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-l-thiogalactopyranoside (IPTG)of different density. Results DNA sequencing showed that the fragment was 99.802％ the same to the sequence published in NCBL Under the best density, IPTG could be highly expressed. Conclusion The GAPDH had been successfully cloned and expressed in E. coli which gets ready for the following experiment to study the immunity of GAPDH and the homologues antibody preparation.%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茵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.
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