猴细小病毒Vp2的克隆、表达和鉴定

The cloning, expression and identification of simian parvovirus Vp2 protein

目的 克隆、表达和鉴定猴细小病毒 (simianparvovirus,SPV)Vp2蛋白。方法 用设计的SPVVp2区的特异性引物 ,PCR法扩增SPVVp2基因DNA片断 ;将获得的基因片断插入 pThioHisA载体中 ,转化大肠杆菌DH5α后挑选阳性克隆 ,利用限制性内切酶酶切和核酸序列分析技术鉴定重组质粒 ;以LB培养液培养转化有插入SPVVp2基因的pThioHisA载体的大肠杆菌 ,以终浓度 1mmol·L -1的IPTG诱导蛋白的表达 ;用SDA PAGE和Westernblot分析和鉴定表达的蛋白。结果 经限制性内切酶酶切和核酸序列分析 ,获得了插入pThioHisA载体框架的SPVVp2基因的表达质粒 ;阳性转化大肠杆菌在IPTG诱导下获得了SPVVp2蛋白的表达 ;SDA PAGE和用抗 Thio及抗 SPVVp2的特异性抗体的Westernblot分析证实了表达蛋白的特异性。结论 获得了SPVVp2基因的表达质粒并在大肠杆菌中得了高效表达 ,为进一步建立SPV感染的检测方法和研究SPV的血清流行病学等奠定了基础。

Objective To clone,express and identify simian parvovirus (SPV) Vp2 protein in E.coli. Methods SPV Vp2 gene was amplified by PCR using specific primers and inserted into the multiple cloning site of pThioHis A vector. The expression vector pThioHis A-Vp2 was constructed and transformed into E.coli DH5α competent cells. The positive clones were identified by restriction enzyme digestion analysis and sequencing analysis. SPV Vp2 protein expression was induced by IPTG and the expressed protein was identified by SDS-PAGE and Western blot analysis using both anti-Thio and anti-SPV Vp2 antibodies. Results The expression vector pThioHis A-Vp2 and positively transformed clones were obtained. SPV Vp2 protein was expressed by the positive clones under the induction of IPTG. Western blot analyses showed that the expressed protein specifically reacted with both anti-Thio and anti-SPV Vp2 antibodies. Conclusion SPV Vp2 can be highly expressed by the constructed pThioHis A-Vp2 transformed E.coli. The successful expression of SPV Vp2 is of great significance for the further investigation of SPV infection and related studies.