Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2013, Vol. 33 Issue (3): 47-53    
研究报告     
重组角质细胞生长因子-1在杆状病毒表达系统中的表达及其生物活性研究
朱小静, 姜潮, 薛萍, 王晓艳, 徐丹, 南佳, 艾君, 李校堃
温州医学院药学院 浙江省生物技术与制药工程重点实验室 温州 325035
Expression and Purification of Biological-active Recombinant Human Keratinocyte Growth Factor-1 Base on Baculovirus Expression Vector System
ZHU Xiao-jing, JIANG Chao, XUE Ping, WANG Xiao-yan, XU Dan, NAN Jia, AI Jun, LI Xiao-kun
Wenzhou Medical College, Key Laboratory of Biotechnology and Pharmaceutical Engineering of Zhejiang Province, Wenzhou 325035,China
 全文: PDF(807 KB)   HTML
摘要： 目的:利用杆状病毒载体表达系统(baculovirus expression vector system,BEVS)表达重组角质细胞生长因子-1(KGF-1),并对其进行鉴定纯化及活性测定。方法:PCR扩增角质细胞生长因子-1基因(KGF-1)序列,克隆到pFastBac杆状病毒转座载体,经酶切测序鉴定出阳性重组转座载体pFastBac-kgf1后,转化含有穿梭载体Bacmid的DH10Bac感受态细胞,蓝白斑筛选出阳性质粒Bacmid-kgf1,转染Spodoptera frugiperda (sf9)昆虫细胞。对收获的蛋白进行SDS-PAGE、Western blot鉴定。利用肝素亲和层析柱和阳离子交换柱对目的蛋白进行纯化,BaF3细胞检测细胞增殖活性,并利用C57BL/6小鼠检测促毛囊增殖活性。结果:分子量约21 kDa的KGF-1在昆虫细胞中实现了表达。病毒侵染细胞48 h时开始表达目的蛋白,到96 h时表达量达到最高;且感染复数multiplicity of infection(MOI)为4时表达量较好。经过纯化之后,蛋白纯度达到90%以上,蛋白产量达2 mg/L。纯化之后的蛋白对转染FGFR2Ⅲb的BaF3细胞在0.10 ng/ml-1.56 ng/ml之间有促增殖活性,呈剂量依赖关系。并且KGF-1处理后小鼠毛囊增殖效果有所加强。
关键词： 角质细胞生长因子-1;  sf9细胞;  杆状病毒表达系统    
Abstract: Objective: the keratinocyte growth factor-1 (KGF-1) was expressed base on baculovirus expression vector system (BEVS). Methods: KGF-1 gene was amplified by PCR and then the product was cloned into the pFastBac vector at the NotⅠ and PstⅠ sites. The recombinant plasmid pFastBac-kgf1 was identified by enzyme digestion and sequencing. The recombinant pFastBac-kgf1 was transformed into E coli DH10Bac cells which contains a baculovirus shuttle vector (bacmid) and a helper plasmid. The recombinant Bacmid-kgf1 was identified by Blue-white selection and then transfected sf9 insect cells. P3 baculoviral stock was used to product KGF-1 protein at varying MOIs (1,2,4,8,16) and different times after infection (24,36,48,60,72,84,96,120 hours after infection). The protein KGF-1 was identified by SDS-PAGE and Western Blot and purified with heparin affinity column and cation-exchange column. And its activity was analyzed by BaF3 cells. Conclusion: After infection happened 48 h, KGF-1 was expressed by sf9 cells and at 96 h the expression yield reached the highest level. And the optimum multiplicity of infection (MOI) was 4 pfu/cell. The purity of KGF-1 was more than 90% and the protein production was about 2 mg/L after purification. Mitogenic activity assay demonstrated that KGF-1 could significantly stimulate the proliferation of mouse BaF3 cells in a dose-dependent manner, ranging from 0.1 to 1.56 ng/ml. Besides, subcutaneously injection of KGF-1 could significantly stimulated the growth of hair follicles.
Key words: Keratinocyte growth factor-1    sf9 insect cells    Baculovirus expression vector system
收稿日期: 2012-11-20 出版日期: 2013-03-25
ZTFLH:  Q78  
通讯作者: 姜潮     E-mail: proflxk@163.com
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
朱小静
姜潮
薛萍
王晓艳
徐丹
南佳
艾君
李校堃

引用本文:

朱小静, 姜潮, 薛萍, 王晓艳, 徐丹, 南佳, 艾君, 李校堃. 重组角质细胞生长因子-1在杆状病毒表达系统中的表达及其生物活性研究[J]. 中国生物工程杂志, 2013, 33(3): 47-53.

ZHU Xiao-jing, JIANG Chao, XUE Ping, WANG Xiao-yan, XU Dan, NAN Jia, AI Jun, LI Xiao-kun. Expression and Purification of Biological-active Recombinant Human Keratinocyte Growth Factor-1 Base on Baculovirus Expression Vector System. China Biotechnology, 2013, 33(3): 47-53.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/    或    https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I3/47

