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目的构建盆腔炎患者感染大肠埃希菌重组质粒pGEX-SrtA△N59并进行测序鉴定,为寻找盆腔炎患者感染大肠埃希菌的治疗新方法和新药研发奠定基础。方法分别提取pMD20-SrtA和pGEX-4T-1质粒并进行鉴定。然后根据SrtA基因序列来设计特异性引物,以pMD20-SrtA为模板进行SrtA△N59基因PCR扩增。将目的片段克隆至pGEX-4T-1中,构建pGEX-SrtA△N59重组质粒,进行酶切和测序分析。结果 pMD20-SrtA酶切目的基因片段约为618bp,与预期SrtA基因片段大小相符合,可作为模板扩增SrtA△N59。pGEX-4T-1经单、双酶切均获得5 000bp片段,大小与预期值(4 900bp)相符合,该质粒可应用于构建重组质粒。PCR扩增产物约460bp,与SrtA△N59基因的大小符合。构建的pGEX-SrtA△N59重组质粒经双酶切,获得460bp目的基因片段,表明SrtA△N59基因成功插到载体pGEX-4T-1中;基因测序显示,其与AF162687基因序列100%匹配。SrtA△N59基因大小应为441bp,但本实验设计引物时在其两端分别加有酶切位点以及保护性基团,所以所得扩增产物的目的片段大小约为460bp。结论构建的pGEX-SrtA△N59中成功插入SrtA基因,重组质粒构建正确。
Objective To construct and identify the recombinant plasmid pGEX-SrtA △ N59 of Escherichia coli in patients with pelvic inflammatory disease and lay a foundation for the new treatment of Escherichia coli in patients with pelvic inflammatory disease. Methods Plasmid pMD20-SrtA and pGEX-4T-1 were extracted and identified respectively. Then specific primers were designed according to the sequence of SrtA gene and PCR amplification of SrtAΔN59 gene was carried out using pMD20-SrtA as a template. The target fragment was cloned into pGEX-4T-1 and the recombinant plasmid pGEX-SrtAΔN59 was constructed and digested and sequenced. Results The fragment of pMD20-SrtA was about 618bp, which was consistent with the expected size of SrtA gene and could be used as a template to amplify SrtA △ N59. The 5 000 bp fragment of pGEX-4T-1 was obtained by single digestion and double digestion. The size of pGEX-4T-1 was in accordance with the expected value (4 900 bp). This plasmid can be used to construct recombinant plasmids. The PCR product was about 460 bp, which was consistent with the size of SrtA △ N59 gene. The recombinant plasmid pGEX-SrtAΔN59 was double-digested to obtain a 460 bp target gene fragment, indicating that the SrtA △ N59 gene was successfully inserted into the vector pGEX-4T-1. The gene sequence showed that it was 100% identical to the AF162687 gene sequence. SrtA △ N59 gene size should be 441bp, but in this experiment, primers were added to both ends of the restriction enzyme sites and protective groups, so the size of the resulting amplified fragment of about 460bp. Conclusion SrtA gene was successfully inserted into pGEX-SrtA △ N59, and the recombinant plasmid was constructed correctly.