禽呼肠孤病毒σNS非结构蛋白基因的克隆和表达

Cloning and expression of nonstructural protein gene σNS of avian reovirus

采用RT-PCR技术扩增了禽呼肠孤病毒(ARV)S1133株与广西分离株R2 σNS非结构蛋白基因,将σNS基因克隆到质粒表达载体pGEX-4T-1上,获得的重组质粒经PCR、酶切鉴定以及测序分析,σNS基因的插入位置、大小和阅读框均正确,将阳性克隆命名为pGEX-S1133-σNS和pGEX-R2-σNS.构建好的重组质粒经37 ℃ 1 mmol/L IPTG诱导、SDS-PAGE分析超声裂解后的上清和沉淀,显示目的蛋白以包涵体方式表达,其蛋白质分子质量约为66.2 ku,约占菌体总量的31.5%～34.8%.Western-blotting分析表明,目的蛋白能够与ARV阳性血清发生特异性反应,表明该重组蛋白具有较好的抗原性.

The σNS gene of ARV S1133 and R2 isolates from Guangxi were amplified by RT-PCR, the amplicon was inserted into a bacterial plasmid pGEX-4T 1 vector, and a recombinant plasmid containing σNS gene was identified by PCR amplification and digestion with restriction endonucleases, and then sequenced. Result showed that the insert fragment was the σNS gene of ARV. The recombinant plasmid was induced by 1 mmol/L of IPTG. SDS-PAGE analysis showed that the expressed fusion protein with a molecular weight of 66. 2 ku was soluble in the form of inclusion bodies. The fusion protein represented 31.5%--34.8% of the total bacterial protein. Western-blotting analysis with ARV antibodies against the fusion protein showed that the recombinant protein had good antigenicity.