摘要
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。
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.
出处
《中国病毒学》
CSCD
2006年第5期477-480,共4页
Virologica Sinica
关键词
鸡贫血病毒
衣壳蛋白
表达
纯化
多克隆抗体
Chicken anemia virus
Capsid protein
Expression
Purification
Polyclonal antibody
