| 大肠杆菌质粒复制子在巴氏杆菌中传代的稳定性研究 |
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| 引用本文： | 李三建,荣俊.大肠杆菌质粒复制子在巴氏杆菌中传代的稳定性研究[J].长江大学学报,2007,4(1):48-50,55. |
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| 作者姓名： | 李三建 荣俊 |
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| 作者单位： | 长江大学生命科学学院 湖北荆州434025 |
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| 基金项目： | 湖北省教育厅资助项目(2001Z1405) |
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| 摘 要： | 首先将多杀性巴氏杆菌谷氨酸脱氢酶的基因gdhA启动子及鸡传染性法氏囊病病毒VP2基因插入pMD18-T质粒载体的多克隆位点中构建巴氏杆菌表达质粒pMD18T-PGDH-VP2,用该质粒转化禽多杀性巴氏杆菌G190E40株。为了解重组质粒在巴氏杆菌中传代的稳定性以判断其作为重组活疫苗的可能性,将转化菌种在含氨苄青霉素及血清的平板固体培养基上连续传代至第9代,肉眼观察下的菌落形态和显微镜下见到菌体形态与原始菌种无明显差异;每隔3代提取质粒DNA用限制性内切酶切割检查,酶切图谱没有改变;用扩增PGDH的引物,将提取3、6、9代质粒进行PCR检查,均可见约300bp的PGDH基因片段。结果表明,pMD18-T中的大肠杆菌复制子能在巴氏杆菌中稳定传递至少9代,这为构建能在禽巴氏杆菌和大肠杆菌中复制和表达外源基因的穿梭表达质粒打下了基础。
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| 关 键 词： | 禽多杀性巴氏杆菌 穿梭载体 启动子 遗传稳定性 复制子 |
| 文章编号： | 1673-1409（2007）01-S048-03 |
| 修稿时间： | 2007-02-14 |
Study on Stability of E.coli Plasmid Replicon in the Passing of Pasteurella multocida Generations |
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| LI San-jian,RONG Jun.Study on Stability of E.coli Plasmid Replicon in the Passing of Pasteurella multocida Generations[J].Journal of Yangtze University,2007,4(1):48-50,55. |
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| Authors: | LI San-jian RONG Jun |
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| Abstract: | The glutamate dehydrogenase(gdhA)promoter of Pasteurella multoeida and the VP2 gene of Infectious bursal disease virus(IBDV)were inserted into the multiple cloning sites of pMD18T to create an expressing plasmid pMD18T-PGDH-VP2.The recombinant plasmid was transformed into Avian Pasteurella multocida G190E40.In order to know the stability of the recombinant plasmid in the passing of Pasteurella multocida generations and estimate that if it is possible to use it as recombinant live bacterial vaccines.The strain of Avian Pasteurella multocida/pMD18T-PGDH-VP2 had been subcultured consecutively from the original to the 9th generations on LB plates with AMP and plasma.The morphology of bacterial colony and bacteria did not differ distinctly with the original strain of the bacteria if observed with naked eyes or under microscope.The DNA bands of digestion product of pMD18T-PGDH-VP2 did not change much between the original and the ninth generations in the electrophoresis map.The PGDH segments of 300 bp could be amplified by PCR with the corresponding primers in the plasmids of 3rd,6th,9th generations of recombinant bacteria.The results showed that the replicon of E.coli in pMD18T had a high hereditable stability in the passing of Pasteurella multocida at least for 9 generations,which provided a basis for the construction a shuttle expressing plasmid which could replicate and express foreign gene in E.coli and Pasteurella multocida. |
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| Keywords: | Avian Pasteurella multocida shuttle vector promoter genetic stability replicon |
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