卡介苗(BCG)D2株分泌型抗原85A基因的克隆与序列分析(英文)

Cloning and sequence analysis of the gene encoding the secreted form of Ag85A in strain D2 of Mycobacterium bovis BCG

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 。

In the present study, the gene encoding the secreted form of Ag85A in strain D2 of Mycobacterium bovis BCG was isolated,and its DNA sequence was determined in order to provide the basis for the development of novel vaccine against tuberculosis. This gene was isolated from the genomic DNA of strain D2 of M.bovis BCG by means of PCR technique, and the amplified products were inserted into pUCm T vectors. The recombinant clones were identified by single colony PCR screening, double digestion with restriction endonuclease BamHⅠ and SacⅠ, and DNA sequencing. It was found that the gene encoding the secreted form of Ag85A in strain D2 of M. bovis BCG isolated and sequenced in the present study consisted of 1041 bp, which was consistent in DNA sequence with the same gene from strain 1173P2 as detected by Luk De Wit et al.This result suggests that the Ag85A gene is highly conserved in M. bovis. The successful cloning and sequence analysis of the gene encoding the secreted form of Ag85A in M. bovis BCG would be helpful for the further studies on this gene, and for the development of new vaccine against tuberculosis.