猪繁殖与呼吸综合征病毒JL07SW株Nsp9基因的序列分析及原核表达  

PROKARYOTIC EXPRESSION AND SEQUENCE ANALYSIS OF NSP9 GENE OF PRRSV JL07SW STRAIN

作　　者：黄志强[1] 张学东[1] 丁壮[1] 张泉鹏[1] 宣华 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]广东省农科院兽医研究所,广州510000

出　　处：《内蒙古农业大学学报（自然科学版）》2011年第1期7-12,共6页Journal of Inner Mongolia Agricultural University(Natural Science Edition)

基　　金：广东省科技攻关重大项目(2008A020100020)

摘　　要：根据猪繁殖与呼吸综合征病毒BJ-4株及变异毒株(HB-1、SY0608、HN-HW、HuN4等)的核苷酸序列,利用Primer软件设计合成针对PRRSV Nsp9基因保守区域的特异性引物。用RT-PCR方法扩增出猪繁殖与呼吸综合征病毒JL/07/SW株Nsp9基因片段,克隆到pMD18-T载体并测序,基因克隆至表达载体pET-28a(+),得到重组表达载体pET-28a-Nsp9,转化BL21(DE3)细胞。经IPTG诱导,Nsp9蛋白以包涵体形式表达,SDS-PAGE分析表明重组蛋白的分子量约为34.8 kD,表达量占菌体蛋白的40.32%。Western-Blotting结果表明重组蛋白可被PRRSV阳性血清所识别。表达的重组蛋白为进一步制备抗Nsp9单抗及建立区分野毒和灭活毒感染的ELISA方法提供物质基础。According to the porcine reproductive and respiratory syndrome virus BJ -4 plant and the mutation strains （HB - 1, SY0608 ,HN - HW,HuN4） , a pair of specific primers of Nsp9 gene was designed. Amplified the specific fragment of JL/07/SW by RT - PCR, and sequenced after being cloned directly into the pMD18 - T. The obtained recombinant expression vector pET - 28a - Nsp9 was transformed into Escberichia coli BL21 （DE3） . Induced by IPTG, the recombinant protein was highly expressed as the form of inclusion body. Analyzed by SDS - PAGE, it showed that the molecular weight of recombinant protein was 34.8kD and could amount to 40.32% of the total bacterial protein. Western - Blotting analysis indicated that the purified recombinant protein Nsp9 could be recognized by the positive serum from the pig infected with PRRSV.

关 键 词：猪繁殖与呼吸综合征病毒 Nsp9基因 克隆序列分析 原核表达 

分 类 号：S858.286.3[农业科学—临床兽医学]