Isolation and plant expression plasmid construction of 32-kDa secretary protein gene of mycobacterium bovis BCG strain D2

  Objective   To clone and sequence the 32-kDa secretary protein gene(fbpA gene)and construct a recombinant plant expression plasmid of pBI 121-fbpA researching foredible vaccine by transgenic plants against tuber culosis.

  Methods   PCR amplification of the fbpA gene from BCG D2 genomic DNA was performed.Characterization of the fbpA gene cloned in pUCm-T vectors was carr ied out by PCR screening individual bacterial colonies, single and double digestion with restriction endonuclease BamH Ⅰ, Sac Ⅰ and Hind Ⅲ as well as DNA sequence analysis.After identification, the excised fbpA insert was subcloned in vector pBI121 to obtain recombinant plant expression plasmid of pBI121-fbpA.Then, agrobacterium tumefaciens strain.EHA 105 was transformed and seeded on YEP plate.After that, colonies were selected by PCR.

  Results   The 1041-bp nucleotide sequence derived from recombinant vector pUCm-T-fbpA was represented.The DNA sequence contained a 1014-bp open reading frame.The DNA region upstream of this sequence encoded a signal peptide containing 129 base pairs required for the secretion of the 32-kDa protein.The mature protein gene thus presumably consisted of 885 base pairs.The fbpA geng was identical to that from M.bovis BCG strain 1173P2.Moreover, the recombinant plant expression plasmid of pBI121-fbpA was constructed and tr ansformed in Agrobacterium tumefaciens EHA105.

  Conclusion   The fbpA gene encoding secreated form of 32-kDa protein from M.bovis BCG D2 was achieved.The DNA sequence of the fbpA gene was strongly conserved in mycobacterium.It may be possible to further refinements to the study of the fbpA gene and transgenic plants vaccine against tuberculosis.