摘要
为获得高抗病毒活性的猪α亚型干扰素(porcine interferon α,poIFN-α),将天然的poIFN-α17进行突变和合成,并将突变后的猪poIFN-α17基因命名为poIFN-α17m,合成poIFN-α17和poIFN-α17m 基因后将其克隆入pVB220大肠杆菌表达载体,将该载体转化DH5α感受态细胞,进行温度诱导表达后,收集菌体进行SDS-PAGE.将表达的蛋白采用Ni琼脂糖凝胶纯化后,采用 Western blot检测重组poIFN-α17和poIFN-α17m的反应原性,在PK-15细胞上检测其对VSV和PRV的抗病毒活性,检测重组poIFN-α17和poIFN-α17m蛋白对下游干扰素刺激基因(interferon stimulating genes,ISGs)Mx1、OAS1、ISG15 的诱导激活作用.结果显示,成功构建了 pVB220-IFN-α17 和pVB220-IFN-α17m表达载体,实现了 poIFN-α17和poIFN-α17m在大肠杆菌中的表达.该蛋白具有良好的反应原性,且重组poIFN-α17m在PK-15细胞上对水泡性口炎病毒(vesicular stomatitis virus,VSV)和猪伪狂犬病病毒(pseudorabies virus,PRV)的抗病毒活性明显高于天然的poIFN-α17,重组poIFN-α17m能有效激活ISGs的表达.
Abstract
The aim of this study is to obtain the high antiviral activity of porcine interferon α(poIFN-α).Porcine IFN-α17(poIFN-α17)gene was mutated and synthesized,and mutated poIFN-α17 was named as poIFN-α17m,then the gene was further digested and cloned into the pVB220 plasmid to construct E.coli expression vectors pVB220-IFN-α17 and pVB220-IFN-α17m.The ex-pression vectors were transformed into E.coli DH5α and recombinant poIFN-α17 and poIFN-α17m were expressed with temperature change.The recombinant protein was purified using affinity chro-matography and identified by Western blot.The antiviral activity of recombinant poIFN-α17 and poIFN-α17m was tested in PK-15 cells against PRV and VSV.The expression of interferon stimu-lating genes(ISGs)induced by recombinant poIFN-α17 and poIFN-α17m was further detected and compared.The results demonstrated that the pVB220-IFN-α17 and pVB220-IFN-α17m expression vector were successfully constructed,and inclusion expression of poIFN-α17 and poIFN-α17m protein in E.coli DH5α was obtained after the induction with temperature change,and the recom-binant poIFN-α17 and poIFN-α17m could react with porcine interferon polyclonal antibody.Fur-thermore,the result demonstrated that the antiviral activity of poIFN-α17m against PRV and VSV was superior to poIFN-α17.Moreover,recombinant poIFN-α17m could effectively induce the ex-pression of ISGs.
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