卡介苗D2株分泌蛋白基因植物表达载体的构建
Isolation and plant expression plasmid construction of 32-kDa secretary protein gene of mycobacterium bovis BCG strain D2
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摘要:目的 克隆卡介苗(BCG)D2株32-kDa分泌蛋白基因(fbpA基因), 构建重组植物表达载体pBI121-fbpA, 为研制转基因植物口服疫苗奠定基础。方法 采用PCR法从BCGD2株基因组DNA中分离fbpA基因, 构建重组克隆载体pUCm-T-fbpA, 经过单菌落PCR法、BamHⅠ、SacⅠ和HindⅢ单/双酶切、DNA序列分析鉴定后, 将fbpA基因亚克隆入植物表达载体pBI121, 得到重组载体pBI121-fbpA, 并转化根癌农杆菌株EHA105, 用PCR法对其进行鉴定。结果 重组载体pUCm-T-fbpA测序后证实fbpA基因由1041bp组成, 包括1个1014bp的开放阅读框。DNA序列上游由编码一段信号肽的129bp组成, 并对32-kDa蛋白的分泌起决定作用。相应的编码成熟蛋白的序列由885个碱基组成。fbpA基因与来源于BCG1173P2株的同一基因序列完全一致。此外, 构建的重组植物表达载体pBI121-fbpA成功转入根癌农杆菌株EHA105。结论 编码BCGD2株32-kDa分泌蛋白的fbpA基因分离成功, DNA序列分析证实fbpA基因在分枝杆菌中高度保守, 为进一步深入分析fbpA基因以及研制抗结核病转基因植物疫苗奠定了基础。Abstract: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.
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