pfs基因原核表达载体的构建、表达及蛋白纯化
Construction and expression on prokaryotic expression vector of pfs gene together with recombinant protein purification
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摘要: 以植物乳杆菌KLDS1.0391的基因组DNA为模板,采用PCR方法扩增得到pfs基因,与Gen Bank中的序列对比发现同源性为99%。将目的基因与表达载体p QE-30连接,并将重组质粒p QE-30-pfs转化到E.coli M15中。异丙基-β-D-硫代吡喃半乳糖苷（IPTG）诱导表达重组蛋白,经SDS-PAGE分析,在全菌体蛋白中含有一条明显的特异性蛋白条带,大小约为27.4ku。经Ni-NTA纯化系统纯化后,在SDS-PAGE凝胶电泳图上获得纯化产物的条带。结果表明,原核表达载体p QE-30-pfs构建成功,且Pfs蛋白在大肠杆菌中成功表达,采用Ni-NTA纯化系统成功纯化了重组蛋白Pfs,为体外合成AI-2（Autoinducer-2）奠定了基础。Abstract: The pfs gene was amplified by polymerase chain reaction( PCR) using the DNA of Lactobacillus plantarum KLDS1.0391 as template in this study. The nucleotide sequence of the cloned DNA shared 99%homology with pfs gene from Lactobacillus plantarum JDM1 and WCFS1. The target gene was connected with expression vector p QE-30 and the recombinant plasmid p QE-30-pfs was transformed into E.coli M15. The recombinant expressed proteins induced by IPTG were analyzed and identified with SDS-PAGE. SDS-PAGE results showed that there was one main protein band with molecular weight 27.4ku in the total protein. The recombinant protein was purified using Ni-NTA Purification System and identified with SDS-PAGE. The results showed that the prokaryotic expression vector p QE-30-pfs was constructed successfully. Pfs protein was expressed successfully in E.coli M15. Recombinant protein Pfs was purified successfully using Ni-NTA Purification System. This results will be helpful for the synthesis of AI-2 in vitro.
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