[1] Rubin J S, Osada H, Finch P W, et al. Purification and characterization of a newly identified growth factor specific for epithelial cells. Proc Natl Acad Sci USA, 1989, 86(3):802-806.
[2] Rubin J S, Bottaro D P, Chedid M, et al. Keratinocyte growth factor as a cytokine that mediates mesenchymal-epithelial interaction. Exs, 1995, 74:191-214.
[3] Kelley M J, Pech M, Seuanez H N, et al. Emergence of the keratinocyte growth factor multigene family during the great ape radiation. Proc Natl Acad Sci USA, 1992, 89(19):9287-9291.
[4] Finch P W, Rubin J S, Miki T, et al. Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth. Science, 1989, 245(4919):752-755.
[5] Wen J, Hsu E, Kenney W C, et al. Characterization of keratinocyte growth factor binding to heparin and dextran sulfate. Archives of biochemistry and biophysics, 1996, 332(1):41-46.
[6] Blijlevens N, Sonis S. Palifermin (recombinant keratinocyte growth factor-1):a pleiotropic growth factor with mutiple biological activities in preventing chemotherapy- and radiotherapy-induced mucositis. Annals of Oncology, 2007, 18(5): 817-826.
[7] Smith G E, Fraser M J, Summers M D. Molecular engineering of the Autographa californica nuclear polyhedrosis virus genome: deletion mutations within the polyhedrin gene. Journal of Virology, 1983, 46(2):584-593.
[8] Slominski A, Paus R. Melanogenesis is coupled to murine anagen: toward new concepts for the role of melanocytes and the regulation of melanogenesis in hair growth. Journal of Investigative Dermatology, 1993, 101, 90S-97S.
[9] Zong X L, Cai J L, Jiang D Y, et al. Research progress on keratinocyte growth factor. Chinese Journal of Reparative and Reconstructive Surgery, 2009, 23(2):188-193.
[10] Robinson C J, Das R G, Maile P. The World Health Organization reference reagent for keratinocyte growth factor, KGF. Growth Factors, 2006, 24(4):279-284 .
[11] Ron D, Bottaro D P, Finch P W, et al. Expression of biologically active recombinant keratinocyte growth factor. Structure/function analysis of amino-terminal truncation mutants. The Journal of Biological Chemistry, 1993, 268(4):2984-2988.
[12] Zhuo S L, Sun J P, Chen P, et al. High-cell-density cultivation of Escherichia coli expressing human keratinocyte growth factor. Journal of Xiamen University (Natural Science), 2011(01):82-87.
[13] Xue P, Zhu X J, Liu X J, et al. Studies on recombinant truncated human keratinocyte growth factor1 (rhKGF1D23) expression in insect cells. Journal of Jilin University, 2012, 38(4):633-639.
[1] 苏晓蕊, 李伟国, 王延辉, 高晓静, 闪伊红, 谭菲菲, 李向东, 田克恭. 重组杆状病毒细小VP2蛋白40L生物反应器放大工艺研究[J]. 中国生物工程杂志, 2017, 37(10): 60-64.
[2] 刘爱平, 李诚, 刘书亮, 王小红, 陈福生. 抗黄曲霉毒素B1单链抗体在Sf9昆虫细胞中的表达与性质分析[J]. 中国生物工程杂志, 2016, 36(5): 40-45.
[3] 王晓艳, 陈娜子, 艾君, 赵央, 吴美玉, 黄金枝, 姜潮, 李校堃. HBVpre-c-Fc融合蛋白在杆状病毒表达系统中的表达及其生物学活性研究[J]. 中国生物工程杂志, 2015, 35(4): 42-47.
[4] 徐丹, 刘敏, 孔菊, 李校堃, 姜潮. CFP10-ESAT-6-MPB64在杆状病毒系统中的表达纯化及免疫原性鉴定[J]. 中国生物工程杂志, 2014, 34(06): 23-30.
[5] 郭莉莉, 欧霞, 米锴, 孙茂盛, 李鸿钧. EV71类病毒颗粒的表达和免疫原性的初步评价[J]. 中国生物工程杂志, 2013, 33(1): 8-13.
[6] 冯佩平, 张蕾, 柴秀丽, 张海玲, 赵建军, 胡博, 白雪, 高晗, 邵西群, 闫喜军, 赵权, 徐磊. 犬瘟热病毒N基因在昆虫杆状病毒表达系统中的表达及应用[J]. 中国生物工程杂志, 2012, 32(04): 60-66.
[7] 张梅, 付远辉, 何金生, 陆燕燕, 郑娴娴. 增强型绿色荧光蛋白的真核表达和纯化[J]. 中国生物工程杂志, 2011, 31(5): 99-103.
[8] 郭海红, 杨辛, 郭芳, 胡红刚. DEK蛋白的真核表达纯化及其活性检测[J]. 中国生物工程杂志, 2011, 31(12): 1-9.
[9] 张梅 付远辉 何金生 陆燕燕 郑娴娴. 增强型绿色荧光蛋白的真核表达和纯化[J]. 中国生物工程杂志, 2011, 31(05): 0-0.
[10] 龚业莉1,王琳2,耿建玲2,刘颖2,夏焕章1. 密码子优化型HPV16L1基因在两种昆虫细胞中的表达[J]. 中国生物工程杂志, 2009, 29(07): 27-32.
[11] 徐剑文,林建银,林旭,齐元麟,张晓艳,朱进伟,胡建石. 蛇毒金属蛋白酶抑制剂抗小鼠黑色素瘤肺转移[J]. 中国生物工程杂志, 2009, 29(05): 28-32.
[12] 刘高强, 章克昌, 王晓玲, 魏美才. 昆虫杆状病毒表达系统的研究与应用进展[J]. 中国生物工程杂志, 2004, 24(7): 40-44.
[13] 陈璟, 方宏清, 周长林, 陈惠鹏. 昆虫杆状病毒系统表达外源蛋白的糖基化[J]. 中国生物工程杂志, 2004, 24(3): 1-6.
[14] 王长松, 刘友生, 葛晓冬, 李红, 陈燕平. 用杆状病毒-昆虫细胞系统表达、纯化重组的人源化氨基末端LBP[J]. 中国生物工程杂志, 2004, 24(12): 69-73.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会