hK1-L-Fc融合蛋白在CHO细胞中的表达及其活性研究

中国生物工程杂志

2011, Vol. 31

Issue (03): 23-28

研究报告

hK1-L-Fc融合蛋白在CHO细胞中的表达及其活性研究

邓春平¹, 陶建军², 侯永敏², 钟翎¹

1. 中山大学药学院广州 510006;
2. 广东天普生化医药股份有限公司广州 510520

Expression of hK1-L-Fc Fusion Protein in CHO Cells and Analysis of its Bioactivity

DENG Chun-ping¹, TAO Jian-jun², HOU Yong-min², ZHONG Ling¹

1. School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China;
2. Techpool Bio-pharma Co. Ltd., Guangzhou 510520, China

全文: PDF(606 KB) HTML

摘要：

为进一步改造重组人激肽释放酶1(hK1),以期提高其生物活性,制备了通过柔性接头相连接的重组人激肽释放酶1-L-IgG1 Fc融合蛋白(hK1-L-Fc)。采用重叠延伸PCR技术构建hK1-L-Fc融合基因,克隆至表达载体pcDNA3.1,在中国仓鼠卵巢细胞(CHO-S)中表达。利用Protein A 亲和层析柱纯化融合蛋白,SDS-PAGE、Western blotting、飞行时间质谱(MALDI-TOF-MS)、HPLC检测表达产物,底物法检测融合蛋白的体外活性。结果显示:成功构建pcDNA3.1-hK1-L-Fc重组表达载体;获得稳定表达融合蛋白的细胞株;无血清悬浮批式培养的表达量在0.7 mg/L以上;纯化的蛋白其纯度在95%以上,分子量约60 kDa;活性检测显示其比活性在9.2 U/mg以上,较hK1-Fc蛋白提高了18%以上。

关键词： 激肽释放酶1; 融合蛋白; CHO细胞

Abstract:

To further modify the recombinant human kallikrein 1(hK1) for higher activity, new form of recombinant human kallikrein 1(hK1-L-Fc) was constructed by fusion of the human kallikrein1 gene and the coding sequences for Fc fragment of human IgG1 with a flexible linker peptide. The fusion gene hK1-L-Fc was constructed by PCR, then inserted into expression vector pcDNA3.1, and expressed in Chinese Hamster Ovary cells. The chimeric protein was purified by Protein A affinity chromatography, and further analyzed by SDS-PAGE、Western blotting、MALDI-TOF-MS and HPLC. The bioactivity of fusion protein in vitro was determined by the reaction with its substrate. The results showed that recombinant expression vector pcDNA3.1-hK1-L-Fc was constructed successfully and the cell lines stably secreting fusion protein were obtained; The expression output of the fusion protein was higher than 0.7 mg/L in batch culture; The purity of the protein with about 60 kDa molecular weight was higher than 95%, and the biological activity of the fusion protein was more than 9.2 U/mg, which increased more than 18% than that of hK1-Fc fusion protein.

Key words: Kallikrein 1 Fusion protein CHO cell

收稿日期: 2010-10-13 出版日期: 2011-04-01

ZTFLH:

Q78

基金资助:

国家自然科学基金( 30873457 )、广东省科技计划(2008A060202010)资助项目

通讯作者: 钟翎 E-mail: lsszhl@mail.sysu.edu.cn

	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
	邓春平
	陶建军
	侯永敏
	钟翎

引用本文:

邓春平, 陶建军, 侯永敏, 钟翎. hK1-L-Fc融合蛋白在CHO细胞中的表达及其活性研究[J]. 中国生物工程杂志, 2011, 31(03): 23-28.

