摘要
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.
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 DH5ot 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 BI21 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 BI21 of B. coli as an inclusion body at 18 ℃ and 28 ℃ ,but not at 37 ℃ ,when pYUB28b works as expression vector.
出处
《云南大学学报（自然科学版）》
CAS
CSCD
北大核心
2014年第4期600-605,共6页
Journal of Yunnan University(Natural Sciences Edition)
基金
国家自然科学基金(31160425)
新西兰健康研究基金(grant 06/441)~~
