制负调控因子编码基因的克隆及在BCBL-1细胞和NIH/3T3细胞中的表达

Clone and expression of the Negative factor encoding gene of human immunodeficiency virus 1 in BCBL-1 cells and NIH/3T3 cells

目的:分离克隆人类免疫缺陷病毒1型(HIV-1)编码的病毒复制负调控因子(Negative factor,Nef)基因,并将其导入BCBL-1细胞和NIH/3T3细胞中进行表达。方法:根据GenBank登记的Nef基因核苷酸序列设计PCR引物,在其5′端分别引入EcoRⅠ和SalⅠ酶切位点;以含有Nef基因的pCINL质粒为模板,扩增Nef基因,经双酶切后克隆进真核表达载体pCI-neo中。为了便于蛋白检测,在下游引物5′端引入Flag序列,构建重组真核表达质粒。重组质粒经酶切鉴定、核酸序列测定和分析后分别瞬时转染BCBL-1细胞和NIH/3T3细胞,用RT-PCR、Western blot分别从核酸和蛋白水平检测Nef基因的表达情况。结果:成功构建了含Nef基因的重组真核表达质粒,核酸序列分析显示,克隆的Nef基因序列全长651bp,与GenBank中登记的Nef基因序列100%同源,引入的Flag序列完全正确;RT-PCR和Western blot都在Nef预期位置检测到特异性条带。结论:HIV-1Nef基因在BCBL-1细胞和NIH/3T3细胞中获得了正确表达。

Objective：To isolate and clone the Negative factor（Nef） gene of human immunodeficiency virus 1 （HIV-1）,and to investigate its expression in the BCBL-1 cells and NIH/3T3 cells. Methods：A pair of PCR primers for Nef gene with EcoR Ⅰ and Sol Ⅰ restriction enzyme cut sites was designed according to the sequence registered in GenBank. Then the genome of plasmid PCINL was taken as template and Nef gene was amplified using PCR. Subsequently, amplified gene fragments were digested with the two enzymes mentioned above,and then cloned into pCI-neo vector to create recombinant eukaryotic expression plasmid designated as pNefF, which was introduced with Flag sequence to facilitate the detection of protein. After identification with enzyme digestion and nucleotide sequences analysis,recombinant pNefF DNA was transient transfected into BCBL-1 cells and NIH/3T3 cells. The expression of pNefF mRNA and protein in BCBL-1 cells was detected by RT-PCR and Western blot, respectively. Results. Nucleotide sequences analysis indicated that the isolated and cloned pNefF sequence length was 651 bp. The isolated K9 sequence was 100% homology with Nef gene previously registered in GenBank. The specific bands were both detected at the expected place by RT-PCR and Western blot. Conclusion： Nef gene could be correctly expressed in BCBL-1 cells and NHT/3T3 cells.