猪乙型脑炎病毒E蛋白的原核表达与鉴定

Expression and Identification of Japanese Encephalitis Virus Truncated E Protein in E.coli

参照已发表的日本乙型脑炎病毒(Japanese encephalitis virus,JEV)基因组序列,设计合成1对特异性引物,PCR扩增长约972bp的E基因片段。将目的片段定向克隆到pET30a表达载体中,酶切及测序鉴定均正确后,转化BL21表达菌,经IPTG诱导得到了以包涵体形式表达的重组蛋白。将重组蛋白变性、纯化和复性后,免疫印迹检测证明纯化的重组蛋白具有良好的抗原性和特异性。

A fragment of about 972 bp long was amplified by RT-PCR technique with a pair of specific primers based on published Japanese encephalitis virus（JEV） genome sequence. Then the target fragment was direetionally cloned into pET30a vector. After identifying with enzyme cutting and sequencing, the recombinant plasmid was transformed into E. coli BL21 （DE3）. The recombinant protein E was expressed in inclusion body form in E. coli after induction with IPTG. After denaturation, purification and renaturation, the purified protein was analyzed by Western blotting, the results showed that the purified recombinant protein retained better antigenicity and specificity.