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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 the 32-kDa secretory protein gene (fbpA gene) from Bacillus Calmette Guerin (BCG) D2 strain and construct the recombinant plant expression vector pBI12 1-fbpA, which lays the foundation for the development of oral vaccine for transgenic plants. Methods The fbpA gene was isolated from the genomic DNA of BCGD2 by PCR, and the recombinant cloning vector pUCm-T -fbpA was constructed. After single colony PCR, BamHⅠ, SacⅠand HindⅢ single / double digestion and DNA sequence analysis, the fb pA The gene was subcloned into the plant expression vector pBI12 1 to obtain the recombinant vector pBI12 1-fbpA, which was then transformed into the Agrobacterium tumefaciens strain EHA10 5 and identified by PCR. Results The recombinant vector pUCm-T -fbpA confirmed that the fbpA gene consisted of 104bp, including a 1014bp open reading frame. Upstream of the DNA sequence consists of a 12 9 bp encoding a signal peptide and is responsible for the secretion of the 32-kDa protein. The corresponding sequence encoding the mature protein consists of 885 bases. The fbpA gene was identical to the same gene from BCG1173P2 strain. In addition, the constructed recombinant plant expression vector pBI12 1-fbpA was successfully transformed into Agrobacterium tumefaciens strain EHA10 5. Conclusion The fbpA gene encoding 32-kDa secreted protein of BCGD2 strain was successfully isolated. DNA sequence analysis confirmed that fbpA gene was highly conserved in mycobacteria, which lays the foundation for further analysis of fbpA gene and development of anti-TB transgenic vaccine.