人免疫缺陷病毒Ⅰ型Vif蛋白的原核表达、纯化及单克隆抗体制备

Prokaryotic Expression,Purification of Regulatory Protein Vif of HIV-1 and Preparation of Monoclonal Antibody of Vif Protein

目的:克隆、表达、纯化人免疫缺陷病毒Ⅰ型(HIV-1)Vif蛋白,制备其单克隆抗体。方法:提取感染了HIV的细胞基因组DNA,PCR扩增vif基因,插入表达载体pET32a,转化大肠杆菌BL21(DE3)获得工程菌株,IPTG诱导蛋白表达,Western印迹鉴定目的蛋白,亲和层析纯化目的蛋白;免疫BALB/c小鼠,制备单克隆抗体。结果:构建了Vif蛋白的原核表达载体vif-pET32a,并在大肠杆菌中获得高表达,目的蛋白以包涵体形式存在;纯化获得高纯度的重组Vif蛋白,蛋白浓度可达0.56mg/mL;建立了抗Vif蛋白单克隆抗体细胞株,制备了腹水,滴度可达1∶16×106,抗体纯化后保持了活性和特异性。结论:在原核表达系统中表达、纯化了重组Vif蛋白,制备了针对Vif蛋白的单克隆抗体,为研究Vif蛋白的功能和抗原性奠定了基础。

Objective： To prokaryotic express, purify the regulatory protein Vif of HIV-1 and prepare monoclonal antibody against Vif. Methods： vif gene of HIV-1 was amplified by PCR and sub-cloned into prokaryotic expression vector pET32a. The Vif protein expression vector was then transformed into E.coli BL21（DE3）. The expression of recombinant Vif protein was induced by adding IPTG and identified by SDS-PAGE and Western blot. Vif protein was subsequently purified with Ni-NTA affinity chromatograph. BALB/c mouse was immunized and monoclonal antibody against Vif was prepared. Results： Vif expressing vector v/f-pET32a was constructed, and the recombinant Vif protein was expressed in high concen- tration in endosome and purified with the concentration of 0.56 mg/mL. Cell lines secreting anti-Vif monoclonal antibody was developed by cellular fusion. Antibodies in peritoneal fluid showed positive reaction with antigen when being diluted by 1：16×106 times. Anti-Vif monoclonal antibody was purified with activity and specificity retained. Conclusion： Recombinant Vif protein was successfully expressed and purifed, anti-Vif monoclonal antibody was prepared. This research provid- ed materials and foundation for further study on the function and antigen characterization of Vif protein.