【摘要】 目的构建人Toll样受体1(TLR1)胞外段原核表达质粒。方法采用逆转录-聚合酶链式反应(RT-PCR)从人外周血单个核细胞(PBMC)mRNA中扩增人TLR1胞外区cDNA;将扩增的目的基因与原核表达载体pET30a(+)质粒经NcoⅠ和NotⅠ双酶切后连接,转化E.coli BL21(DE3),筛选转化子,菌落PCR及测序验证克隆序列的正确性。结果测序结果表明,成功构建pET30a(+)-TLR1胞外区基因重组质粒,并转化感受态E.coli BL21(DE3)。结论 TLR1胞外段在大肠杆菌BL21中正确克隆,为TLR1信号通路的进一步研究打下了基础。更多还原

【Abstract】 Objective To construct the prokaryotic expression plasmid of extracellular domain of human Toll-like receptor 1(eTLR1).Methods Human TLR1 extracellular cDNA codon domain was amplified by RT-PCR and digested by NcoⅠand NotⅠ,and then cloned into pET30a(+) plasmid.The constructed recombinant plasmid pET30a(+)-eTLR1 was transfered into E.coli BL21(DE3)and the positive clones were determined by PCR.Results The eTLR1 was proved to be correct by DNA sequencing.Conclusion The gene of human TLR1 extracellular cDNA codon domain is successfully cloned into prokaryotic expression plasmid pET30a(+) of E.coli BL21(DE3),which will provide the basis for the advanced study of TLR1 signaling pathway.更多还原