鲤鱼春季病毒血症病毒磷蛋白基因的克隆与原核表达

【目的】克隆鲤鱼春季病毒血症病毒（SVCV）的磷蛋白（P蛋白）基因，并对其进行原核表达，为进一步制备SVCV-P蛋白单克隆抗体及建立新的SVCV诊断方法奠定基础。【方法】采用RT-PCR方法扩增SVCV P蛋白基因，将其克隆入pET-28a(＋)载体中，获得原核重组表达质粒pET28-P，进行原核表达。将该重组原核表达质粒转化至大肠杆菌Rosetta感受态细胞中，用0.4 mmol/L IPTG进行诱导表达，用镍亲和层析试剂盒对表达产物进行纯化，分别用SDS-PAGE和Western Blotting方法对表达产物进行鉴定。【结果】成功克隆了SVCV-P蛋白基因，该基因长度为933 bp。成功构建了原核重组表达质粒pET28-P，其诱导表达产物分子质量约为37 ku；表达的重组P蛋白可被SVCV阳性血清所识别。【结论】成功克隆了SVCV P蛋白基因，获得了分子质量约为37 ku的重组P蛋白。

Cloning and prokaryotic expression of phosphoprotein of spring viremia of carp virus

【Objective】 The research cloned and expressed phosphoprotein(P) gene of spring viremia of carp virus(SVCV),to produce monoclonal antibody and lay the basis for further exploring the infection mechanism of SVCV.【Method】 The P gene was amplified by RT-PCR and cloned into the pET-28a(+) vector.The resultant recombinant plasmid was transformed into E.coli Rosetta to express with IPTG induction.The SDS-PAGE analysis showed that the expressed product was about 37 ku.Specificity was detected by Western blot.【Result】 The results show that P gene is 933 bp in length,and the molecular weight of the recombinant protein about 37 ku.【Conclusion】 Phosphoprotein genes are successfully cloned,and the molecular weight of the recombinant protein is about 37 ku.