上海地区1株SARS-Cov N基因序列分析及表达
Cloning, sequencing and expressing in E.coli for a SARS-Cov N gene founed in Shanghai
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摘要:目的 通过基因克隆、序列分析以及在大肠埃希菌中的表达和分离纯化, 研究上海地区1株SARS-CovN蛋白编码基因变异情况, 为SARS-Cov N蛋白应用于检测SARS感染作基础.方法 用RT-PCR法从患者血清样品中扩增获取SARS-Cov N蛋白编码基因, 并重组于质粒pGEX-2T.双脱氧法双向测定N基因序列, 与GenBank公布的89株SARS-Cov N基因序列比对分析; 重组N蛋白编码基因在E.coli中表达, 亲和层析法分离纯化表达产物.结果 RT-PCR扩增获取SARS-Cov N基因1 269 bp, 测定的核酸序列与GenBank中公布SARS-Cov N基因序列比对, 变异位点数1~4 bp, 其中导致1~3个编码氨基酸突变.重组N基因的表达质粒pGEX-2T/SARS-Cov N转化大肠埃希菌JM109, 经异丙基--D-硫代半乳糖苷(IPTG)诱导, N基因在大肠埃希菌中高效表达, 经亲和层析, 得纯度 > 90%的表达产物, 表达的融合蛋白分子量为74000 Da.结论 上海地区1株SARS-Cov N蛋白编码基因与已公布的89株毒株核酸同源性为99.92%~99.68%, 编码氨基酸同源性为99.8%~99.3%;该基因在E.coli中表达, 经分离纯化得到高纯度表达产物, 为进一步应用于SARS-Cov感染检测打下基础.Abstract:Objective To analysis the mutation of SARS-Cov Ngene and its application in diagnostic kits by cloning, sequencing and expressing of Ngene from a Shanghai SARS-Cov strain.Methods The SARS-Cov Ngene was amplified by RT-PCR fr om serum in a Shang hai SARS patientand was cloned into plasmid pGEX-2T.The cloned Ngene was sequenced and compared with 89 SARS strains in GenBank.The cloned gene was expressed in E.coli.The products was purified by affinty chromatog raphy of Glutathion-e Sepharose 4B and estimated by SDS-PAGE.Results SARS-Cov Ngene 1269 bp was amplified and cloned.Sequencing analisis founded 1~4 bp mutation and 1~3 putative amino acid changed among amplified SARS-Cov Ngene and 89 SARS strains in GenBank.In E.coli, the Nprotein was expressed in 74000 Da fusion protein.The purified products was purity over 90%.Conclusion 99.92~99.68% nucleotides, 99.8~99.3% of putative amino acid were homologous among amplyfied Ngene and 89 SARS-Cov strains in GenBank.The basic data and protein products with high purity for diagnostic kitwas obtainded.
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