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 pBI12 1-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fb pA基因亚克隆入植物表达载体 pBI 12 1,得到重组载体pBI 12 1-fbpA ,并转化根癌农杆菌株EHA10 5 ,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 10 4 1bp组成 ,包括 1个 10 14bp的开放阅读框。DNA序列上游由编码一段信号肽的 12 9bp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 pBI12 1-fbpA成功转入根癌农杆菌株EHA10 5。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。
Objective To clone and sequence the 32-kDa secretary prot ein gene (fbpA gene) and construct a recombinant plant expression plasmid of pBI 121-fbpA researching for edible vaccine by transgenic plants against tuberculosi s.Methods PCR amplification of the fbpA gene from BCG D2 ge nomic DNA was performed.Characterization of the fbpA gene cloned in pUCm-T vecto rs was carried out by PCR screening individual bacterial colonies,single and dou ble digestion with restriction endonuclease BamHⅠ,SacⅠ and HindⅢ as well as D NA sequence analysis.After identification,the excised fbpA insert was subcloned in vector pBI121 to obtain recombinant plant expression plasmid of pBI121-fbpA.T hen,agrobacterium tumefaciens strain.EHA105 was transformed and seeded o n YEP plate.After that,colonies were selected by PCR. Results The 1041-bp nucleotide sequence derived from reco mbinant 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 pep tide 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 gen g was identical to that from M.bovis BCG strain 1173P2.Moreover,the recombinant plant expression plasmid of pBI121-fbpA was constructed and transformed in Agrobacterium tumefaciens EHA105.Conclusion The fbpA gene encoding secreated form of 32-kD a 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.
出处
《中国公共卫生》
CAS
CSCD
北大核心
2004年第10期1193-1195,共3页
Chinese Journal of Public Health
