虎源猫泛白细胞减少症病毒VP2基因主要抗原表位区的原核表达和蛋白纯化

Prokaryotic Expression of VP2 Antigenic Epitope Gene of Feline Panleukopenia Virus and Its Purification

利用PCR技术从FPV-HLJ株病毒细胞培养物中扩增VP2基因主要抗原表位区片段VP2’。扩增片段克隆至pMD18-T载体,经核苷酸序列测定确认后,亚克隆于pGEX-6p-1原核表达载体,构建pGEX-6p-VP2’重组原核表达质粒。将该质粒转化至表达菌BL21(DE3)中用IPTG进行诱导表达。表达蛋白采用切胶法纯化,并用SDS-PAGE和Westernblot检测纯化蛋白纯度和抗原特异性。结果表明,VP2基因全长1200bp,编码400个氨基酸。重组菌可表达相对分子量约为66kD的融合蛋白,包括目的蛋白40kD和GST标签26kD。该蛋白以包涵体形式存在,且GST融合蛋白具有良好抗原特异性。

The gene encoding antigenic epitope VP2 was amplified from feline panleukopenia virus （ FPV） FPV-HLJ strain isolated from tiger feces. The amplified DNA was cloned into the prokaryotic expression vector pGEX-6p-1 after its sequence was confirmed by sequencing. The expression vector was then transferred into E. coli BL21（ DE3） for expression under IPTG treatment. The recombinant protein was isolated using SDS-PAGE and recovered from the gel. A second SDS-PAGE was performed to confirm its purity and detect its antigenic specificity by Western blotting. Results indicated that VP2 gene of FPVHLJ was 1 200 bp in length encoding 400 amino acids. A fusion protein of about 66 kD was obtained from the prokaryotic expression system,including 40 kD VP2 protein and 26 kD GST-tag protein. The VP2 protein had high antigenic specificity.