NNMT基因重组表达质粒的构建以及在大肠杆菌中的表达

Construction of recombinant NNMT plasmid and expression in E. coli

目的利用pGEX-4T-2表达系统表达制备尼克酰胺N-甲基转移酶(NNMT)抗原。方法根据NCBI核苷酸数据库编码为gi:12652950人NNMTcDNA序列,设计合成编码NNMT的DNA引物,并分别在5'端和3'端设计BamHI和XhoI位点,利用RT-PCR从人肝组织中克隆NNMT基因,经T-A克隆、亚克隆至pGEX-4T-2表达载体,重组质粒转化到大肠杆菌BL-21-STAR(DE3),经异丙基-β-D-硫代乳糖苷诱导表达,Glu-tathioneSepharose4B亲和层析制备融合蛋白(GST-NNMT)。通过DNA测序来证实质粒pGEX-4T-2DNNMT的正确性,SDS-PAGE电泳以及Western-blot判断GST-NNMT蛋白的准确性。结果限制性内切酶和DNA测序结果表明,编码NNMT蛋白的DNA序列准确插入到pGEX-4T-2多克隆位点,成功构建表达质粒pGEX-4T-2DNNMT。菌液超声破碎后,融合蛋白主要存在于裂解上清液中,表达量约占菌体总蛋白表达量的20%,每升菌液可表达融合蛋白约4.5mg。Western-blot证实制备的蛋白为GST-NNMT。结论成功地利用基因重组技术制备了NNMT,为制备抗NNMT单克隆抗体和ELISA试剂盒打下基础。

Objective To prepare Nicotinamide N-methyltransferase （NNMT） antigen with fusion protein of GST. by pGEX-4T-2 expression system. Methods According to the nueleotide bank of NCBI （gi：12652950）,DNA primers with BamHI/XhoI site were designed. Total RNA was isolated from the human liver tissue using TRlzol reagent and was reverse transcripted into the cDNA. The polymerase chain reaction （PCR） was used to amplify the NNMT sequence with its cDNA as template and primers designed on the basis of the NNMT DNA sequence. The PCR product was ligated into the vector pGEX-4T-2 after T-A clone and subclone. The recombinnat plasmid was transferred into E.coli BL-21-STAR（DE3） and the GST-NNMT fusion protein was expressed by inducing with IPTG. The fusion protein was purified by glutathione sepharose 4B affinity chromatography. DNA sequencing, SDS-PAGE and Western-blot were used to verify the plasmid and the fusion protein. Results Restriction analysis and DNA sequencing confirmed the correct sequence and insertion site of the recombinant pGEX-4T-2/NNMT. Expressed fusion protein was mainly in soluble supernatant and accounted for about 20% of the total bacterial proteins. SDSPAGE and Western-blot results indicated that the fusion protein was GST-NNMT. Conduslon The expression system of pGEX-4T-2/NNMT was successfully constructed.