HIV-1病毒感染因子基因克隆、表达、纯化及其抗体制备的研究

Cloning,expression and purification of HIV-1 Vif,and its antibodies production

目的制备HIV-1病毒感染因子(Vif)及其抗体。方法用PCR技术从HIV-1NL4·3cDNA质粒中扩增病毒感染因子(vif)基因,vif基因全长为579nt(核苷酸),翻译成含192个氨基酸的蛋白质。将测序鉴定过的vif基因克隆到原核表达载体pET-32a上,以包涵体的形式在大肠埃希菌BL21(DE3)中高效表达,Vif蛋白C端融合6×His标签便于纯化及鉴定。应用酶切鉴定、SDS-PAGE及WesternBlot等方法确保基因片段的正确性及表达蛋白的特异性。间接ELISA法测定兔多克隆抗体滴度。结果成功地获得了高纯度的Vif融合蛋白,纯度可达80%以上。用其制备多克隆抗体滴度可达1∶204800。结论获得高纯度的Vif融合蛋白及其高效价的抗体。

Objective To prepare HIV-I Vif and to produce its antibodies. Methods The full length gene fragment of HIV-I vif was amplified by PCR from plasmid of HIV-I NIA.3 cDNA. The length of the HIV-I vif gene fragment was 579 nt, and encodes 192 amino acid residues. The resulting DNA construct was cloned into a prokaryotic expression vector （pET-32a） and recombinant pET-vif was expressed in Eserichia coli BL21 （DE3） as an insoluble protein. The vector also contained a six-histidine （His6） tag at the C-termlnus for convenient purification and detection. To express and purify the HIV-I vif gene in E. coli cells, the accuracy of inserted gene and specificity of HIV-1 Vif proteins were detected by two enzymes digestion technology, SDS-PAGE, and Western Blot （WB）. Serum samples were tested by enzyme-linked immunosorbent assays （ELISAs） to determine the level of antibodies. Results The purity of Vif was above 80%. The titer of the antibodies was 1：204 800. Conclusion HIV-I Vif fusion protein with high purity was obtained and its corresponding polyclonal antibodies with high titer were produced.