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目的 分离BCGD2株分泌型抗原 85A基因并测定DNA序列 ,为研制抗结核病新型疫苗奠定基础。方法 PCR法从BCGD2株基因组DNA中分离Ag85A基因 ,PCR产物连接入 pUCm -T载体 ,重组克隆用单菌落PCR法、BamHⅠ和SacⅠ双酶切以及DNA测序进行鉴定。结果 分泌型Ag85A基因经过分离和测序后发现 ,它有 10 4 1bp组成 ,与LukDeWit等人测定的来自于BCG1173P2株的同一基因的DNA序列是一致的 ,表明Ag85A基因在分枝杆菌中是高度保守的。 结论 BCGD2株分泌型抗原 85A基因的成功克隆与序列分析将会促进对Ag85A基因深入的研究 ,以及为研制抗结核病候选疫苗奠定基础
Objective To isolate the 85A gene of BCGD2 secreting antigen and determine its DNA sequence, which laid the foundation for the development of a new anti-TB vaccine. Methods The Ag85A gene was isolated from the genomic DNA of BCGD2 by PCR. The PCR product was ligated into pUCm-T vector. The recombinant clones were identified by single colony PCR, restriction endonuclease BamHⅠand SacⅠ and DNA sequencing. Results The secreted Ag85A gene was isolated and sequenced and found to have a 104bp composition, consistent with the DNA sequence of the same gene from BCG1173P2 strain as determined by LukDeWit et al., Indicating that the Ag85A gene is highly conserved in mycobacteria of. Conclusion The successful cloning and sequence analysis of the 85A gene of BCGD2 secreting antigen will promote the further study of Ag85A gene and lay a foundation for the development of candidate vaccine against tuberculosis