鸡传染性支气管炎病毒变异株(793/B)M基因与2种截短M基因的原核表达及鉴定

Prokaryotic expression and identification of M gene and two truncated M gene of avian infectious bronchitis virus(793/B)

运用生物信息学技术推测鸡传染性支气管炎病毒(IBV)变异株(793/B)M基因的抗原位点,应用DNAStar软件针对M基因及其抗原表位片段设计3对引物,PCR扩增并成功构建了M基因的pGEX6p-M和2种截短M基因的pGEX6p-M1,pGEX6p-M2原核表达质粒,SDS-PAGE检测分别在50000,32000,29000处有特异性表达条带,表明GST-M,GST-M1,GST-M2重组蛋白获得成功表达;Westernblotting检测显示,GST-M和GST-M1重组蛋白能被鼠抗IBV(793/B)血清识别,而GST-M2重组蛋白不能被识别。GST-M与GST-M1重组蛋白具有良好的抗原性,初步证实M蛋白存在抗原表位,这为进一步研究IBV(793/B)M蛋白的免疫原性和制备基因工程疫苗提供了重要依据。

To infer the antigen site of variant avian infectious bronchitis virus （793/B） M gene by bioinformatics technology,the primers of the M gene and the two epitope fragments were designed with DNAStar,and these fragments amplified by PCR were cloned into prokaryotic expression plasmid to construct the pGEX6p-M,pGEX6p-M1 and pGEX6p-M2,respectively.The expressed recombinant proteins GST-M,GST-M1 and GST-M2 could be detected at bands of 50 000,32 000 and 29 000 by SDS-PAGE,and the results of Western blotting confirmed that GST-M and GST-M1,not GST-M2 recombinant proteins could specifically react to the serum samples of mouse anti-IBV （793/B）,respectively.The results showed that GST-M and GST-M1 recombinant proteins possessed a good antigenicity which initially confirmed the existence of the M protein epitopes.The study also laid an important foundation for further study on immunogenicity of 793/B （IBV） M protein and preparation of genetically engineering vaccines.