伪狂犬病病毒Ea株gE基因主要抗原区在巴斯德毕赤酵母中的表达

Expression of Major Antigenic Domain of Glycoprotein E of Pseudorabies Virus Ea Strain in Pachia pastoris

伪狂犬病病毒gE蛋白是一种重要的诊断抗原 ,可用于伪狂犬病根除计划中的鉴别诊断。为了获得大量的抗原 ,将编码gE主要抗原区域的基因片段插入毕赤酵母表达载体中 ,得到的重组表达载体转化GS1 1 5酵母 ,通过G41 8抗性筛选得到一株多拷贝的重组酵母 ,经甲醇诱导表达了截短的gE蛋白 ,并分泌到胞外。Westernblotting分析显示表达的蛋白大小为3 3kD。ELISA结果表明表达蛋白具有良好的抗原性 ,重组酵母的诱导培养上清不需纯化可直接作为抗原检测gE的抗体 。

Glycoprotein E of Pseudorabies Virus is known to be an important diagnostic antigen in pseudorabies eradication campaign. In order to obtain the antigen in large quantity, the truncated gE gene encoding the major antigenic domains of gE was expressed and secreted in the methylotrophic yeast, Pichia pastoris. The truncated gE gene was amplified by PCR from pSK1^78K+ plasmid harboring gE full length DNA. The gE fragment was then inserted into pPIC9K vector and fused with the α-factor signal sequence to construct an expression plasmid p9KGE696. After transforming p9KGE696 into GS115 cell by LiCl method, the transformants were screened by G418 for multi-copy recombinants. By using methanol as inducer the truncated gE protein was secreted into culture medium. Western-blotting analysis showed that the molecular mass of the expression product was a 33kD. ELISA indicated that the recombinant protein had good antigenicity and could be used as diagnostic antigen in the serological detection of gE antibodies in swine.