DENG Chun-ping, TAO Jian-jun, HOU Yong-min, ZHONG Ling. Expression of hK1-L-Fc Fusion Protein in CHO Cells and Analysis of its Bioactivity. China Biotechnology, 2011, 31(03): 23-28.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I03/23

[1] Chan H, Springman E B, Clark J M, et al. Expression and characterization of human tissue kallikrein variants. Protein Expr Purif, 1998, 12(3):361-370.
[2] Ling L, Hou Q, Xing S, et al. Exogenous kallikrein enhances neurogenesis and angiogenesis in the subventricular zone and the peri-infarction region and improves neurological function after focal cortical infarction in hypertensive rats. Brain Res, 2008, 1206:89-97.
[3] Liu L, Zhang R, Liu K, et al. Tissue kallikrein protects cortical neurons against in vitro ischemia-acidosis/reperfusion-induced injury through the ERK1/2 pathway. Exp Neurol, 2009, 219(2):453-456.
[4] 周同,陶建军,李林国,等.hK-Fc融合蛋白的改良、表达及其生物活性的分析.生物工程学报,2009,25(11):1697-1704. Zhou T, Tao J J, Li L G, et al. Chin J Biotech, 2009, 25(11):1697-1704.
[5] 边雷,石屹峰.蛋白质药物长效化技术的现状与进展.中国生物工程杂志,2009,29(1):114-118. Bian L, Shi Y F. China Biotechnology, 2009, 29(1):114-118.
[6] 杨建军,张昕洞,周洁,等.rhEPO-Fc融合蛋白的表达、纯化及质量研究.中国生物工程杂志,2007,27(6):6-9. Yang J J, Zhang Q D, Zhou J, et al. China Biotechnology, 2007, 27(6):6-9.
[7] Peters R T, Low S C, Kamphaus G D, et al. Prolonged activity of factor IX as a monomeric Fc fusion protein. Blood, 2010, 115(10):2057-2064.
[8] 王磊,何剑,肖卫华.人干扰素α2b和IgG Fc片段融合蛋白显著延长体内半衰期.生物工程杂志,2008,24(1):53-62. Wang L, He J, Xiao W H. Chin J Biotech, 2008, 24(1):53-62.
[9] Subramanian G M, Fiscella M, Lamousé-Smith A, et al. Albinterferon alpha-2b: a genetic fusion protein for the treatment of chronic hepatitis C. Nat Biotechnol, 2007, 25 ( 12 ):1412-1419.
[10] Osbron B L, Olsen H S, Nardelli B, et al. Pharmacokinetic and pharmacodynamic studies of a human serum albumin-interferon-alpha fusion protein in cynomolgus monkeys. J Pharmacol Exp Ther, 2002, 303(2):540-548.
[11] Huang Y S, Chen Z, Chen Y Q, et al. Preparation and characterization of a novel exendin-4 human serum albumin fusion protein expressed in Pichia pastoris. J Pept Sci, 2008, 14(5):588-595.
[12] Zhao H L, Yao X Q, Xue C, et al. Increasing the homogeneity, stability and activity of human serum albumin and interferon-a2b fusion protein by linker engineering. Protein Expr Purif, 2008, 61(1):73-77.
[13] Huang Y S, Chen Z, Yang Z Y, et al. Preparation and characterization of a potent long-lasting recombinant human serum albumin-interferon-α2b fusion protein expressed in Pichia pastoris. Eur J Pharm Biopharm, 2007, 67 (2):301-308.
[14] Bai Y, Shen W C. Improving the Oral Efficacy of Recombinant Granulocyte Colony-Stimulating Factor and Transferrin Fusion Protein by Spacer Optimization. Pharm Res, 2006, 23(9):2116-2121.
[15] Gustavsson M, Lehti J, Denman S, et al. Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris. Protein Eng, 2000, 14(9):711-715.
[16] Arai R, Ueda H, Kitayama A, et al. Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng, 2001, 14(8):529-532.
[17] Black G W, Rixon J E, Clarke J H, et al. Evidence that linker sequences and cellulose-binding domains enhance the activity of hemicellulases against complex substrates. Biochem J, 1996, 319:515-520.
[18] 田硕,姚文兵,周敏毅,等.新型集成干扰素-人血清白蛋白融合蛋白在毕赤酵母中的分泌表达和纯化.中国生物工程杂志,2010,30(6):34-37. Tian S, Yao W B, Zhou M Y, et al. China Biotechnology, 2010, 30(6):34-37.
[19] 宋小红,于婷,李冰,等.促进CHO细胞生长及其产物hNGF表达的培养条件的初步研究.中国生物工程杂志,2010,30(4):13-19. Song X H, Yu T, Li B, et al. China Biotechnology, 2010, 30(4):13-19.

