摘要
通过overlap PCR扩增了含有bp26基因上游片段、bp26基因下游片段及牛分枝杆菌esat6-cfp10基因的目的片段Δbp26-ec,利用含有sacB基因的自杀质粒pRE112为载体,通过等位基因交换的方法,将构建好的自杀质粒电转入已经构建好的缺失bp26基因的流产布氏杆菌减毒活疫苗S19突变株中,构建了具有非抗性基因标记的突变株S19-Δbp26-ec,并对构建好的新型基因突变株进行了生物学特性及毒力的鉴定及分析。PCR及核苷酸序列测序结果显示,S19-Δbp26-ec构建成功。与亲本株相比,疫苗株S19-Δbp26-ec的生长特性没有显著性差异;体外连续传至20代后,菌落PCR和核苷酸测序结果显示,S19-Δbp26-ec株具有良好的遗传稳定性。被感染小鼠脾重和脾中菌落数表明,S19-Δbp26-ec的毒力最弱,S19和S19-Δbp26次之。上述结果表明,本试验获得了毒力减弱的基因突变株S19-Δbp26-ec,为牛布氏杆菌病-结核病二联疫苗的研制奠定了基础。
The upstream and downstream fragments of bp26 gene were amplified fromBrucella abortus S19 strain,respectively,and esat6-cfp10 was amplified from Mycobacterium bovis.Then,a DNA fragment was amplified from these three fragments through fusion PCR and subcloned into the suicide plasmid pRE112.The modified plasmid was transferred into the B.abortus S19-Δbp26mutant strains.Under the selection of chloramphenicol and sucrose,a novel mutant strain S19-Δbp26-ec was constructed through homologous recombination.The results of PCR amplification and sequencing showed that the mutant was constructed successfully.Compared to the parental strain S19 and S19-Δbp26,S19-Δbp26-ec mutant strains had similar growing characteristics and the sequence of S19-Δbp26-ec was verified after 20 passages,which indicated that it has stable genetic generation.The animal experiments based on the weight and counts of spleen of BALB/c showed that S19-Δbp26-ec exhibited the lowest virulence compared with S19 and S19-Δbp26strains.In conclusion,we have constructed a novel mutant strain S19-Δbp26-ec with less virulence,and it might be developed to a new candidate vaccine.At the same time,the above-mentioned result might offer the basic theory for the development of vaccines against both bovine brucellosis and tuberculosis.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2015年第4期395-401,共7页
Chinese Veterinary Science
基金
广东省教育部产学研结合项目(2011B090400259)
公益性行业(农业)科研专项经费资助项目(201203056)
