重组人肿瘤坏死因子(hTNF-α)在大肠埃希菌[E.coli BL21(DE3)]中表达条件的优化  

Optimization of Expression of Recombinant Human Tumor Necrosis Factor(hTNF-α) in Escherichia coli[E. coli BL21(DE3)]

作　　者：靳占奎[1] 王曦 杨乐[3] 徐翠香[4] 张丽洁[5] 

机构地区：[1]陕西省人民医院骨科,西安710068 [2]西安市红会医院骨科,西安710054 [3]西安市儿童医院,西安710003 [4]陕西省人民医院陕西省临床检验中心,西安710068 [5]陕西省人民医院血液病研究室,西安710068

出　　处：《现代检验医学杂志》2017年第5期100-103,107,共5页Journal of Modern Laboratory Medicine

摘　　要：目的构建人肿瘤坏死因子(hTNF-α)表达质粒,并对其进行鉴定,优化hTNF-α蛋白表达条件使之在大肠埃希菌中实现高效表达。方法应用PCR扩增技术获得hTNF-α基因片段并进行相应的鉴定,将鉴定后的hTNF-α基因克隆到pET24a载体中,获得pET24a-hTNF-α表达质粒。将此质粒转化入大肠埃希菌BL21(DE3)中,并对其表达条件进行优化。结果成功构建了pET24a-hTNF-α质粒,PCR和酶切技术进行鉴定,结果显示与目的片段一致。将此质粒转入大肠埃希菌BL21(DE3)中,得到最佳表达条件为:M9+LB培养基,37℃,0.5 mmol/L IPTG,pH值=7.5,诱导时间为5 h。优化后的菌体干重增高了2.56倍,hTNF-α产率增加3.68倍,hTNF-α表达率从9.38%增加到32.74%,增高了3.49倍。结论优化了pET24a-hTNF-α质粒在大肠埃希菌BL21(DE3)中的表达条件,使hTNF-α蛋白得到高效表达。Objective To construct a human tumor necrosis factor （hTNF-α） plasmid and identify it to optimize the fermenta tion conditions of hTNF-α protein so as to achieve high expression in Escherichia coli. Methods The gene of hTNF-α was cloned into pET24a vector to obtain the pET24a-hTNF-α expression plasmid that was transformed into Escherichia coli BL21（DE3）,and the expression conditions of BL21（DE3） were optimized. Results The plasmid of pET24a hTNF-α was successfully constructed and identified by PCR and digestion,which was consistent with the target fragment hTNF-a. The plasmid was transformed into Escherichia coli BL21 （DE3）,the best induced expression conditions of Escherichia coli BL21 （DE3） were as follows： M9 ＋ LB medium, 37℃, 0.5 rnmol/L IPTG, pH - 7.5, and induction time was 5 h. The results showed that dry weight of the cells and the rate of TNF were increased by 2.56 times and 3.68 times,respectively,and the expression rate of hTNF-α was increased by 3.49 times from 9.38% to 32.74%. Conclusion The optimal conditions for the expression of plasmid pET24a-hTNF-α in Escherichia coli were determined.

关 键 词：人肿瘤坏死因子Α 质粒 大肠埃希菌 基因克隆 

分 类 号：R378.21[医药卫生—病原生物学] Q784[医药卫生—基础医学]