摘要
目的克隆牙龈卟啉单胞菌外膜蛋白3-磷酸甘油醛脱氢酶(GAPDH),并将其置于大肠杆菌中作融合表达。方法利用PCR技术,克隆GAPDH,插入克隆载体pMD18-T中得到克隆重组子pMD18-T-GAPDH。克隆重组子与表达载体pET-32a双酶切后连接构建表达质粒pET-32a-GAPDH。重组原核表达质粒经酶切鉴定后转化大肠杆菌BL21感受态细胞,以不同浓度异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达融合蛋白。结果核酸序列测定与分析表明:表达重组子pET-32a-GAPDH与NCBI核酸数据库中收录的GAPDH序列同源性达99.802%;在最佳浓度的IPTG诱导下,GAPDH可高效表达。结论本实验成功克隆了牙龈卟啉单胞菌GAPDH基因,并在大肠杆菌中表达了GAPDH蛋白,为后续实验研究GAPDH的免疫学性能及相应的抗体制备奠定了基础。
Objective To clone the glyceraldehydes 3-phosphate dehydrogenase（GAPDH） gene of Porphyromonas gingivalis（P.gingivalis） and to induce its fusion expression in E.coli.Methods GAPDH was obtained by PCR and was inserted into cloning vector pMD-18-T to construct clone recon.Double enzymes digest the recon pMD18-T-GAPDH and the prokaryotic expression vector pET-32a and then connect to get the expressing recon pET-32a-GAPDH.The recombinant expression plasmid which had been confirmed by enzymes digestion was transformed to E.coli competent cells BL21 and induced the expression of GAPDH with isopropyl β-D-1-thiogalactopyranoside（IPTG） of different density.Results DNA sequencing showed that the fragment was 99.802% the same to the sequence published in NCBI.Under the best density,IPTG could be highly expressed.Conclusion The GAPDH had been success-fully cloned and expressed in E.coli which gets ready for the following experiment to study the immunity of GAPDH and the homologues antibody preparation.
出处
《华西口腔医学杂志》
CAS
CSCD
北大核心
2011年第2期199-202,共4页
West China Journal of Stomatology
基金
国家自然科学基金资助项目(30500560)
陕西省自然科学基础研究计划基金资助项目(2006C242)
西安市科技攻关计划基金资助项目(GG05159)
关键词
牙龈卟啉单胞菌
3-磷酸甘油醛脱氢酶
原核表达
Porphyromonas gingivalis
glyceraldehydes 3-phosphate dehydrogenase
prokaryotic expression
