原核表达载体pET28a-3×flag-LukS-PV的构建与表达鉴定

Construction and identification of prokaryotic expression vector pET28a-3×flag-LukS-PV

目的 构建原核表达载体pET28a-3×flag-LukS-PV,并在大肠杆菌中进行表达纯化及鉴定。方法 通过PCR扩增得到LukS-PV基因和3×flag基因,经琼脂糖凝胶电泳分离切胶回收后与pMD-18T载体连接。通过NdeⅠ和Bam HⅠ对pMD-18T-3×flag载体和pET28a载体进行双酶切后,连接得到pET28a-3×flag载体。使用Bam HⅠ和XhoⅠ分别对pET28a-3×flag载体和pMD-18T-LukS-PV进行双酶切,连接后得到pET28a-3×flag-LukS-PV重组质粒并测序鉴定,随后转化入大肠杆菌BL21中。利用SDS-PAGE电泳对表达产物进行分析并使用抗His单克隆抗体和抗flag多克隆抗体进行Western blot检测。结果 PCR获得了LukS-PV和3×flag基因,构建的pET28a-3×flag-LukS-PV重组质粒经测序鉴定无碱基突变。SDS-PAGE电泳表明重组蛋白主要以可溶性蛋白形式存在,且重组蛋白浓度和纯度均较高。Western blot表明使用抗His单克隆抗体和抗flag多克隆抗体在约40 kD均可产生特异性浓集条带。结论 成功纯化得到了高浓度和高纯度的3×flag-LukS-PV重组蛋白,为后续研究LukS-PV的生物学功能和具体机制奠定了基础。

Objective To construct a prokaryotic expression vector pET28a-3×flag-LukS-PV,and to purify and identify the expression vector in Escherichia coli.Methods LukS-PV gene and 3×flag gene were amplified by PCR.After double digestion of pMD-18T-3×flag vector and pET28a vector by NdeⅠand Bam HⅠ,the pET28a-3 flag vector was obtained.The pET28a-3×flag vector and pMD-18T-LukS-PV were double digested using Bam HⅠand XhoⅠ,respectively.After ligation,the recombinant plasmid pET28a-3×flag-LukS-PV was obtained and verified by sequencing,which was subsequently transformed into Escherichia coli BL21.The expression products were analyzed by SDS-PAGE electrophoresis and Western blot using anti-His monoclonal antibody and anti-flag polyclonal antibody.Results LukS-PV and 3×flag genes were obtained by PCR,and pET28a-3×flag-LukS-PV recombinant plasmid was identified by sequencing and showed no base mutation.SDS-PAGE electrophoresis showed that the recombinant protein was mainly in the form of soluble protein,and the concentration and the purity of recombinant protein were high.Western blot showed that specific concentration bands were generated at about 40 kD using both anti-His monoclonal antibody and anti-flag polyclonal antibody.Conclusion The 3×flag-LukS-PV recombinant protein is successfully purified with high concentration and high purity,which lay a foundation for the subsequent study on the biological function and specific mechanism of LukS-PV.