ltB-TpN47融合基因原核表达系统的构建

The construction of prokaryotic expression product of ltB-TpN47 fusion gene

目的分别从梅毒螺旋体(Treponema pallidum,Tp)临床菌株和大肠杆菌DNA中扩增ltB和TpN47基因,构建ltB-TpN47融合基因及其原核表达系统。方法采用PCR分别从上述菌株DNA中扩增TpN47基因和ltB基因片段、构建ltB-TpN47融合基因,T-A克隆后测定核苷酸序列。采用质粒pET32和宿主菌E.coliBL21DE3构建ltB-TpN47融合基因原核表达系统,并用不同浓度的IPTG诱导表达。结果所构建的ltB-TpN47融合基因核苷酸序列与从GeneBank中查询并处理得出的数据基本一致。所构建的表达系统pET32-ltB-TpN47的目的重组蛋白(rltB-TpN47)表达量高达细菌总蛋白的1/3左右。结论本文成功地构建了ltB-TpN47融合基因高效原核表达系统。

Objective To amplify ltB gene and TpN47 gene from DNAs of a clinical strain Treponema pallidum and Eseheriehia eoli strain, respectively, and construct ltB-TpN47 fusion gene and its prokaryotie expression system. Methods TpN47 and ltB genes from DNAs of the bacteria mentioned above were amplified and ltB-TpN47 fusion gene was construeted by PCR. Amplification fragments of the target genes were sequenced after T-A cloning. Plasmid pET32 and E.eoli strain BL21 DE3 were applied to construct expression system of the fusion gene. This prokaryotie expression system was expressed under induce- ment with different dosages of IPTG. Results In comparison with the corresponding se- quences registered in GenBank, the nueleotide and putative amino acid sequences of the constructed ltB-TpN47 gene were nearly same, respectively. Expression output of the target recombinant protein （rltB-TpN47） by the constructed expression system pET32-ltB-TpN47- BL21 DE3 was as high as approximate 1/3 of the total bacterial proteins. Conclusion A prokaryotic expression systems with high efficiency of ltB-TpN47 fusion gene is successfully established in this study.