PRRS病毒核衣壳蛋白在大肠杆菌中的高效表达

Highly Efficient Expression of Nucleocapsid Protein of PRRS Virus in E.coli

用表达非融合蛋白的原核表达载体 p BV2 2 0 ,通过 PCR技术的引物设计对猪繁殖与呼吸综合征( PRRS)病毒 BJ-4株核衣壳蛋白 ( N)基因起始密码子上游进行了改造和修饰 ,成功地构建了含有 PRRS病毒 N基因的原核重组表达载体 p BV-N。 p BV-N转化大肠杆菌 DH5α,温度诱导后菌体裂解物经 SDS-PAGE分析发现 ,在约 1 5 0 0 0处出现 1条诱导前后的 p BV2 2 0载体对照和诱导前的 p BV-N中均缺少的特异性的蛋白条带 ,分子量大小与 PRRS病毒 N蛋白相符 ,并随着诱导时间的延长而变化 ,在诱导后 4 h达到高峰。薄层扫描结果显示 ,重组 N蛋白表达量最多可占菌体总蛋白的 2 7.4 %。 Western-blotting检测 ,此 1 5 0 0 0的蛋白带可为 PRRS病毒 N蛋白的单克隆抗体所识别 ,表明该重组

Nucleocaspid protein ( N) gene c DNA of PRRS virus was modified by PCR technique,the restriction enzyme Eco R site was added to the upstream of the initial code ATG of N gene.The modified c DNA was cloned into the downstream of PLPRpromotor and S-D sequence of the E.coliexpression vector p BV2 2 0 .The recombinant PRRS virus nucleocaspid protein expression vector( p BV-N) was successfully constructed.After p BV-N was transformed into E.colistrain DH5 α and inducted at4 2℃ ,an expected1 5 0 0 0 protein was found in the induced total cell lysate which was lack in the control E.coli by SDS-PAGE analysis.The recombinant PRRS virus N protein expression was varied with the induction time and reached2 7.4 % of the total mass of bacterial proteins4 hours afterinduction.The1 5 0 0 0 recom- binant protein expressed in E.coliwas recognized by Mc Ab for nucleocaspid protein of PRRS virus and showed a good antigencity when analysis by Western-blotting.