摘要
目的克隆鸡MxA基因,构建其原核表达质粒,并诱导其在大肠杆菌中表达融合蛋白。方法采用RT-PCR方法从鸡成纤维细胞(CEF)中扩增MxA,将其克隆至克隆载体pMD18-T中,筛选阳性克隆后回收目的片段,将其克隆入原核表达质粒pGEX-6p-1,构建其重组表达质粒pGEX-MxA,以IPTG诱导表达,经SDS-PAGE、鸡胚新城疫病毒干扰试验和VSV-CEF微量细胞抑制试验进行分析、鉴定。结果经RT-PCR扩增获得的MxA序列与GeneBank报道的序列一致,SDS-PAGE和干扰试验证实重组质粒可以表达出相应分子量为45000的蛋白,与GST-MxA融合蛋白分子量一致。结论成功完成了鸡MxA基因的克隆及其原核表达质粒的构建,在大肠杆菌DH5α中经IPTG诱导表达了融合蛋白GST-MxA,为进一步探讨MxA的生物学活性,探索抗病毒药物的研发奠定了基础。
Objective：To clone chickens MxA gene, construct its recombinant expression plasmid and induce the expression of fusion protein using a prokaryotic expression system, Methods：The MxA gene fragment was amplified by RT-PCR from CEF cells and subcloned into the pMDI 8-T vector, filtrated the positive clone and reclaimed the MxA. Subcloned the MxA into the prokaryotic expres- sion plasmid pGEX-6p-1. After recombinant plasmid was induced by IPTG, the expressed proteins were analyzed by SDS-PAGE, the NDV intervence experiment and VSV-CEF restrain experiment. Results：The sequence of MxA gene amplified by RT-PCR was the same as the sequence in gene map of Genebank ; SDS-PAGE, the NDV intervence experiment and VSV-CEF restrain experiment showed that a protein was expressed, the molecular weight of this protein was 45 000, which was the same as the fusion protein GST-MxA. Conclusion：The MxA is cloned and its recombinant expression plasmid is constructed successfully. The fusion protein GST-MxA is successfully expressed in the prokaryotic expression system E. coli DH5α induced by IPTG. This research lay a foundation for further studying on ints antiviral effects and exploring new way of antiviral medication.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2006年第11期1018-1020,1024,共4页
Chinese Journal of Immunology
基金
山东农业大学博士后启动基金资助项目
关键词
鸡MxA基因
基因克隆
原核表达
生物活性
Chickens MxA gene
Gene clone
Protein expression
Biological activity
