共表达ETEC K99、GFP重组大肠杆菌的构建及表达  

Coexpression of the recombinant ETEC K99,GFP in E.coli

作　　者：刘卫贞 窦速林 侯喜林[2] 王珊珊[2] 余丽芸[1,2] 

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,黑龙江大庆163319 [2]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《生物技术》2017年第6期522-526,共5页Biotechnology

基　　金：黑龙江省自然科学基金项目("ETEC K99特异性s Ig A-抗原复合物反向转运的黏膜免疫机制";No.C2017047);黑龙江省垦区科研课题("规模化猪场病毒性腥泻新型黏膜疫苗的应用与示范";No.HNK135-04-06-04);黑龙江八一农垦大学研究生创新科研项目("针对ETEC K99的特异性SIg A免疫排除功能研究";No.YJSCX2015-Y59)

摘　　要：[目的]构建K99、GFP基因重组质粒并在大肠杆菌BL21(DE3)感受细胞中表达。[方法]利用PCR扩增技术扩增K99基因和GFP基因,经连接和转化将目的片段克隆到pLA载体上,利用双酶切技术及PCR技术对重组质粒进行鉴定并测序分析,利用Western Blot技术分析蛋白质表达情况。[结果]测序分析结果表明,基因具有正确的阅读框;双酶切和PCR鉴定得到498 bp和720 bp的特异性条带;荧光检测表明重组菌表达了蛋白;Western Blot结果表明重组菌表达为融合蛋白。[结论]成功构建了大肠重组菌,并表达外源融合蛋白。[Objective]Construction of recombinant plasmid of K99 and GFP gene and expression in E.coli BL21（DE3） sensory cells.[Methods]The K99 gene and GFP gene were amplified by PCR amplification.The purpose of the fragment were cloned into pLA vector by ligated and transformed.The recombinant plasmids were identified and sequenced.The expression of protein was analyzed by Western Blot.[Results]Sequencing analysis showed that the fusion gene had the correct reading frame.The specific bands of 498 bp and 720 bp were obtained by double digestion and PCR.Fluorescence showed that the recombinant strain expressed the protein.Western Blot showed that the recombinant strain was expressed as fusion protein.[Conclusion]Successful construction of colorectal recombinants,and the expression of exogenous fusion protein.

关 键 词：ETEC K99 GFP 融合蛋白 

分 类 号：Q786[生物学—分子生物学]