hilA基因两端51bp序列对示踪标记性绿色荧光蛋白在禽肠炎沙门氏菌中倍增表达效率的鉴定  

Multiple enhanced expression of the green fluorescent protein as an indicator in avian Salmonella enteritidis by 51 bp sequences from both termini of hilA gene

作　　者：王高玲[1] 李涛[1,2] 史冰田 司微[1] 王斌[1,2] 刘恒贵[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/动物细菌病研究室,黑龙江哈尔滨150001 [2]福建农林大学动物科技学院,福建福州350000

出　　处：《中国预防兽医学报》2015年第6期409-413,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：兽医生物技术国家重点实验室基本科研业务费(SKLVBP201422)

摘　　要：为构建表达绿色荧光蛋白(GFP)作为示踪标记的重组禽肠炎沙门氏菌(S.enteritidis)并鉴定其遗传稳定性,本研究通过重叠延伸PCR构建5'端含有T7启动子和核糖体结合位点(RBS),3'端含有T7终止子序列的增强型GFP(e GFP)表达盒,并将e GFP表达盒插入p MD18-T载体中构建p MD-e GFP原核表达重组质粒,将其转化于S.enteritidis。结果显示e GFP能够在S.enteritidis中持续表达。进一步将S.enteritidis hil A基因两端的51 bp序列分别添加到p MD-e GFP中e GFP表达盒的两端,构建p MD-He GFP重组质粒。荧光显微镜观察和流式细胞仪分析与p MD-e GFP相比,p MD-He GFP转化S.enteritidis后,其e GFP的荧光强度提高约30倍。通过在无抗生素选择压力下连续传20代次,重组S.enteritidis中e GFP仍然能够稳定表达。本研究结果为进一步研究S.enteritidis在动物体内的分布和定植规律等提供了一种具有荧光示踪标记的目标菌,并为其他细菌的同类研究提供了借鉴。To construct the recombinant avian Salmonella enteritidis expressing enhanced green fluorescent protein （eGFP） as an indicator and identify its expression stability, the expression cassette of eGFP containing T7 promoter and ribosome binding site （RBS） at the 5′ terminus and T7 terminator at the 3′ terminus, respectively, was generated by overlap extension PCR, which was inserted into pMD 18-T vector to construct the prokaryotic expression recombinant plasmid pMD-eGFP. The eGFP was showed to constantly express after transforming into S.enteritidis SM6 strain. In addition, the 51 bp fragments from both terminals of hilA gene from S.enteritidis were fin＇ther added to each side of the eGFP cassette to construct the pMD-HeGFP, which enhanced the expression of eGFP more than 30 times comparing to pMD-eGFP. Furthermore, the eGFP expression in S.enteritidis was detected constantly even at 20th passage in non-antibiotic selective condition. These results provided a new technique using eGFP as an indicator to monitor the bacterium distribution and colonization in organ tissues or cells of the bacteria infected animal.

关 键 词：肠炎沙门氏菌 绿色荧光蛋白 荧光标记 hilA基因 

分 类 号：S852.61[农业科学—基础兽医学]