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目的:利用重叠延伸PCR合成风疹病毒E1基因的抗原肽段,构建其原核表达载体并表达蛋白,为进一步获得高质量的rE1重组抗原肽奠定基础。方法:利用软件对风疹病毒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,以重叠延伸PCR法合成相应抗原肽段,克隆后测序鉴定。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;利用IPTG诱导抗原肽段的表达,SDS-PAGE和West-ern blot法分析表达产物。结果:经过6轮重叠延伸PCR扩增,成功获得与预期目的序列一致的风疹病毒E1基因抗原肽并构建获得重组质粒pET32-rE1;在37℃下分别以终浓度为1 mmol/L和0.5 mmol/L的IPTG诱导抗原肽表达,SDS-PAGE和Western blot法检测到预期的蛋白条带;且以IPTG终浓度为1 mmol/L诱导6 h后表达量最高。结论:成功合成了密码子优化的风疹病毒E1基因抗原肽段,构建其原核表达载体pET32-rE1并表达该抗原肽。
OBJECTIVE: To synthesize the antigenic fragment of Rubella virus E1 gene by overlap extension PCR, construct its prokaryotic expression vector and express the protein, which lays the foundation for further obtaining high quality rE1 recombinant antigen peptide. Methods: The bioinformatics analysis of E1 gene of rubella virus was carried out by software, and its codon was optimized according to codon preference of E. coli. Multiple pairs of oligonucleotide primers were designed and the corresponding antigenic peptides were synthesized by overlap extension PCR. Clones After sequencing identification. The recombinant peptide was cloned into the prokaryotic expression vector pET32a by restriction enzyme digestion. The expression of antigenic peptide was induced by IPTG. The expressed products were analyzed by SDS-PAGE and Western-blot. Results: After six rounds of overlap extension PCR amplification, the antigenic peptide of the rubella virus E1 gene was successfully obtained and the recombinant plasmid pET32-rE1 was obtained. The recombinant plasmids pET32-rE1 were obtained at a final concentration of 1 mmol / L and 0.5 mmol / L IPTG induced antigen peptide expression, SDS-PAGE and Western blot detected expected protein bands; and IPTG final concentration of 1 mmol / L induced 6 h after expression was the highest. Conclusion: The codon-optimized rubella virus E1 gene fragment was successfully synthesized and its prokaryotic expression vector pET32-rE1 was constructed and expressed.