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虹鳟肿瘤坏死因子（TNFα）基因体外表达与纯化的研究
In vitro expression and purification of rainbow trout (oncorhynchus mykiss)TNFα

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蔡中华 1   宋林生 1   Zou Jun 2   C J Secombes 2   董双林 3  
文摘 将虹鳟两种肿瘤坏死因子TNFα1和TNFα2基因的成熟肽编码区域，用带有BamHI和HindIII酶切位点的基因特异性引物进行PCR扩增。扩增片段用限制性内切酶消化并连接到pQE30表达载体上，连接产物转化到大肠杆菌JM109感受态细菌中。转化子经PCR筛选，质粒测序，完成了虹鳟两种TNFα基因重组子的构建。重组子经体外培养和诱导后，获得了高效表达的TNFα重组蛋白。高效表达的重组TNFα不受诱导剂IPTG的影响，并且由于重组子高效表达而形成了包涵体；重组蛋白产量约占菌体蛋白总量的25％－30％。应用Ni-NTA和固定金属亲和层析（IM－PC）技术，在变性条件下获得了高度纯化的重组蛋白，纯化重组蛋白的产量约为0.5-1mg/L。
其他语种文摘 There are two distinct Tumor necrosis factor alpha (TNFa 1 and TNFa2)genes in the genome of rainbow Iroul (On-corhynchus mykiss) . The two TNF a genes share 94% and 92% identity in nueleolide and amino acid, respectively. This phe-nomena is different from thai in mammals which containing only one copy of TNFa gene in the genome. In order to investigate the biological functions of the two TNFαhomologues in immune response, these two TNFα genes were recombined and expressed in prokaryotes to produce recombinanl proteins in vitro. Both of the two TNFα mature peptide coding fragments were amplified from plasmid pTl and pT2 which contained TNFa gene, using gene specific primers covered BamHI and Hindlll sites. The PCR products and pQE30 expression vector were di-gested by BamHI and Hindlll, respectively, and then purified by gel purification. The purified products were pooled together and ligated by T4 ligase at 14℃ overnight. Subsequently, the ligation products were transformed into E. coli strain JM109 and cul-tured al 37℃ overnight. The Iransformants were screened by vector primers and gene specific primers, and then identified by DNA sequencing. The recombinants TNF (rTNFs)were cultured and induced to express the recombinant proteins, then deter-mined by SDS-PAGE. Expression results showed that both of the two rTNFs were expressed at high level and formed insoluble ag-gregates (inclusion bodies) in the cytoplasm of E, coli with or without inducement of IPTG, the yield of expression rTNFs was about 25%-30% of gross bacterial protein. Due to the inclusion body, the recombinant proteins could only be purified under denature condition using 8M urea to solubilize the inclusion bodies, and then purified with the techniques of immobilized-metal affinity chromatography (1MAC) and Ni-NTA matrices. The 6-His tag with the recombinanl proteins can attach the Ni-NTA ma-trices to separate the other proteins in high pH condition, and can be eluted in low pH condition. About 0.5-lmg/L purified recombinant proteins could be obtained from the cultured bacteria with this approach.
来源 水生生物学报 ,2003,27(6):596-601 【核心库】
关键词 肿瘤坏死因子 ; 重组 ; 纯化
地址

1. 中国科学院海洋研究所, 青岛, 266071  

2. Department of Zoology, University of Aberdeen, Aberdeen, AB242TZ  

3. 青岛海洋大学, 266003

语种 中文
文献类型 研究性论文
ISSN 1000-3207
学科 水产、渔业
基金 国家863计划 ;  中国科学院王宽诚教育基金
文献收藏号 CSCD:1172109

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引证文献 4 篇

1 董伟仁 河豚白介素2基因鉴定及其生物信息学分析 浙江大学学报. 理学版,2006,33(6):688-694,701
被引 1 次

2 王海林 包涵体纯化技术 生物技术通报,2007(1):78-79,88
被引 1 次

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