Abstract: 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.