肠致病性大肠杆菌eae基因的克隆及其原核表达

发布时间：2018-04-14 18:25

本文选题：肠致病性大肠埃希菌 + eae基因　；参考：《长春理工大学》2010年硕士论文

【摘要】： 根据肠致病性大肠埃希菌(EPEC)eae基因序列,设计引物,并通过PCR方法从肠致病性大肠埃希菌(EPEC)总DNA中扩增eae基因的高度保守序列,获得约1800bp的eae基因片段。通过TA克隆技术,将PCR产物克隆至pMD18-T Vector中,转化至受体菌DH5α中,通过质粒提取、限制性内切酶酶切、PCR、序列测定等方法鉴定构建出重组阳性克隆质粒pMD18T-eae。经DNASTAR软件分析核苷酸和氨基酸序列,结果显示与GenBank上EPEC eae基因比较,核苷酸同源性均在99.8%以上,氨基酸同源性均在99.5%以上。应用基因体外重组技术,将回收纯化的EPEC eae基因融合入大肠杆菌-乳酸菌穿梭表达载体pW425t中,通过质粒提取、限制性内切酶酶切、PCR等方法鉴定表明构建出重组阳性克隆质粒pW425t-eae,并在DH5α内表达此eae蛋白,通过SDS-PAGE检测,pW425t-eae在重组大肠杆菌DH5α内表达eae蛋白。
[Abstract]:According to the sequence of EPEC-eae gene of Escherichia coli, primer was designed, and the highly conserved sequence of eae gene was amplified from the total DNA of Escherichia coli by PCR method, and the eae gene fragment of about 1800bp was obtained.The PCR product was cloned into pMD18-T Vector and transformed into DH5 伪. The recombinant plasmid pMD18T-eaee was identified by plasmid extraction, restriction endonuclease digestion and sequencing.The nucleotide and amino acid sequences were analyzed by DNASTAR software. The results showed that the nucleotide homology was over 99.8% and the amino acid homology was over 99.5% compared with the EPEC eae gene on GenBank.The purified EPEC eae gene was fused into Escherichia coli-lactic acid bacteria shuttle expression vector pW425t by in vitro recombination technique.The recombinant plasmid pW425t-eaewas constructed and expressed in DH5 伪 by restriction endonuclease digestion. The recombinant plasmid pW425t-eae was detected by SDS-PAGE to express eae protein in DH5 伪 of recombinant Escherichia coli.
【学位授予单位】：长春理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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