水貂肠炎病毒VP2基因原核表达及鉴定

Prokaryotic Expression and Identification of Mink Enteritis Virus VP2 Gene

采用PCR技术扩增MEV VP2基因,将该基因片段定向克隆原核表达载体PET-30a,构建MEV VP2基因原核表达重组质粒PET-30a-VP2。将该重组质粒转化到宿主菌BL21(DE3),IPTG诱导后进行SDSPAGE和Western-blotting分析。结果表明:该基因全长1 755 bp,其目的基因序列完全正确,能编码584个氨基酸。利用载体上的His标签对其进行纯化,经SDS-PAGE鉴定该重组蛋白以包涵体的形式存在,大小约为70 ku;Western-blotting验证该重组蛋白具有被抗MEV血清识别的特性,具有良好的反应原性,可为MEV基因工程亚单位疫苗、免疫学诊断方法等提供参考。

The gene fragment of mink enteritis virus VP2 protein was amplified by PCR from MEV genome and cloned into PET-30a vector to construct recombinant prokaryotic expression plasmid PET-30a-VP2. The recombinant plasmid was transformed into Escherichia. coil BL21 （DE3） and ex- pression protein was analyzed by SDS-PAGE and Western-blotting. The result showed that the full length of VP2 gene included 1 755 nucleotides and encoded 584 amino acids, the target gene se- quence was completely correct. And the expression product was purified by His-tag of vector. SDS- PAGE analysis showed that the recombinant protein was about 70 ku and mainly expressed in the form of an inclusion body. Western-blotting analysis proved the recombinant protein had good reactive ability against MEV positive serum. The protein could be used for developing subunit vaccine and antigen of immunological diagnostic methods.