猪瘟病毒Erns蛋白在毕赤酵母中的表达及鉴定

为制备猪瘟病毒(CSFV)特异性诊断抗原,根据国内CSFV流行毒株的序列合成了其主要的保护性抗原Erns蛋白的基因序列,将合成的基因按设计的酶切位点与毕赤酵母表达载体pPIC9K连接,构建了重组表达质粒pPIC9K-Erns.用SalI将重组表达质粒线性化后,转化GS115酵母菌.经筛选、鉴定,成功获得了表达CSFV的Erns蛋白的阳性重组酵母菌.该阳性重组酵母菌经甲醇诱导,在培养液上清中可检测到目的蛋白.Western-blot结果显示,该蛋白能特异识别CSFV抗体,且具有很好的反应原性.

Expression and Identification of Erns Protein of Classical Swine Fever Virus in Pichia pastoris

In order to produce the specifc diagnostic antigens of classical swine fever virus(CSFV),the gene sequence of CSFV's main protective antigen-Erns protein was synthesized according to the sequence of the prevalent CSFV Cstrain.Then the purified products were cloned into pPIC9K expression vector to construct recombinant expression plasmid(Ppic9k-Erns).After recombinant expression plasmid linearized by Sal I,the microzyme GS115 was transformed by Sal I.Through screening and identification,the positive recombinant micmzyme of Erns protein to express CSFV was obtained successfully.This positive recombinant microzyme induced by methanol,then,the target protein could be examined in the superuatant culture solution.The results of Western-blot showed that this target protein could specifically recognize the antigens of CSFV and it had a great reactionogenicity.