【摘要】：[目的]基于重疊延伸PCR技術(shù),構(gòu)建串聯(lián)親和層析標(biāo)簽(TAP)標(biāo)記的幽門螺桿菌骨架蛋白Mre B的重組質(zhì)粒。[方法]將幽門螺桿菌Mre B基因終止密碼子TAA前DNA序列(Mre Ba)、TAP和終止密碼子TAA后的DNA序列(Mre Bb),通過重疊延伸PCR進(jìn)行連接,形成大小約3.1 kb的融合片段Mre BCF;Mre BCF片段經(jīng)XhoⅠ酶切、純化后,克隆到經(jīng)SmaⅠ和XhoⅠ雙酶切的線性載體p K18mob Sac B上。[結(jié)果]PCR、酶切及DNA測序的結(jié)果表明,重組質(zhì)粒p K18Mre BCF包含大小約為740 bp、1 400 bp和1 000 bp的三個片段(Mre Ba、TAP和Mre Bb),并且這三個片段的接頭連接及核苷酸序列完全正確。[結(jié)論]利用重疊延伸PCR可對多個片段進(jìn)行無縫連接,簡便、高效地構(gòu)建重組質(zhì)粒;成功構(gòu)建了重組質(zhì)粒p K18Mre BCF,為將來幽門螺桿菌Mre B蛋白功能復(fù)合體的分離和鑒定奠定了基礎(chǔ)。
[Abstract]:[objective] to construct the recombinant plasmid of Mre B, a cytoskeleton protein of Helicobacter pylori labeled by tandem affinity chromatography (TAP), based on the overlap extension PCR technique. [methods] the pre-TAA DNA sequence (Mre Ba), TAP of Helicobacter pylori Mre B gene and the DNA sequence (Mre Bb), after terminating codon TAA were connected by overlapping extended PCR to form a fusion fragment Mre BCF; of about 3.1 kb in size. The Mre BCF fragment was digested by Xho 鈪，

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