重组猪IFN-α原核表达及其活性分析

为了表达猪重组α干扰素(rIFN-α)并对它的生物学特性进行研究,本研究采用RT-PCR从猪脾淋巴细胞中扩增出猪IFN-α基因,以pPROExHTa为载体构建了表达重组质粒polFN-α,转化宿主菌BL21,经IPTG诱导猪rIFN-α获得了表达,其分子量约22 ku.该重组蛋白以包涵体形式存在,其表达量占总菌体蛋白7%.粟用Ni-Ni金属螯合亲和层析方法纯化,其纯度达到80%以上,包涵体经复性后,在WISH/VSV系统上,采用细胞病变抑制法测定表明,其稀释度在小于1:103时才有抗病毒活性.本实验表达的猪rIFN-α具有一定的抗病毒活性,为研究具有广谱抗病毒效应的猪干扰素具有重要意义.

Expression of recombinant porcine alpha-interferon in E.coli and the anti-virus activity analysis

Porcine alpha interferon (IFN-α) gene was amplified by using RT-PCR from pig spleen lymphocytes and subcloned into pPROExHTa expression vector, transformed into host bacteria BL21. Recombinant porcine IFN-αwas about 22 ku expressed by IPTG-induction, accounted for up to 7 % of total bacterial protein. Porcine rIFN-α achieved purity to above 80 % by Ni-Ni metal chelate affinity chromatography purification, The rIFN-α possessed a anti-vesicular stomatitis virus activity at 2 900 IU/mg.