乳酸菌表达CRISPR gRNA载体的构建与鉴定  

Construction and verification of lactic acid bacterial vector expressing CRISPR gRNA

作　　者：邸洋 王茂鹏[2] 李昌[2,4,5] 潘荣荣[2] 荣凤君 杜寿文[2] 朱羿龙[2] 郭焱[3] 金宁一[2,4,5] 

机构地区：[1]长春中医药大学药学院,吉林长春130117 [2]军事医学科学院军事兽医研究所,省部共建吉林省人兽共患病预防与控制重点实验室,吉林长春130122 [3]长春中医药大学基础医学院,吉林长春130117 [4]温州大学病毒病研究所,浙江温州325035 [5]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《生物技术》2017年第1期9-16,共8页Biotechnology

基　　金：国家自然科学基金项目(“IFITMs限制病毒进入的分子机制及其免疫调控研究”;No.31472197);病原微生物生物安全国家重点实验室开放课题(“宿主限制因子IFITM3抗SFTSV作用及其机制研究”;No.SKLPBS1435);北京市自然科学基金项目(“干扰素诱导跨膜蛋白抵御病毒感染的分子机制研究”;No.5152023)

摘　　要：[目的]构建并筛选重组gRNA(向导RNA)表达质粒p SJCR-LDH和p SJCR-C9PX,并对gRNA在乳酸菌中的表达进行鉴定。[方法]通过PCR技术扩增复制子SH71、氯霉素抗性基因CM和gRNA表达盒片段。将CM片段分别与gRNA表达盒进行融合,再与SH71复制子进行无缝克隆。通过转化,构建重组质粒p SJCR-LDH和p SJCR-C9PX。应用PCR、RT-PCR、Real-Time PCR对重组质粒进行鉴定,gRNA表达验证及拷贝数检测。[结果]PCR及RT-PCR结果显示获得169 bp大小目的条带,表明获得重组质粒载体且该gRNA表达载体在植物乳杆菌WCFS1株、CGMCC1.557株、乳酸乳球菌MG1363株中均成功表达,绝对荧光定量PCR检测获得LDH-gRNA标准曲线为y=-3.480log X+45.34,扩增效率为93.8%;C9PX-gRNA标准曲线为y=-3.327log X+37.07,扩增效率为99.8%。[结论]为利用CRISPR基因编辑技术在乳酸菌中进行基因操作奠定基础,为乳酸菌载体构建提供方法。[Objective] Constructing and screening of recombinant gRNA（guide RNA） expression plasmids p SJCR-LDH and p SJCR-C9 PX,and characterizing gRNA expression efficacy in lactic acid bacteria. [Methods]Replicon SH71,chloramphenicol resistance gene,gRNA expression cassette fragment were amplified by PCR. The CM fragment was fused with gRNA expression cassette,and seamless cloned with SH71 replicon. By transformation,recombinant plasmids p SJCR-LDH and p SJCR-C9 PX were constructed and gRNA was verified by PCR（including RT-PCR,Real-Time PCR）,and their copy number and expression capacity were determined. [Results] PCR and RT-PCR results showed that 169 bp target band was obtained,proved that two recombinant plasmids were successfully transcribed in Lactobacillus plantarum WCFS1,CGMCC1. 557 and Lactococcus lactis MG1363. By absolute quantitative PCR,the standard curve of LDH-gRNA was obtained as y =-3. 480 log X ＋ 45. 34,with an amplification efficiency of 93. 8%; the standard curve of C9PX-gRNA was y =-3. 327 log X ＋ 37. 07,with an amplification efficiency of 99. 8%. [Conclusion]The results will lay the foundation for gene manipulation in acid bacteria which using CRISPR gene editing technology,and a method was provided for the vector construction of lactic acid bacteria.

关 键 词：乳酸菌 基因编辑 重组表达质粒 gRNA CRISPR 

分 类 号：Q785[生物学—分子生物学]