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根据Gen Bank中鲤春病毒血症病毒(SVCV)糖蛋白全基因序列设计特异性引物,以SVCV欧洲株病毒核酸为模板,通过RT-PCR方法获得欧洲株SVCV-G基因,克隆至p PICZαA,构建p PICZαASVCV1540表达质粒。以限制性内切酶Sac I线性化p PICZαASVCV1540后通过化学法转化毕赤酵母感受态细胞X33和GS115,添加1%甲醇进行诱导表达,SDS-PAGE电泳分析表达产物显示其分子质量为70 ku。Western Blot分析显示该蛋白可以被SVCV山羊多抗识别,表明目的蛋白具有反应原性。
Specific primers were designed based on the complete gene sequence of the glycoprotein of the carp virus viremia virus (SVCV) in Gen Bank. SVCV European strain of viral nucleic acid was used as a template to obtain the SVCV-G gene of European strain by RT-PCR and cloned into p PICZαA, The p PICZα ASVCV1540 expression plasmid was constructed. After linearization of p PICZαASVCV1540 with restriction enzyme Sac I, the competent cells of Pichia pastoris X33 and GS115 were transformed by chemical method, and 1% methanol was used for induction. SDS-PAGE analysis showed that the expressed product had a molecular mass of 70 ku. Western Blot analysis showed that the protein could be identified by SVCV goat polyclonal antibody, indicating that the target protein was reactive.