结核分枝杆菌H37Ra FadD3基因克隆与表达研究

为探索FadD3在结核分枝杆菌胆固醇分解代谢中的作用,从结核分枝杆菌H37Ra的全基因组中克隆了FadD3基因,并在大肠杆菌BL21中表达.根据NCBI公布的结核分枝杆菌全基因组序列设计一对引物,PCR扩增FadD3基因.PCR扩增产物与克隆载体pGEM3Zf(+)进行拼接,得到重组基因FadD3-pGEM3Zf(+),再转化到大肠杆菌DH5ɑ得到克隆.PCR检测阳性克隆,再与表达载体pYUB28b拼接,得到重组质粒FadD3-pYUB28b.阳性重组质粒亚克隆到大肠杆菌BL21宿主菌进行自体诱导表达.经过表型筛选及鉴定分析,已成功构建了重组表达质粒FadD3-pYUB28b.SDS-PAGE和Western blotting证实,重组基因FadD3-pYUB28b在大肠杆菌BL21中有表达产物.实验结果表明,以pYUB28b为表达载体,FadD3重组基因在大肠杆菌表达体系于温度分别为18,28 ℃有包涵体形式的表达蛋白,在37 ℃无表达产物.

Cloning and expression of (fatty Acyl-CoA synthetase )gene FadD3 in Mycobacterium tuberculosis H37Ra

In order to explore the role of FadD3 in the cholesterol catabolism of Mycobacterium tuberculosis (Mtb),FadD3 was cloned from the whole-genome sequence of Mtb and expressed in Escherichia coli BL21. A pair of primers for FadD3 was designed based on the genome sequence of Mycobacterium tuberculosis from NCBI.FadD3 was amplified by PCR,and spliced with cloning plasmid pGEM3Zf (+),from which the recombination gene FadD3-pGEM3Zf (+) was obtained.The recombinant was transformed into DH5α of E.coli and cloned.The positive recombinant was picked up by PCR,and spliced with the expression vector pYUB28b,from which the recombinant vector FadD3-pYUB28b was obtained and subcloned into BL21 of E.coli.The recombinant gene expression was auto-induced in the new host cell.By restriction enzyme and phenotype analysis,the recombinant FadD3-pYUB28b was constructed successfully and the expressed protein in BL21 of E.coli was confirmed by SDS-PAGE and Western blotting.The results showed that the FadD3 recombinant protein existed in BL21 of B.coli as an inclusion body at 18 ℃ and 28 ℃,but not at 37 ℃,when pYUB28b works as expression vector.