【作者】 马贞丽；

【导师】 芦春斌；

【作者基本信息】 暨南大学 ， 生物化学与分子生物学， 2011， 硕士

【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。更多还原

【Abstract】 ObjectivesTo research efficient recombinant antigens for serological assays for rubella virus infection, full-length rubella virus E1 gene was synthesized with overlapping PCR-Restriction enzyme ligation method and major E1 epitope gene was amplified by PCR; Full-length E1 gene and E1 epitope gene were constructed into prokaryotic vectors for expression of recombinant antigen, respectively.MethodsBioinformatic analysis of E1 gene was done and the E1 sequence containing Escherichia coli preferred codons was designed. Two restriction enzymes were used to cut the full-length sequence of E1 to generate three fragments. Which were then amplified, respectively. With overlapping PCR using multiple pairs of oligonucleotide primers, a full-length of E1 sequence was assembled via restriction enzyme ligation. The full-length of E1 sequence was cloned into expression vector pET32a. The recombinant plasmid pET32-RV E1 was confirmed by PCR, restriction enzyme digestion and DNA sequencing. The gene of epitope,195-305 amino acid residues of the rubella virus E1 glycoprotein, was amplified from new synthesized E1 sequence, and cloned into expression vector pET32a to construct recombinant plasmid pET32-E1epitope-The expression of the two recombinant proteins was induced by IPTG, and analyzed by SDS-PAGE and Western blotting.Results1. The three fragments were amplified, and verified by DNA sequencing. The full-length sequence was inserted into plasmid pET32a which was confirmed as the designed one. The recombinant protein was expressed with IPTG inducement and identified by western blotting.2. The desired epitope gene fragment was amplified with PCR and confirmed with DNA sequencing. The sequence was cloned into plasmid pET32a, which was confirmed as the desired with PCR, restriction enzyme digestion and DNA sequencing. Western blotting showed the expression of recombinant protein with IPTG inducement. The recombinant protein of high level was expressed at 6 hours after induced with 0.1 mmol/L IPTG.ConclusionsThe rubella virus E1 gene sequence containing Escherichia coli preferred codons was synthesized with overlapping PCR-Restriction enzyme ligation method, and the plasmid pET32-RV E1 was constructed. The plasmid pET32-E1epitope for E1 epitope gene was constructed too. The recombinant full-length El protein and E1 epitope was expressed.更多还原