[1]靳占奎a,王 曦,杨 乐,等.重组人肿瘤坏死因子hTNF-α在大肠埃希菌[E.coli BL21DE3]中表达条件的优化[J].现代检验医学杂志,2017,32(05):100-103,107.[doi:10.3969/j.issn.1671-7414.2017.05.017]
　JIN Zhan-kuia,WANG Xi,YANG Le,et al.Optimization of Expression of Recombinant Human Tumor Necrosis Factor(hTNF-α)in Escherichia coli[E.coli BL21(DE3)][J].Journal of Modern Laboratory Medicine,2017,32(05):100-103,107.[doi:10.3969/j.issn.1671-7414.2017.05.017]
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重组人肿瘤坏死因子hTNF-α在大肠埃希菌[E.coli BL21DE3]中表达条件的优化()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第32卷
期数:
2017年05期
页码:
100-103,107
栏目:
论著
出版日期:
2017-11-02

文章信息/Info

Title:
Optimization of Expression of Recombinant Human Tumor Necrosis Factor(hTNF-α)in Escherichia coli[E.coli BL21(DE3)]
文章编号:
1671-7414(2017)05-100-05
作者:
靳占奎1a; 王 曦2; 杨 乐3; 徐翠香1b; 张丽洁1c
1.陕西省人民医院a.骨科; b.陕西省临床检验中心; c.血液病研究室,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
Author(s):
JIN Zhan-kui1a; WANG Xi2; YANG Le3; XU Cui-xiang1b; ZHANG Li-Jie1c
1a.Department of Orthopedics; 1b.Shaanxi Provincial Clinical Laboratory Center; 1c.Hematology Research Laboratory,Shaanxi Provincial People's Hospital,Xi'an 710068,China; 2.Xi'an Honghui Hospital,Xi'an 710054,China; 3.Xi'an Children's Hospital,Xi'an 710003,China
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; Q784
DOI:
10.3969/j.issn.1671-7414.2017.05.017
文献标志码:
A
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。
Abstract:
Abstract:Objective To construct a human tumor necrosis factor(hTNF-α)plasmid and identify it to optimize the fermentation conditionsof hTNF-α protein so as to achieve high expression in Escherichia coli.Methods The gene of hTNF-α was cloned into pET24a vector to obtainthe 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-α.Theplasmid 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 mmol/L IPTG,pH=7.5,and induction time was 5 h.The results showedthat 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.

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备注/Memo

备注/Memo:
作者简介:靳占奎(1982-),男,博士,主治医师,E-mail:zhankui999@163.com。 通讯作者:徐翠香(1980-),女,在读博士,主治医师,主要从事免疫学研究,E-mail:xucuixiang1129@163.com。
更新日期/Last Update: 1900-01-01