SARS-CoV GDH株N蛋白全长基因克隆及其可溶性表达

目的构建SARS冠状病毒N蛋白的原核表达质粒,诱导重组蛋白表达并纯化,鉴定其抗原性。方法以我国SARS冠状病毒GDH株总RNA为模板,采用RT-PCR技术扩增N蛋白的全长基因,TA克隆后测序。构建pET-23d的N基因表达载体,用IPTG诱导目的蛋白表达,利用硫酸铵沉淀、分子筛层析及离子交换层析纯化重组蛋白,免疫印迹鉴定重组蛋白。结果RT-PCR扩增出1269bpSARS冠状病毒N蛋白的基因片段,其序列分析结果与SARS-CoVGD01、BJ01株的同源性为99.92%;该基因在大肠杆菌表达系统中高效表达,占可溶性蛋白的33.57%,表达产物为非融合的可溶性蛋白,Westernblot结果显示重组N蛋白具有良好的抗原性;纯化后重组N蛋白纯度为92.9%。结论成功构建了SARS冠状病毒N蛋白的重组表达质粒,并在大肠杆菌中以非融合蛋白的形式得到高效的可溶性表达,为SRAS的诊断和疫苗的研制奠定了基础。

Cloning and expression of the full length gene encoding the N-protein of SARS-CoV

To obtain the recombinant N-protein of SARS-CoV, the full length cDNA of N-protein was amplified by RT-PCR from the supernatants of cell cultures of SARS-CoV GHD strain, the aimed gene fragment was cloned into vectors pMD18-T, the positive recombinants were sequenced and subcloned into vector pET-23d. The recombinant plasmid was then transformed into (E.coli) BL21 cells for expression, and the expressed products were purified by deposit of saturated (NH4_)_2SO_4, molecular sieve chromatography and ion exchange chromatography. These products could be detected by Western blotting using the convalescence sera of patients with SARS as the primary antibody. It was found that a 1269 bp gene fragment of N-protein of SARS-CoV was amplified by RT-PCR, and the prokaryotic expression plasmid pET-23d/Nwas constructed by using the sucloning technique from T/A clone pMD18/N. The sequencing result of the cloned N-gene showed that its homology with SARS-CoV GD01, BJ01 strain was 99.92%. The N-protein was highly expressed as a non-fusion soluble protein, accounting to 33.57% of the total soluble protein. As demonstrated by Western blotting, this protein showed excellent antigenicity. The purity of the recombinant N-protein was proved to be (92.9%.)It is concluded that the N-gene of SARS-CoV GDH strain has been cloned and expressed successfully in the present investigation.