摘要
目的:对殊异韦荣菌S-腺苷同型半胱氨酸水解酶(sahH)基因进行克隆、鉴定、表达和纯化,为研究龋病的微生物致病机制提供理论依据。方法:提取殊异韦荣菌基因组DNA,采用PCR方法扩增sahH基因序列,构建pET28a-sahH重组质粒,转化到大肠杆菌(E.coli)BL21(DE3)感受态细胞中,酶切、PCR鉴定和测序后,IPTG诱导蛋白表达,并进行分离纯化。结果:PCR扩增产物全长为1 410bp。测序结果经BLAST软件分析,与GenBank中所报道的绿脓假单胞菌sahH基因序列进行对比分析,其同源性为99%。该基因序列已登陆GenBank,序列号为KC477409。SDS-PAGE分析,重组质粒在E.coli JM109中表达并纯化,得到的融合蛋白相对分子质量约为50 000,诱导5h蛋白表达量最高,浓缩后蛋白浓度为5.4g.L-1。Western blotting法得到的蛋白条带与目的蛋白大小相符。结论:成功克隆了殊异韦荣菌sahH基因,并通过基因序列和氨基酸分析证明其具有完整的阅读框架,且重组载体表达出融合蛋白,分离纯化得到目的蛋白。
Objective To clone,identify,express and purify the S-adenosyl-L-homocysteine hydrolase（sahH）gene of Veillonella dispar and to provide theoretical basis for the mechanism of dental caries disease.Methods The DNA exacted fromVeillonella dispar sahH gene were amplified by PCR method,and the recombinant plasmid PET28a-sahH was constructed and transformed into E.coil BL21.The sequencing was performed after PCR identification and restriction enzyme digestion.Then the protein expression was induced by IPTG and the protein was purified.Results The PCR product was 1 410bp.The sequencing data was analyzed by BLAST software.The homology of the sequencing result was 99 % compared with the sahH gene of Pseudomonas aeruginosa strain PAO1which was reported in GenBank.The sequence had been submitted to the NCBI GenBank,and the accession number was KC477409.With SDS-PAGE electrophoresis,the recombinant plasmid PET28a-sahH was expressed and purified in E.coil JM109,and the molecular weight of 50 000was noted in sahH protein bands.The highest amount of protein expression was generated 5hafter induction,The protein concentration was 5.4g.L-1 after concentrated.The size of protein obtained by Western blotting method was corresponded with target protein.Conclusion The sahH gene of Veillonella dispar is successfully cloned,and it is proved to have a full reading framework through the analysis on gene sequences and amino acids.And the recombinant vector could express fusion protein,and the interest protein is obtained by isolation and purification.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2013年第4期704-709,共6页
Journal of Jilin University：Medicine Edition
基金
吉林省科技厅自然科学基金资助课题(201015204)
