猫免疫缺陷病毒p24基因原核表达载体的构建及表达

The construction and expression of prokaryotic expression vector of FIV p24 gene

为了研究猫免疫缺陷病毒（Feline immunodeficiency virus,FIV）的核衣壳蛋白（core nucleocapsid protein）——p24蛋白的免疫活性,试验根据Gen Bank中FIV的p24基因序列设计了1对引物,对FIV检测阳性的猫血液样本提取总RNA并进行反转录,PCR扩增p24基因,将此目的片段克隆到原核表达载体p ET-28a（＋）中,构建获得重组质粒p ET-p24,并热转化至E.coli BL21（DE3）感受态细胞中,诱导蛋白表达后用SDS-PAGE和Western-blot进行鉴定,用FIV感染的猫阳性血清和重组p24蛋白免疫小鼠得到的多克隆抗体对表达蛋白的免疫原性和反应原性进行检测。结果表明：RNA反转录成c DNA后成功扩增出大小为622 bp的p24基因和构建出原核表达载体;构建成功的含p ETp24重组质粒的大肠杆菌诱导后能大量表达FIV-p24蛋白,并且p24蛋白以包涵体形式存在;p24蛋白具有良好的免疫原性和反应原性。说明p24蛋白是具有生物学活性的蛋白,在猫免疫缺陷病的诊断和防治中具有重要意义。

The aim of the present study was to identify the immunogenicity of core nucleocapsid protein-p24 of Feline immunodeficiency virus（ FIV）. According to the p24 gene sequence of FIV in Gen Bank,a pair of primers was designed. The total RNA extracted from the blood samples of FIV positive cats was reverse transcribed,and the p24 gene was amplified by PCR. The target fragment was ligated into the prokaryotic expression vector p ET-28 a（ ＋）. The prokaryotic expression plasmid p ET-p24 was constructed and transformed into E. coli BL21（ DE3） competent cells. The protein expression was induced by IPTG,and identified by SDS-PAGE and Western blotting. The immunogenicity and reactionogenicity of the expressed protein were detected by using the positive serum of FIV infected cats and the polyclonal antibody of mice immunized with recombinant p24 protein. The results showed that the p24 gene was successfully amplified from the c DNA,which was 622 bp in size. The prokaryotic expression vector containing p24 gene was also successfully constructed. The recombinant expression bacteria containing p ET-p24 could express a large amount of FIV-p24 protein after induction,and the p24 protein existed in the form of inclusion body. The expressed p24 protein had good immunogenicity and reactionogenicity. It was suggested that the p24 protein was a biological active protein. The study of FIVp24 protein has important significance for the diagnosis and prevention of cat immunodeficiency disease.