【摘要】 以结核分枝杆菌H37Rv基因组为模板,采用Gateway重组表达系统,以基因长度200~2500 bp为标准,选择目的基因(n=384),以ORF的保守序列设计引物并加上正反向AttB,通过PCR扩增获得基因片段检测入门克隆,得到的克隆有效率为374/384。通过两次同源重组,这些基因片段被克隆到表达载体中,克隆效率为350/384。随后将这些重组表达质粒分别在2个表达菌株BL21和BL21-codonplus-RP中进行诱导表达,表达得率为270/384,并对其中64个进行了纯化,16个是可溶性表达。这种大规模高效率地克隆表达技术适合各种生物的开放阅读框的克隆表达,是一种高通量的克隆表达方法。更多还原

【Abstract】 Mycobacterium tuberculosis H37Rv genoeme chromatosome as template.We selected Mycobacterium tuberculosis H37Rv protein-encoding open reading frames(ORFs),and developed high-throughtput for recombinant protein expression,applying the Gateway cloning/expression technology,we have carried out heterologous protein expression experiments on 384 ORFs,which lengch within 200-2 500 bp.With one expression vector and two Escherichia coli strain,BL21 and BL21codonplus-RP.374 clone and 350 recombinated clone were obtained,and there were 270 experssed,among them 66 were purificated and 16 were soluble.The technology described here is applicable to high-throughput expression of recombinant proteins of Mycobacterium tuberculosis H37Rv.更多还原