重组人角质化细胞生长因子-2发酵条件的优化及其活性  

Optimization of Fermentation Condition and Determination of Activity of Recombinant Human Keratinocyte Growth Factor-2

作　　者：唐禄[1,2] 田海山[1,3] 王晓杰[1,3] 周鑫[1,2] 王一[1,2] 李校堃[1] 

机构地区：[1]吉林农业大学生物反应器与药物开发教育部工程研究中心,长春130118 [2]吉林农业大学生命科学院,长春130118 [3]长春格鲁斯特生物科技公司,长春130118

出　　处：《中国生物制品学杂志》2010年第10期1090-1094,共5页Chinese Journal of Biologicals

基　　金：长春市应用技术计划研究技术项目(08YZ45);2009年度科技型中小企业技术创新基金项目(09C26212203306)

摘　　要：目的优化重组人角质化细胞生长因子-2(Recombinant human keratinocyte growth factor-2,rhKGF-2)基因工程菌的发酵条件,并对rhKGF-2进行纯化及活性检测。方法优化rhKGF-2基因工程菌接种量、装液量、培养基初始pH值、IPTG浓度、诱导时机和诱导时间等发酵条件;rhKGF-2经离子交换层析和肝素亲和层析后,采用MTT法检测其对NIH-3T3细胞增殖的影响。结果 rhKGF-2基因工程菌的最佳发酵条件为:接种量2.0%,装液量50ml/250ml三角瓶,培养基初始pH值6.5～7.0,IPTG浓度0.10mmol/L,菌体处于对数生长初期时(A600=0.8～1.0)开始诱导,诱导时间4h;纯化的rhKGF-2经HPLC检测纯度达95.0%以上;在1～100μg/ml浓度范围内,rhKGF-2对NIH-3T3细胞的增殖具有促进作用。结论优化了rhKGF-2基因工程菌的发酵条件,获得了纯度较高、具有活性的rhKGF-2蛋白,为其工业化生产奠定了基础。Objective To optimize the condition for fermentation of recombinant E. coli with human keratinocyte growth fac-tor-2 （KGF-2）, purify the fermented product and determine its activity. Methods The conditions for fermentation of recombinant E. coli with KGF-2, such as inoculation quantity, volume of medium, original pH value, IPTG concentration, opportunity and time for induc-tion, were optimized. The rhKGF-2 was purified by ion exchange chromatography and herapin affinity chromatography, then deter-mined for effect on proliferation of NIH-3T3 cells by MTT method. Results The optimal inoculation quantity, volume of medium, original pH value of medium, IPTG concentration, opportunity and time for induction were 2. 0%, 50 ml / 250 ml Erlenmeyer flask, 6. 5 ～ 7. 0, 0. 10 mmol / L, early logarithmic growth phase （A600 = 0. 8 ～ 1. 0）and 4 h respectively. The HPLC purity of purified rhKGF-2 was more than 95%. The rhKGF-2 at a concentration range of 1 ～ 100 μg / ml showed promoting effect on the proliferation of NIH-3T3 cells. Conclusion The conditions for fermentation of rhKGF-2 was optimized, and the rhKGF-2 with activity and high purity was obtained, which laid a foundation of industrial production of rhKGF-2.

关 键 词：角质化细胞生长因子-2 发酵 纯化 活性 

分 类 号：Q78[生物学—分子生物学]