AAV9衣壳蛋白VP的原核表达、纯化及多克隆抗体制备

Prokaryotic expression,purification and polyclonal antibody preparation of AAV9 capsid protein VP

目的:表达腺相关病毒(AAV)9型衣壳蛋白VP,制备抗VP蛋白的多克隆抗体。方法:使用PCR技术从AAV9病毒包装质粒中扩增AAV9衣壳蛋白VP,同源重组法构建衣壳蛋白表达质粒pET30a-AAV9-VP。质粒转化表达菌E.coli BL21(DE3)后,IPTG诱导目的蛋白VP表达,亲和层析法纯化VP蛋白,将纯化后的蛋白免疫日本大耳白兔,3次免疫后获得针对VP蛋白的兔多克隆抗体。以Western blot和细胞免疫荧光法检测抗体的应用,ELISA检测所得抗体的效价。结果:成功构建pET30a-AAV9-VP表达质粒,在大肠杆菌BL21中,IPTG可诱导VP蛋白表达。亲和层析法纯化获得高纯度的VP蛋白。该蛋白在日本大耳兔体内能够诱导产生多克隆抗体,ELISA检测抗血清效价达到1∶512 000。结论:成功原核表达和纯化AAV9衣壳蛋白VP并制备了兔多克隆抗体。制备的抗AAV9 VP抗体能够特异性识别和结合AAV衣壳蛋白,并可有效用于Western blot和细胞免疫荧光分析,为后续深入研究AAV9在基因治疗中的作用及AAV9载体开发提供了研究基础。

Objective:To express the recombinant proteins of adeno-associated virus(AAV)9 capsid VP and prepare the polyclonal antibodies against VP.Methods:AAV9 capsid protein VP coding sequence was amplified from AAV9 plasmid by PCR,and capsid protein expression plasmid pET30a-AAV9-VP was constructed by homologous recombination.After transforming it into E.coli BL21(DE3),IPTG was used to induce expression of VP,and the VP was purified by Ni-NTA resin affinity chromatography,the purified protein was used as antigen to immunize Japanese white rabbits to prepare polyclonal antibodies.The rabbit polyclonal antibody against VP protein was obtained after three times of immunization.Specificity of anti-sera was detected by Western blot and cell immunofluorescence,ELISA was used to detect titer of anti-VP antibody.Results:The pET30a-AAV9-VP expression plasmid was successfully constructed.IPTG can induce the expression of VP in E.coli BL21 bacteria.Ni-NTA resin affinity chromatography can effectively purify VP protein,and the VP can induce polyclones effectively in Japanese white rabbits,the antibody titer detected by ELISA was reaches 1∶512000.Conclusion:Successful expression and purification of AAV9 capsid protein VP and preparation of rabbit polyclonal antibodies.The AAV9 VP antibody can specifically recognize and bind to the AAV capsid protein and can be effec‐tively used for Western blot and immunofluorescence analysis.It provides a research foundation for development of AAV9 vector and the in-depth study of the role of AAV9 in gene therapy.