SARS冠状病毒M蛋白全长基因的克隆、表达与纯化  

Cloning,expression and purification of the M gene from SARS Coronavirus

作　　者：雷明军[1] 吴少庭[2] 戴五星[1] 秦莉[1] 潘晖榕[1] 袁仕善[2] 黄达娜[2] 高世同[2] 张仁利[2] 林绮萍[3] 

机构地区：[1]华中科技大学同济医学院生物化学与分子生物学系,武汉430030 [2]深圳市疾病预防控制中心分子生物室,深圳518020 [3]华南农业大学动物医学系,广州510089

出　　处：《中国人兽共患病杂志》2005年第12期1100-1102,1067,共4页Chinese Journal of Zoonoses

摘　　要：目的克隆SARS冠状病毒M蛋白基因编码序列,构建原核重组表达质粒pET23a-M,并在大肠杆菌BL21(DE3)中进行表达、纯化。表达产物用Western Blot检测其抗原性。方法RT-PCR扩增M编码基因目的片段,胶回收纯化,插入克隆载体pMD-18T载体并转化大肠杆菌DH5α。序列测定后亚克隆至表达质粒载体pET23a,构建重组体pET23a-M,转化大肠杆菌DH5α。筛选阳性克隆,以限制性酶切分析鉴定后,转化大肠杆菌BL21(DE3)以异丙基硫代半乳糖苷(IPTG)进行诱导表达。用SDS-PAGE与免疫印迹分析表达产物。结果PCR扩增出约675bp的特异性片段,与预期片段大小相符,测序鉴定无有义突变;所构建的pET23a-M重组体阳性克隆经PCR与双酶切鉴定,与预期结果一致;SDS-PAGE显示表达产物约27kD;表达产物经金属螯和层析纯化;免疫印迹表明表达产物能被混合的SARS病人恢复期血清识别。结论成功克隆了SARS冠状病毒M蛋白基因编码序列,构建了pET23a-M表达质粒,诱导表达并纯化出了SARS冠状病毒M蛋白,并以免疫印迹鉴定。本研究的成功为进一步进行SARS病毒诊断试剂与疫苗的开发奠定了基础。To clone the M gene of SARS Coronavirus, express in E. coli and purify the expression products, the M gene fragment of SARS Coronavirus was amplified and cloned into the pMD-18T vector. After nucleotide sequencing, the M gene fragment was subcolned into the expression vector pET23a with correct orientation and then transformed into E. coli DH5α. The plasmid from the corrected clone identified by PCR and endonuclease digestion was transformed into E. coli BL21（DE3）and then induced for expression. The expression product was purified by metal-chelated affinity chromatography and analyzed by Western blotting. It was found that the 675bp gene fragment was amplified by RT-PCR. Nucleotide sequencing showed there was no sense mutation. A 27KD protein was expressed after induction and purified by metal-chelated affinity Chromatography. Western blotting result indicated that the expressed products could be recognized by sera of SARS Patinats. M gene of SARS Coronavirus was successively cloned and expressed in E. coil and the expressed gene products were successfully purified and recognized by SARS patient＇s is serum.

关 键 词：SARS M蛋白 基因表达 蛋白纯化 免疫印迹 

分 类 号：R373.1[医药卫生—病原生物学]