摘要
目的 :探讨卡介苗 (BacilleCalmette Guerin ,BCG)分枝杆菌的电穿孔转化方法及其影响因素。方法 :在基因脉冲仪上用电穿孔法对快生长型耻垢分枝杆菌———M .Smegmatismc2 15 5和BCG电转化大肠杆菌 分枝杆菌穿梭质粒ME 6 5。结果 :电穿孔法可以成功地转化两种分枝杆菌 ,筛选出卡那霉素抗性菌株。通过对转化子提取质粒进行酶切鉴定和PCR扩增均证明为阳性重组体。转化效率达 10 4 / μgDNA。重组质粒可以在分枝杆菌中稳定复制。初步结果表明 ,质粒DNA的构象和浓度、初始培养物的浓度和活力、电转化参数包括电压、电阻以及转化前对菌体的冰浴时间等都对转化效率有一定的影响。结论 :BCG重组疫苗研制中分枝杆菌的基因转化问题的解决为研制能表达分枝杆菌及其它感染性疾病或肿瘤抗原的BCG多价重组疫苗提供了实验基础。
Objective: To study the method of electro transformation of Mycobacteria including M.Smegmatis mc 2155 and Bacille Calmette Guerin (BCG), and the factors that affect transformation efficiency. Methods: Electroporation was performed on gene pulser with E.coli Mycobacteria shuttle vector ME 65 accordingly. Results: Electro transformation of two strains of Mycobacteria was successfully accomplished with a high efficiency of 10 4/μg DNA. Transformants were screened with Kanamycin resistance and plasmids extracted were confirmed by restriction endonuclease digestion and PCR amplification of DNA fragment in ME 65. The hybrid plasmids maintained genetically stable in hosts. Primary results showed that variables that affected transformation efficiency were: conformation and concentration of plasmids DNA; age and viability of the Bacteria; electroporation parameters such as voltage, resistance, and incubation time on ice before washing. Conclusion:This study has successfully solved the the problem of BCG transformation in the research of genetic engeneering recombinant BCG vaccines. The introduction and stable expression of foreign DNA in BCG on a plasmid vector have provided a basis for the construction of polyvalent recombinant BCG vaccines that can express not only putative protective mycobacterial antigens, but also antigens for other infectious and malignant diseases. ( J Beijing Med Univ , 2000,32:178 181)
出处
《北京医科大学学报》
CSCD
2000年第2期178-181,共4页
Journal of Peking University(Health Sciences)
基金
国家自然科学基金!(391 70 4 50 )
卫生部科研基金!(94 1 1 4 4 )
关键词
分枝杆菌
卡介苗
电穿孔
基因转化
Mycobacteria
Electro transformation
Transforming genetic
Transforming Bacterial
