摘要
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 a fusion gene gapC-fliC of S. typhimurium tiC gene and S. agalactiae gapC gene, and express in E. coli. Methods ThefliC and gapC genes were amplified by PCR respectively using the genomic DNA of S. typhimurium and plasmid of gapC-pQE30 as template, based on which fusion genefliC-gapC was amplified by overlap PCR using the linker of （Gly4Ser）2 coding sequence, and cloned into vector pQE30 （＋）. The constructed recombinant plasmid fliC-gapC-pQE30 was transformed to E. coli XL1-Blue and induced with IPTG, and the expressed product was identified by SDS-PAGE, purified by nickel ion affinity chromatography, and analyzed for activity by Western blot. Results Restriction analysis and sequencing proved that recombinant plasmidfliC-gapC-pQE30 was constructed correctly. The expressed fusion protein, with a relative molecular mass of about 95 000, contained about 58% of total somatic protein, mainly existed in a form of inclusion body, and showed specific reactions with both mouse polyclonal sera against S. typhimurium and S. agalactiae. Conclusion Recombinant plasmidflic-gapC-pQE30 was successfully constructed, and fusion protein was expressed in E. coli XL1-Blue, which laid a foundation of animal immune protection test in further study.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第2期171-175,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金项目(31072120)
黑龙江省研究生创新科研项目(YJSCX2011-279HLJ)
关键词
链球菌
无乳
沙门菌
鼠伤寒
鞭毛蛋白
重组融合蛋白质类
Streptococcus, agalaetiae
Salmonella, typhimurium
Flagellin
Recombinant fusion proteins
