风疹病毒E1重组蛋白及其抗原肽在大肠杆菌中的表达
发布时间：2018-09-19 09:26
【摘要】：目的 利用重叠PCR合成风疹病毒E1全长基因,同时扩增E1主要抗原表位的基因序列,分别构建它们的原核表达载体并表达蛋白,为研发高灵敏度风疹ELISA诊断试剂奠定基础。 方法 对风疹E1基因进行生物信息学分析,根据大肠杆菌密码子偏爱性对其密码子进行优化；设计多对寡核苷酸引物,以重叠PCR法分别合成该基因的3个片段,再用酶切连接法将各段拼接成全长为1 443 bp的RV E1,将E1基因的全长序列克隆导入原核表达载体pET32a获得pET32-RV E1克隆并测序；选取E1全长基因中195 aa-305 aa抗原肽基因序列进行扩增,再以酶切连接的方法将合成的抗原肽序列克隆导入原核表达载体pET32a,获得重组质粒载体pET32-E1epitope测序鉴定。将重组原核表达载体pET32-RV El和pET32-E1epitope在大肠杆菌BL21(DE3)表达株中利用IPTG诱导蛋白表达并优化蛋白表达量, SDS-PAGE和Western blot分析表达产物。 结果 1.分别进行了8轮、5轮和6轮的重叠PCR扩增,合成风疹E1全长基因3个片段；以酶切连接法将3个片段拼接成全长E1基因并克隆入pET32a,构建载体pET32-RV E1,PCR、酶切和测序鉴定结果表明,合成的E1基因大小、序列与预期相符；构建获得重组质粒pET32-RV E1。在37℃下用终浓度为1 mmol/L的IPTG诱导蛋白表达,Western blot检测到预期的蛋白条带； 2.以PCR扩增成功获得风疹E1抗原肽基因并构建重组质粒pET32-El epitope,测序结果正确；在37℃下以终浓度为1 mmol/L IPTG诱导抗原肽表达,SDS-PAGE和Western blot检测到预期的蛋白条带；且以IPTG终浓度为0.1 mmol/L诱导6小时后表达量达到最高。 结论 成功合成了密码子优化的风疹病毒E1基因并构建其重组质粒pET32-RV E1,表达完整E1蛋白；同时成功扩增风疹病毒E1抗原肽段基因,构建其原核表达载体pET32-E1epitope并表达该抗原肽。
[Abstract]:Objective to synthesize the full-length gene of rubella virus E1 by overlapping PCR and amplify the gene sequence of the major epitopes of E1, and construct their prokaryotic expression vectors and express proteins respectively. It lays a foundation for the development of high sensitivity ELISA diagnostic reagent for rubella. Methods the rubella E1 gene was analyzed by bioinformatics, its codon was optimized according to the codon preference of Escherichia coli, and three fragments of the gene were synthesized by overlapping PCR, and multiple pairs of oligonucleotide primers were designed. The whole sequence of E1 gene was cloned into prokaryotic expression vector pET32a to obtain pET32-RV E1 clone and sequenced, and 195 aa-305 aa antigenic peptide gene sequence of E1 full-length gene was amplified. The recombinant plasmid pET32-E1epitope was sequenced and cloned into prokaryotic expression vector pET32a, by restriction endonuclease ligation. The recombinant prokaryotic expression vectors pET32-RV El and pET32-E1epitope were induced by IPTG in E. coli BL21 (DE3) expression strain and the expression amount was optimized. The expression products were analyzed by SDS-PAGE and Western blot. Result 1. Three fragments of rubella E1 full-length gene were synthesized by overlapping PCR amplification of 8 rounds and 5 rounds and 6 rounds, respectively. The three fragments were spliced into full-length E1 gene by restriction endonuclease ligation and cloned into pET32a, to construct the vector pET32-RV E1 PCR. The results of restriction endonuclease digestion and sequencing showed that, The size of the synthesized E1 gene was consistent with the expected sequence, and the recombinant plasmid pET32-RV E1 was constructed. At 37 鈩，
本文编号：2249721
[Abstract]:Objective to synthesize the full-length gene of rubella virus E1 by overlapping PCR and amplify the gene sequence of the major epitopes of E1, and construct their prokaryotic expression vectors and express proteins respectively. It lays a foundation for the development of high sensitivity ELISA diagnostic reagent for rubella. Methods the rubella E1 gene was analyzed by bioinformatics, its codon was optimized according to the codon preference of Escherichia coli, and three fragments of the gene were synthesized by overlapping PCR, and multiple pairs of oligonucleotide primers were designed. The whole sequence of E1 gene was cloned into prokaryotic expression vector pET32a to obtain pET32-RV E1 clone and sequenced, and 195 aa-305 aa antigenic peptide gene sequence of E1 full-length gene was amplified. The recombinant plasmid pET32-E1epitope was sequenced and cloned into prokaryotic expression vector pET32a, by restriction endonuclease ligation. The recombinant prokaryotic expression vectors pET32-RV El and pET32-E1epitope were induced by IPTG in E. coli BL21 (DE3) expression strain and the expression amount was optimized. The expression products were analyzed by SDS-PAGE and Western blot. Result 1. Three fragments of rubella E1 full-length gene were synthesized by overlapping PCR amplification of 8 rounds and 5 rounds and 6 rounds, respectively. The three fragments were spliced into full-length E1 gene by restriction endonuclease ligation and cloned into pET32a, to construct the vector pET32-RV E1 PCR. The results of restriction endonuclease digestion and sequencing showed that, The size of the synthesized E1 gene was consistent with the expected sequence, and the recombinant plasmid pET32-RV E1 was constructed. At 37 鈩，
本文编号：2249721
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