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[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 aim of the study was to construct recombinant plasmid of Helicobacter pylori scaffold protein Mre B labeled with tandem affinity tag (TAP) based on overlap extension PCR. [Method] The Mre Ba, TAP and Mre Bb sequences of the termination codon TAA of Helicobacter pylori Mre B gene were ligated by overlap extension PCR to form a DNA fragment of about 3.1 kb The fusion fragment Mre BCF; Mre BCF fragment was digested with XhoI, purified, and cloned into the linear vector pK18mob Sac B digested with SmaI and XhoI. [Result] The results of PCR, restriction enzyme digestion and DNA sequencing showed that the recombinant plasmid pK18Mre BCF contained three fragments (Mre Ba, TAP and Mre Bb) of size 740 bp, 1400 bp and 1 000 bp, and these three Fragments of the linker and the nucleotide sequence is correct. [Conclusion] Multiple overlapping fragments could be linked by overlap extension PCR, and the recombinant plasmids could be constructed easily and efficiently. The recombinant plasmid pK18Mre BCF was successfully constructed for the future isolation and identification of the functional complex of Helicobacter pylori Mre B protein The foundation.