禽呼肠孤病毒99G株σB编码基因的克隆及其在大肠杆菌中的高效表达

Cloning of σB gene of avian reovirus 99G strain and its expression in Escherichia coli

采用RTPCR方法扩增禽呼肠孤病毒（ARV）99G株σB编码基因，序列分析表明，99G株σB编码基因与其他禽呼肠孤病毒的同源性为90％～99％，而与我国番鸭呼肠孤病毒S12株的同源性仅有60％；用BamHⅠ和XhoⅠ酶将σB编码基因切下并克隆到表达载体pGEX-6p-1中，构建了重组表达质粒pGEX-σB；鉴定后转化至大肠杆菌BL21（DE3）中，经IPTG诱导，σB基因在大肠杆菌中得到了高效表达，表达的融合蛋白占总蛋白的41．46％，融合蛋白的分子质量为66ku，主要以包涵体形式存在；Western-blot结果显示，该融合蛋白能与抗ARV阳性血清发生特异性反应，表明重组蛋白具有良好的反应原性，可用于禽呼肠孤病毒病诊断试剂盒和基因工程疫苗的研制。

The σB-encoding gene of avian reovirus （ARV） 99G strain was amplified by RT-PCR with primers designed based on the reported avian reoviruses in GenBank. The sequence of the aB-encoding gene was compared with those of other ARVs and Muscovy duck reovirus （DRV）,and the results showed that the nucleotide identities between ARV 99G strain and ARVs ranged from 90 % to 99 %,and the identity between ARV 99G and DRV S12 from China was only 60%. To construct fusion expression vector pGEX-σB, the amplified σB-encoding gene was inserted into the bacterial plasmid vector pGEX-6p-1 after the gene was derived from pMD18-T vector with BamH Ⅰ and Xho Ⅰ and then sequenced. The positive pGEX-σB was transformed into Escherichia coli BL21（DE3） and induced with 1 mmol/L IPTG. The recombinant fusion protein of approximately 66 ku in molecular mass was expressed highly in inclusion body, and made up 41.46% of the total proteins. Western-blot analysis showed that the recombinant fusion protein was recognized specifically by the antiserum against ARV,confirming that the recombinant fusion protein had good reactogenicity and could be used to develop diagnostic kit for ARV and genetic engineering vaccine against ARV.