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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JM10 9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 2 5 %— 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。
The mature peptide coding regions of two tumor necrosis factor TNFα1 and TNFα2 genes of rainbow trout were amplified by PCR using gene-specific primers with restriction sites of BamHI and HindIII. The amplified fragment was digested with restriction enzymes and ligated into the pQE30 expression vector, and the ligation product was transformed into E. coli JM109 competent bacteria. The transformants were screened by PCR and sequenced, and the construction of two recombinant TNFα genes of rainbow trout was completed. Recombinant was cultured and induced in vitro, and highly expressed TNFα recombinant protein was obtained. The highly expressed recombinant TNFα was not affected by the inducer IPTG, and the inclusion bodies were formed due to the high expression of the recombinant protein. The yield of the recombinant protein was about 25% -30% of the total bacterial protein. Using Ni NTA and immobilized metal affinity chromatography (IM PC) technology, a highly purified recombinant protein was obtained under denaturing conditions. The yield of purified recombinant protein was about 0.5-5mg.