重组鸡α-干扰素的表达与抗病毒活性分析

Expression and antiviral activity analysis of recombinant chickenα-interferon

为了表达具有抗病毒活性的重组鸡α-干扰素(IFN-α),试验根据大肠杆菌密码子的偏好性,对鸡IFN-α的成熟肽基因序列进行优化,合成后连接至原核表达载体pET30a-ELP,经体外诱导后表达重组蛋白ELP-ChIFN-α,借助重复可逆相变循环(ITC)法进行纯化;纯化蛋白经过变性、复性处理后进行安全性评价,采用微量细胞病变抑制法在犬肾细胞(MDCK)/水疱性口炎病毒(VSV)系统中进行蛋白抗病毒活性的检测。结果表明:合成的重组鸡IFN-α成熟肽基因能在原核表达系统中成功表达;不同浓度的重组蛋白ELP-ChIFN-α对细胞的生长抑制率为0;重组蛋白ELP-ChIFN-α在MDCK/VSV系统中具有抗病毒活性,活性为1.2×10^(6)IU/mg。说明原核表达的重组鸡IFN-α有较好的抗病毒活性,可作为鸡的抗病毒生物药物进一步开发研究。

In order to express the recombinant chickenα-interferon(IFN-α)with antiviral activity,according to the codon preference of Escherichia coli,the mature peptide gene sequence of chicken IFN-αwas optimized.After synthesis,it was connected to the prokaryotic expression vector pET30 a-ELP and induced in vitro to express the recombinant protein ELP-ChIFN-α.Purification was carried out by means of repeated reversible phase transition cycles(ITC)method;the purified protein was denatured and renatured for safety evaluation.The antiviral activity of the protein was detected in the canine kidney cell(MDCK)/Vesicular stomatitis virus(VSV)system by the microcytopathic inhibition method.The results showed that the synthesized recombinant chicken IFN-αmature peptide gene was successfully expressed in the prokaryotic expression system,and the cell growth inhibition rate of recombinant protein ELP-ChIFN-αat different concentrations was 0.The recombinant protein ELP-ChIFN-αhad antiviral activity in the MDCK/VSV system,with an activity of 1.2×10^(6)IU/mg.The results suggested that the recombinant chicken IFN-αexpressed in prokaryotic had good antiviral activity,and could be used as an antiviral biological drug in chicken for further development and research.