文献类型：期刊文献

中文题名：山羊α-干扰素基因克隆、原核表达及其抗病毒活性研究

英文题名：Study on Cloning,Prokaryotic Expression and Antiviral Efficiency of Recombinant Goatα-interferon Gene

作者：徐菁昱[1];刘艳芬[1];陈绍红[2];刘铀[2]

机构：[1]广东海洋大学滨海农业学院,广东湛江524088;[2]广东海洋大学食品科技学院,广东湛江524088

年份：2023

卷号：32

期号：8

起止页码：1151

中文期刊名：西北农业学报

外文期刊名：Acta Agriculturae Boreali-occidentalis Sinica

收录：CSTPCD、、CSCD2023_2024、北大核心、CSCD、北大核心2020

基金：广东省南方现代草牧业产业体系(2019K127)。

语种：中文

中文关键词：雷州山羊;α-干扰素(IFN-α);序列分析;原核表达;抗病毒活性

外文关键词：Leizhou goat;Interferon-α;Sequence analysis;Prokaryotic expression;Antiviral efficiency

中文摘要：为研究雷州山羊α-干扰素基因的遗传变异及重组α-干扰素的抗病毒活性,采用RT-PCR扩增雷州山羊α-干扰素基因,分析其核苷酸序列;采用PCR方法扩增山羊α-干扰素基因成熟肽编码序列,构建原核表达载体pET-mIFN-α,并在大肠杆菌BL21(DE3)诱导表达;采用SDS-PAGE和Western-blot检测重组蛋白rG-mIFN-α;通过细胞病变抑制法测定其抗小反刍兽疫病毒(PPRV)和山羊痘病毒(GPV)活性。结果表明,雷州山羊α-干扰素全基因由570个核苷酸组成,编码189个氨基酸;与GenBank上发表的山羊、林麝、弯角大羚羊、黄牛、瘤牛、绵羊、猪、人和鼠的核苷酸序列同源性分别为99.8%、95.6%、96.8%、94.7%、94.7%、96.8%、81.6%、77.8%和70.0%;氨基酸序列同源性分别为100%、92.6%、94.7%、92.1%、93.2%、93.7%、67.9%、61.6%和55.3%,提示反刍动物α-干扰素基因的核苷酸和氨基酸序列高度保守;rG-IFNα主要以包涵体形式存在,分子质量约32 ku;重组山羊α-干扰素能显著抑制PPRV和GPV感染引起的细胞病变,其抗PPRV和GPV效价分别为1.6×10^(4)U/mg和1.35×10^(4)U/mg。

外文摘要：To investigate the genetic variation ofα-interferon gene of Leizhou goat and the antiviral efficiency of recombinantα-interferon,α-interferon gene of Leizhou goat was amplified by RT-PCR and its nucleotide sequence was analyzed.The nucleotide fragment coding for the mature peptide of goatα-interferon gene was amplified by PCR and inserted into the prokaryotic expression vector pET32a(+)for constructing the prokaryotic expression plasmid pET-mIFN-α.Then,the recombinant plasmid was conducted into the Escherichia coli BL21(DE3)competent cells and the recombinant protein rG-mIFN-αexpression was induced by IPTG and detected by SDS-PAGE and Western-blot.The antiviral activity against peste des petits ruminants virus(PPRV)and goat pox virus(GPV)were determined by cytopathic inhibition assay.The results showed that theα-interferon gene of Leizhou goat consisted of 570 nucleotides coding for 189 amino acids.The homology of the nucleotide sequences of theα-interferon genes in Leizhou goat,Capra hircus,Moschus berezovskii,Oryx dammah,Bos taurus,Bos indicus,Ovis aries,Sus scrofa,Homo sapiens and Mus musculus was 99.8%,95.6%,96.8%,94.7%,94.7%,96.8%,81.6%,77.8%and 70.0%,respectively,and the homology of the deduced amino acid sequences was 100%,92.6%,94.7%,92.1%,93.2%,93.7%,67.9%,61.6%and 55.3%,respectively.The results showed that the nucleotide and amino acid sequences ofα-interferon gene in ruminants were highly conserved.The recombinant protein rG-IFN-αmainly existed in the form of inclusion body in BL21(DE3)cells with molecular mass of 32 ku.The cytopathic effect induced by PPRV and GPV was significantly inhibited by rG-IFN-αtreatment,and the anti-PPRV and anti-GPV titers were 1.6×10^(4)U/mg and 1.35×10^(4)U/mg,respectively.This study provided a new way for the prevention and treatment of goat viral diseases.