鸡贫血病毒衣壳蛋白的表达纯化及多克隆抗体制备

利用PCR技术,以pPrpo-VP1为模板扩增得到鸡贫血病毒的衣壳蛋白基因(VP1),以T4多聚核苷酸激酶磷酸化处理、纯化后,克隆至表达载体pET-30a( )中,从而构建了原核表达质粒pET30-VP1。将pET30-VP1转化至感受态细胞E.coliBL21(DE3)中,经IPTG诱导后,SDS-PAGE分析,可见约45kDa的目的蛋白获得表达。该蛋白经亲和层析纯化后,免疫6-8w的雌性Balb/c鼠,三次免疫后,采血分离血清,制得抗VP1的多克隆血清。以纯化的VP1为包被抗原,用ELISA方法检测,制备的血清效价达12800×以上。以Westernblot检测,该血清可与目的蛋白发生特异性反应,证明其具有良好的免疫原性。VP1蛋白的成功表达及其多克隆抗体的制备为进一步研究VP1蛋白的功能及开展CAV疫苗及诊断制剂的研制奠定了基础。

Expression and Purification of the Capsid Protein of Chicken Anemia Virus and Preparation of Antibody

The coding region of capsid protein gene from Chicken Anemia virus(CAV) was amplified from pPro-VP1 by PCR and cloned into pET-30a( ). E.coli BL21(DE3)were transformed by the recombinant plasmid pET30-VP1. Analysis by SDS-PAGE and Western blot showed that the target gene was expressed successfully in the form of inclusion body when induced with IPTG. The protein was then purified by Ni2 -affinity chromatography and used to immunize female Balb/c mice. After three rounds of immunizations, antiserum was collected and used to detect the specificity of recombinant protein. ELISA showed that the titer of antiserum was above 1:12800. Moreover, antiserum reacted specifically with purified recombinant protein in Western blot. This study laid a foundation for the development of CAV diagnostic kit and vaccine.