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目的构建无乳链球菌(Streptococcus agalactiae,S.agalactiae)gapC基因和鼠伤寒沙门菌(Salmonella typhimu-rium,S.typhimurium)fliC基因的融合基因gapC-fliC,并在大肠杆菌中表达融合蛋白。方法应用PCR技术分别扩增鼠伤寒沙门菌鞭毛蛋白fliC基因和无乳链球菌gapC基因片段,通过柔性肽(Gly4Ser)2编码序列将二者串联并克隆至质粒pQE-30上,构建重组原核表达质粒fliC-gapC-pQE30,转化E.coli XL1-Blue,IPTG诱导表达,并进行SDS-PAGE分析。表达产物经镍离子亲和层析柱纯化后,进行Western blot分析及融合蛋白活性检测。结果重组表达质粒fliC-gapC-pQE-30经双酶切和测序证明构建正确;表达的融合蛋白FliC-GapC相对分子质量约95 000,表达量占菌体总蛋白的58%,主要以包涵体形式存在,且可与小鼠抗鼠伤寒沙门菌和抗无乳链球菌多抗血清发生特异性反应,具有较好的甘油醛-3-磷酸脱氢酶(GAPDH)活性。结论成功构建了重组表达质粒fliC-gapC-pQE-30,并在E.coli XL1-Blue中表达了融合蛋白,为下一步动物免疫保护性试验的研究奠定了基础。
Objective To construct the gapC-fliC fusion gene of Streptococcus agalactiae gapC gene and S. typhimurium fliC gene and express the fusion protein in E. coli. Methods The fliC gene of S. typhimurium flagella and the gapC gene fragment of Streptococcus agalactiae were amplified by polymerase chain reaction (PCR) and inserted into the plasmid pQE-30 by the flexible peptide (Gly4Ser) 2 coding sequence to construct recombinant prokaryotic expression vector The plasmid fliC-gapC-pQE30 was transformed into E.coli XL1-Blue and induced by IPTG, and analyzed by SDS-PAGE. The expressed product was purified by nickel ion affinity chromatography, and then detected by Western blot and fusion protein activity. Results The recombinant plasmid fliC-gapC-pQE-30 was constructed correctly by double enzyme digestion and sequencing. The relative molecular mass of FliC-GapC was about 95 000 and accounted for 58% of total bacterial proteins, Form and can react specifically with mouse anti-Salmonella typhimurium and Streptococcus agalactiae polyclonal antiserum, and has good glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. Conclusion The recombinant plasmid fliC-gapC-pQE-30 was successfully constructed and the fusion protein was expressed in E. coli XL1-Blue, which laid the foundation for the further study of animal immunoprotection.