[1]	陈英,肖海鹏,张晓焰,龚庆伟,马利,李文佳,陈小锋. GLP-1-IgG4-Fc融合蛋白的表达与鉴定 ^*[J]. 中国生物工程杂志, 2018, 38(7): 58-66.
[2]	邵莉, 马晓慧, 王相阳, 徐寒梅. 长效化融合蛋白药物及其药动学分析技术的研究进展[J]. 中国生物工程杂志, 2017, 37(4): 83-88.
[3]	曹荣月, 俞敏霞, 张昕黎, 李曼曼, 苗梓韬, 金亮. VEGFⅡ/GRP融合蛋白的构建、表达、纯化及其抗小鼠RM-1前列腺癌的作用研究[J]. 中国生物工程杂志, 2016, 36(8): 9-15.
[4]	陈华新, 武静, 赵瑾, 姜鹏. 抗人AFP单链抗体与藻胆蛋白融合蛋白的构建、表达与活性分析[J]. 中国生物工程杂志, 2016, 36(5): 74-80.
[5]	高相雷, 林树珊, 龚庆伟, 潘兰, 马利, 冯艳, 林小鹊, 曾剑, 李文佳, 陈小锋, 陈英. 重组人胰高血糖素样肽-1类似物的分离纯化和鉴定[J]. 中国生物工程杂志, 2016, 36(12): 15-20.
[6]	钱建瑛, 许正宏, 窦文芳. 融合蛋白GGH粉针剂制备工艺研究[J]. 中国生物工程杂志, 2016, 36(11): 48-53.
[7]	温家明, 聂艳峰, 梁翰章, 黄雯茜, 雷云, 谢秋玲, 裴运林, 熊盛. MG-132提高TNFR-Fc融合蛋白在CHO细胞中表达的研究[J]. 中国生物工程杂志, 2015, 35(9): 1-6.
[8]	龚隆财, 罗镇明, 杨雁青, 王振宇, 向军俭, 王宏. cTnI-linker-TnC融合蛋白的原核表达及鉴定[J]. 中国生物工程杂志, 2015, 35(4): 48-53.
[9]	晏丽, 王康, 李瑞建, 白义, 白先宏, 周海平. 一种新型高糖基化免疫融合蛋白NESP-Fc的研制[J]. 中国生物工程杂志, 2015, 35(4): 80-85.
[10]	艾君, 姜潮, 刘敏, 王晓艳, 田海山, 李校堃. 拟南芥双油体蛋白融合表达KGF-2及其生物学活性研究[J]. 中国生物工程杂志, 2015, 35(1): 21-26.
[11]	邵妤, 曲国龙, 金晶, 王微, 谭俊杰, 阚乃鹏, 李玉霞, 刘刚, 陈惠鹏. 利用BglBrick方法进行HSA/Exendin-4长效融合蛋白的快速组装[J]. 中国生物工程杂志, 2014, 34(4): 59-64.
[12]	杨凡, 黄虎, 李屹晨, 邓衡露, 吴茂柏, 钟翎, 侯永敏. 新型长效重组促卵泡素的纯化和性质及药代动力学研究[J]. 中国生物工程杂志, 2014, 34(2): 45-51.
[13]	王启帆, 薛莹, 冯新为, 张革. 靶向抗肿瘤蛋白iRGD-CDD的原核表达及生物活性鉴定[J]. 中国生物工程杂志, 2014, 34(12): 1-9.
[14]	李晨, 顾华, 郭佳, 孙慧, 黄经纬, 蒋明, 靳令经, 房健民. Tat-MANF融合蛋白的制备及生物活性研究[J]. 中国生物工程杂志, 2014, 34(06): 7-15.
[15]	孙静, 王斌, 段志青, 胡凝珠, 李建芳, 李彦涵, 胡云章. 重组人LIF融合蛋白表达纯化及其活性鉴定[J]. 中国生物工程杂志, 2013, 33(5): 50-55.

Viewed

Full text

Abstract

Cited

Shared

Discussed