犬干扰素α2的原核表达及抗病毒活性分析

PROKARYOTIC EXPRESSION AND ANTIVIRAL ANALYSIS OF CANINE INTERFERON α 2

本研究将人工合成的犬干扰素α2成熟区序列插入原核表达载体p ET-28a(+)中,构建重组表达质粒p ET-28a-Ca IFN-α2;然后将该质粒转化至大肠杆菌Rosetta感受态中进行IPTG诱导表达,经SDS-PAGE和Western blot分析鉴定,分子量约为23 k Da的目的蛋白表达,表达的重组蛋白主要以包涵体的形式存在,表达量约占菌体总蛋白的52.5%。包涵体蛋白经变性、复性和纯化处理后,获得的重组Ca IFN-α2纯度为92%;用MDCK/VSV微量细胞病变抑制法检测重组蛋白的抗病毒活性为3.16×106/m L。本研究结果为进一步研制新型犬用干扰素制品奠定了物质基础。

In this study,the mature encoding sequence of canine interferonα2 subtype was synthesized and inserted into the prokaryotic expression vector pET28a(+)to construct the recombinant plasmid pET-28a-CaIFN-α2.The resulting pET-28a-CaIFN-α2 was transformed into E.coli Rosetta competent cells for protein expression with induction of IPTG.The recombinant CaIFN-α2 was determined to be 23 kDa of molecular mass in SDS-PAGE and Western blot.In addition,the recombinant CaIFN-α2 mainly existed in a form of inclusion body and accounted for 52.5%of the total bacterial proteins.The recombinant CaIFN-α2 with the purity of 92%was obtained through a series of denaturation,renaturation and purification treatments.The antiviral activity of the recombinant CaIFN-α2 was determined by CPE reduction of vesicular stomatitis virus infection in MDCK cells.The results showed that the antiviral activity of the recombinant CaIFN-α2 was 3.16×106 U/mL,which laid the foundation for the development of novel canine interferon agent.