基于重叠延伸PCR构建幽门螺杆菌MreB重组载体  

Construction of Helicobacter pylori MreB recombinant vector through overlapping extension PCR

作　　者：李芳[1] 高婧[1] 马金成[1] 范聪聪[1] 付亚娟[1,2] 侯晓强[1,2] Fang Li Jing Gao Jincheng Ma Congcong Fan Yajuan Fu Xiaoqiang Hou(College of Life Science, Langfang Normal University,Langfang 065000, China Edible and Medicinal Fungi Research and Development Center of Hebei Universities, Langfang Normal University, Langfang 065000, China)

机构地区：[1]廊坊师范学院生命科学学院,河北廊坊065000 [2]河北省高校食药用菌应用技术研发中心,河北廊坊065000

出　　处：《生物技术》2016年第5期415-420,共6页Biotechnology

基　　金：河北省大学生创新创业计划项目("基于PCR sewing高效快速地构建幽门螺杆菌Mre B重组质粒";No.201510100035)

摘　　要：[目的]基于重叠延伸PCR技术,构建串联亲和层析标签(TAP)标记的幽门螺杆菌骨架蛋白Mre B的重组质粒。[方法]将幽门螺杆菌Mre B基因终止密码子TAA前DNA序列(Mre Ba)、TAP和终止密码子TAA后的DNA序列(Mre Bb),通过重叠延伸PCR进行连接,形成大小约3.1 kb的融合片段Mre BCF;Mre BCF片段经XhoⅠ酶切、纯化后,克隆到经SmaⅠ和XhoⅠ双酶切的线性载体p K18mob Sac B上。[结果]PCR、酶切及DNA测序的结果表明,重组质粒p K18Mre BCF包含大小约为740 bp、1 400 bp和1 000 bp的三个片段(Mre Ba、TAP和Mre Bb),并且这三个片段的接头连接及核苷酸序列完全正确。[结论]利用重叠延伸PCR可对多个片段进行无缝连接,简便、高效地构建重组质粒;成功构建了重组质粒p K18Mre BCF,为将来幽门螺杆菌Mre B蛋白功能复合体的分离和鉴定奠定了基础。[ Objective ] The recombinant plasmid of Helicobacter pylori cytoskeletal protein MreB that was tagged with tandem affinity purification （TAP tag）was constructed, based on overlapping extension PCR. [ Methods ] The upstream and downstream fragments of TAP insertion site on MreB gene （MreBa, and MreBb ） , and TAP were successfully fused together by overlapping extension PCR, and the size of final fusion product was 3.1 kb, named as MreBCF. MreBCF fragment was digested by Xho I and purified by gel extraction. The purified fragment was then inserted into linear pK18mobSacB vector digested by Sma I and Xho I. [ Resnlts ] The results from PCR amplification and restriction enzyme digestion revealed that the recombinant plasmid pK18MreBCF contained inserting fragments of MreBa, TAP and MreBb, and their size was approximate 740 bp, 1 400 bp, and 1 000 bp, respectively. The sequence analysis indicated that insertion direction and nucleotide sequence of fusion fragments on pK18MreBCF plasmid were completely correct. [ Conclusion] Combination overlapping extension PCR with one - step restriction enzyme digestion, ligation and transformation ,the recombinant plasmid could he rapidly constructed. The recombinant plasmid pK18MreBCF constructed in the study would be beneficial for the isolation and identification of MreB protein complex of H. pylori in the future.

关 键 词：幽门螺杆菌 骨架蛋白 MREB 重叠延伸PCR 重组载体 

分 类 号：Q784[生物学—分子生物学]