牛流行热病毒糖蛋白G1抗原表位在毕赤酵母中的表达和抗原性鉴定

Expression and Antigenic Characterization of the Epitope-G1 of Bovine Ephemeral Fever Virus Glycoprotein in Pichia pastoris

摘要:

从包含牛流行热病毒G蛋白基因的质粒pMD-G中克隆G₁抗原表位区基因，亚克隆进表达载体pPIC9K，构建重组载体pPIC9K-G₁，线性化后电转化毕赤酵母GS115，通过G418压力和PCR法筛选阳性重组酵母进行诱导表达。经SDS-PAGE、脱糖基化分析、Western blot、ELISA、兔体免疫实验和特异性分析，表明该基因在GS115中表达并进行了适度的糖基化，表达蛋白有良好的生物学活性和特异性，可作为包被抗原，开发ELISA诊断试剂盒。

Abstract:

The epitope-G₁ gene, cloned from the pMD-G plasmid including G protein gene of bovine ephemeral fever virus, was subcloned into expression vector pPIC9K to construct pPIC9K-G₁ recombinant plasmid successfully, and the recombinant plasmid linearized was transformed into Pichia pastoris GS115 by electroporation. The recombinant Pichia pastoris strains were screened by G418 and PCR, and induced by methanol. The expressed products were analyzed by SDS-PAGE, deglycosylation, Western blot, ELISA, immunizing rabbits and specificity experiments. The results indicated that the gene was expressed successfully in GS115 and glycosylated moderately, and the target protein had nicer biological activity and specificity. The protein is able to be used as coating antigen to develop ELISA Kit for diagnosing bovine ephemeral fever.