牛γ-干扰素基因的克隆及在大肠杆菌中的表达

Cloning and Expression of Bovine IFN-γ Gene in E.coli

根据GenBank发表的牛γ-干扰素基因设计1对特异性引物,从牛脾脏淋巴细胞提取基因组总RNA,采用反转录?聚合酶链式反应(reverse transcriptase-PCR,RT-PCR)技术扩增出IFN-γ基因,与pMD18-T载体连接后进行酶切、PCR鉴定及序列测定和分析,并将其连接到pQE-30原核表达载体中,命名为pQE30-IFN-γ,转化到大肠杆菌x-LI Blue感受态细胞,IPTG(1.0 mmol/L)37℃诱导表达。结果显示,对重组质粒进行PCR鉴定,扩增到510 bp片段,重组蛋白大小为22 ku,与预期大小相符;Western blotting分析结果表明,重组蛋白能与抗His-tag单克隆抗体反应,为下一步研究干扰素的功能奠定了基础。

A pair of special primers was designed according to the sequence of IFN-γ in GenBank.Total RNA was extracted from cow spleen lymphocyte,IFN-γ gene was amplified by the reverses transcription polymerase chain reaction.After linked with pMD18-T vector,it was digested by enzyme,PCR and gene sequencing were carried.And this fragment was cloned into the multiple cloning sites of pQE-30 vector.The recombinant plasmid was designated pQE30-IFN-γ and was transformed into E.coli x-LI Blue competent cells.Expression of the recombinant protein was induced by IPTG under 37 ℃ condition.The length of PCR product of recombinant plasmid was 510 bp,the size of recombinant protein was 22 ku.The results of Western blotting showed that it could be recognized by anti-His tagged mouse monoclonal antibody.These results were benefit to research for function of IFN-γ.