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中国农学通报 ›› 2010, Vol. 26 ›› Issue (7): 6-10.

所属专题: 生物技术

• 畜牧 动物医学 蚕 蜂 • 上一篇    下一篇

布鲁氏菌omp25基因真核表达载体的构建与分析

王勇1,陈创夫2,张辉2,郭乾2,任艳2,蒋松2   

  • 收稿日期:2009-11-18 修回日期:2009-12-01 出版日期:2010-04-05 发布日期:2010-04-05
  • 基金资助:

    国际科技合作项目

Construction and analysis of eukaryotic expression vector for gene omp25 of Brucella

  • Received:2009-11-18 Revised:2009-12-01 Online:2010-04-05 Published:2010-04-05

摘要:

摘 要: 【目的】 构建表达流产布鲁氏菌外膜蛋白基因omp25的酿酒酵母表达菌并进行验证。 【方法】 对流产布鲁氏菌疫苗株S19外膜蛋白基因omp25设计特异引物进行PCR扩增,连接到pGM-T载体上,获得pGM-T-omp25,进行PCR扩增、酶切、测序鉴定。将质粒pGM-T-omp25与pGBKT7载体进行EcoRⅠ/SalⅠ双酶切,回收DNA片段,并进行连接,构建pGBKT7-omp25后,转化酿酒酵母菌Y187,并进行PCR鉴定、自激活实验和毒性检测。 【结果】 克隆了流产布鲁氏菌外膜蛋白基因omp25,PCR、酶切、测序表明成功构建了pGBKT7-omp25真核表达载体,获得的转化子酵母Y187菌株无自激活活性,无毒性。 【结论】 成功构建了表达流产布鲁氏菌外膜蛋白基因omp25的酿酒酵母表达菌,为揭示流产布鲁氏菌感染与母畜流产间的分子机制奠定基础。关键词:流产布鲁氏菌;基因omp25;酿酒酵母Y187;真核表达

关键词: 马拉硫磷, 马拉硫磷, 植株, 水样, 土壤, 残留动态

Abstract:

Abstract: 【Objective】 To construct and identify Saccharomyces cerevisiae expression strain Y187 containing outer membrane protein gene omp25 in B.abortus. 【Method】 The gene omp25 was amplified and obtained from B. abortus vaccine strain S19 by PCR with a pair of specific primers and ligated into pGM-T vector,pGM-T-omp25 is then obtained and identified by PCR、restriction analysis and sequencing. pGM-T-omp25 and vector pGBKT7 were digested by EcoRⅠ/SalⅠ, purify and ligate gene omp25 and pGBKT7. Constructed pGBKT7-omp25 was transformed into Y187 and identified by PCR、transcription activation and toxicity testing. 【Result】 Outer membrane protein gene omp25 was cloned successfully. Through PCR、restriction analysis and sequencing, it was confirmed that bait vector pGBKT7-omp25 was successful constructed and transformed into Y187. Obtained transformant yeast Y187 is inactive and nontoxic,too. 【Conclusion】 Saccharomyces cerevisiae expression strain Y187 containing outer membrane protein gene omp25 in B.abortus was constructed successfully and can facilitate study on the molecular mechanism between infection and female livestocks abortion . Key words: B. abortus;gene omp25;Saccharomyces cerevisiaeY187;eukaryotic expression