猪瘟病毒囊膜蛋白E2基因真核分泌型表达载体的构建及其表达

Construction of eukaryotic expression vector of CSFV E2 genes and its secretory expression

【目的】构建猪瘟病毒囊膜蛋白(E2 protein)基因的真核表达载体,并将其在猪脐静脉血管内皮细胞中进行表达。【方法】采用PCR技术扩增出E2蛋白全长基因(E2qc)序列和去除跨膜基因(E2sh)序列,并将其克隆入真核表达载体pSecTag2(A)中,构建pSecTag-E2qc和pSecTag-E2sh表达载体,分别进行PCR、双酶切及测序鉴定。用脂质体法将阳性克隆瞬时转染猪脐静脉血管内皮细胞,对转染细胞进行RT-PCR检测目的基因的转录情况,同时对转染细胞及细胞上清液进行SDS-PAGE和Western-blot分析,以检测目的蛋白的表达情况。【结果】成功克隆了E2蛋白基因全长序列1119bp和去除E2蛋白跨膜基因序列999bp的目的基因。构建的表达载体经PCR、双酶切法及测序鉴定均无误。转染后细胞的RT-PCR结果显示,目的基因被成功转录。转染后细胞及细胞上清液的SDS-PAGE和Western-blot结果显示,目的基因被成功表达。【结论】成功构建了猪瘟病毒E2蛋白的真核表达载体,转染猪脐静脉血管内皮细胞后,分泌表达了猪瘟病毒E2重组蛋白。

[Objective] CSFV envelope protein genes were subcloned into a eukaryotic expression vetor which could make the protein secret into medium,and then swine umbilical vein endothelical cell（SUVECs） was used to express this protein. [Method] CSFV E2 full-length genes （E2qc）and E2 genes without transmembrane sequence（E2sh） were amplified from plasmid pMD19T-E2 by PCR, then the two genes were subcloned into pSec Tag 2（A）;PCR digestion and sequencing were used to identify positive plasmidithe identified recombined plasmid was transiently transfected into SUVECs by Lipofectamine 2000 and expression products in SUVECs were determined by RT-PCR.SDS-PAGE and Western blot. [Result] The fragments of E2 full-length genes and E2 genes without transmembrane sequence were examined by 10 g/L agarose electrophoresis consistent with predicted size. PCR digestion and sequencing showed that the recombinant plasmids were accurate;E2 protein expressing in SUVECs was confirmed by RT-PCR and Western-blot. The results showed that the E2 protein was secteted into the medium. [Conclusion] Recombinant plasmid pSec Tag-E2 containing CSFV envelope protein genes was successfully constructed,and E2 protein made secretory expression in SUVECs.