重组RPLP0蛋白的表达、鉴定及评价

目的:构建重组RPLP0蛋白的原核表达载体并诱导表达,对表达产物进行纯化鉴定及评价。方法:设计重组RPLP0蛋白基因,经PCR扩增获得目的基因;在其5′和3′端引入BamHⅠ和XhoⅠ酶切位点,构建pET41a-GST-RPLP0-6*His融合基因表达载体;对该质粒载体进行酶切鉴定和测序验证,将鉴定正确的重组质粒载体转化至大肠杆菌并以IPTG诱导表达;经过尿素溶液纯化及复性以后得到复性的重组RPLP0蛋白;采用Western blot分析重组RPLP0蛋白的免疫原性;采用ELISA法对其抗体结合性、特异性进行分析。结果:酶切及测序结果显示,本实验成功克隆出重组RPLP0蛋白基因,将获得的目的基因构建至原核表达载体pET41a,经IPTG诱导后成功表达目的蛋白。SDS-PAGE电泳结果显示重组RPLP0蛋白为包涵体表达蛋白,分子量约为61 kD。Western blot检测显示重组RPLP0蛋白具有完全的免疫原性;ELISA检测显示重组RPLP0蛋白具有良好的抗体结合性和特异性。结论:表达、鉴定并分析了重组RPLP0蛋白,为深入研究RPLP0蛋白的功能及应用奠定基础。

Expression,identification and evaluation of recombinant RPLP0 protein

Objective: To construct the recombinant prokaryotic expression vector of RPLP0 protein,and to express,purify and identify this RPLP0 protein. Methods: The gene of recombinant RPLP0 protein was designed and amplified by PCR.BamH Ⅰ and XhoⅠ were introduced into the 5′ends and 3′ends of the gene to construct the expression vector.The recombinant plasmid vectors were identified by enzyme digestion and sequencing analysis.Finally,the recombinant plasmid vector was identified as correct and express in E.coli,and the expression was induced by IPTG.Then refolded and purified with urea solution to gain refolded recombinant RPLP0 protein.The immunogenicity and antibody binding ability of recombinant RPLP0 protein were analyzed by Western blot and ELISA. Results: The results of enzyme digestion and sequencing showed that the recombinant RPLP0 protein gene was successfully cloned,and the target gene was constructed into prokaryotic expression vector pET41a.The target protein was successfully expressed after IPTG induction.SDS-PAGE analysis showed that the recombinant RPLP0 protein was an inclusion body expression protein with a molecular weight of about 61 kD.Western blot and ELISA analysis displayed that the recombinant RPLP0 protein had complete immunogenicity,good antibody binding and specificity. Conclusion: The recombinant RPLP0 protein was expressed,identified and analysised,which could be used to further study the function and application of RPLP0 protein.