抗癌胚抗原纳米抗体与人Fc融合蛋白在毕赤酵母和HEK293细胞中的表达、纯化和性质比较
|
摘要
通过对不同系统表达的肿瘤标记物癌胚抗原(CEA)纳米抗体与人Fc融合蛋白(11C12-Fc)的表达、纯化及抗体性质等的比较分析,比较不同表达系统的特点及其对融合抗体性质的影响;从而为CEA融合纳米抗体在体内检测方面的应用提供理论依据。构建两种11C12-Fc表达系统(Pichia pastoris和HEK293)相应的表达载体和菌株,分别进行诱导表达、分离纯化;并对其纯化产物的生物学活性等进行初步研究。成功构建了毕赤酵母、HEK293细胞两种CEA纳米抗体融合蛋白(11C12-Fc)表达系统并实现11C12-Fc的胞外分泌表达;通过protein A柱及阴离子交换层析纯化,获得了较高纯度和浓度的11C12-Fc样品;通过突变糖基化位点的方式有效去除了毕赤酵母表达11C12的过度糖基化作用;并且其抗体亲和力较突变前未发生明显改变,与HEK293细胞表达的11C12-Fc都表现出较高的亲和力。毕赤酵母和HEK293系统均能够表达具有较高生物活性的11C12-Fc,后续的科研或生产可根据实际需要和条件来合理选择表达系统。为后续的纳米抗体融合蛋白规模化生产、体内诊断与靶向治疗的应用提供了理论和技术参考。
Expression,purification and functional characterization of anti-CEA nanobody-Fc fusion protein( 11 C12-Fc) in Pichia pastoris and HEK293 Cell were studied,and their effects on the properties of fusion antibodies were compared and sunmarized. These results provided theoretical basis of CEA fusion nanobodies in vivo detection. Two expression vectors and strains were constucted corresponding to expression system( Pichia pastoris and HEK293) of 11 C12-Fc. Subsequently,11 C12-Fc of different expression system were expressed,purified respectively,and their biological activities of the purified products were studied preliminarily. Expression systems( Pichia pastoris and HEK293) of anti-CEA nanobody fusion proteins( 11 C12-Fc) were successfully constructed,and11 C12-Fc samples of high purity and concentration were obtained with purified by protein a column and anion exchange chromatography; The hyperglycosylation of 11 C12 in Pichia pastoris was removed effectively by glycosylation sites mutation,and its antibody affinity did not change significantly compared to original antibody. In addition,these 11 C12-Fc expressed by Pichia pastoris and HEK293 cell showed higher affinity. Both Pichia pastoris and HEK293 system are able to express 11 C12-Fc with high biological activity so that follow-up research or production can be based on actual needs and conditions to choose a reasonable expression system. This study provides a theoretical and technical reference for the subsequent mass production,in vivo diagnosis,and targeted therapy of nanobody fusion protein.
Expression,purification and functional characterization of anti-CEA nanobody-Fc fusion protein( 11 C12-Fc) in Pichia pastoris and HEK293 Cell were studied,and their effects on the properties of fusion antibodies were compared and sunmarized. These results provided theoretical basis of CEA fusion nanobodies in vivo detection. Two expression vectors and strains were constucted corresponding to expression system( Pichia pastoris and HEK293) of 11 C12-Fc. Subsequently,11 C12-Fc of different expression system were expressed,purified respectively,and their biological activities of the purified products were studied preliminarily. Expression systems( Pichia pastoris and HEK293) of anti-CEA nanobody fusion proteins( 11 C12-Fc) were successfully constructed,and11 C12-Fc samples of high purity and concentration were obtained with purified by protein a column and anion exchange chromatography; The hyperglycosylation of 11 C12 in Pichia pastoris was removed effectively by glycosylation sites mutation,and its antibody affinity did not change significantly compared to original antibody. In addition,these 11 C12-Fc expressed by Pichia pastoris and HEK293 cell showed higher affinity. Both Pichia pastoris and HEK293 system are able to express 11 C12-Fc with high biological activity so that follow-up research or production can be based on actual needs and conditions to choose a reasonable expression system. This study provides a theoretical and technical reference for the subsequent mass production,in vivo diagnosis,and targeted therapy of nanobody fusion protein.
引文
1张静.CEA和CA19-9联合检测在直肠癌诊断及疗效评估中的应用价值.实用检验医师杂志,2016;8(2):91-93Zhang Jing.The value of CEA and CA19-9 in rectal cancer diagnosis and therapeutic effect evaluation.Journal of Clinical Pathologist,2016;8(2):91-93
2王志忠,张婷,王钦媛,等.癌胚抗原对直结肠偶发高代谢灶良恶性判断.科学技术与工程,2016;16(22):162-164Wang Zhizhong,Zhang Ting,Wang Qinyuan,et al.The application value of serum CEA in incidental focal hypermetabolic colorectal lesions.Science Technology and Engineering,2016;16(22):162-164
3 Holliger P,Hudson P J.Engineered antibody fragments and the rise of single domains.Nat Biotechnol,2005;23(9):1126-1136
4 Revets H,De Baetselier P,Muyldermans S.Nanobodies as novel agents for cancer therapy.Expert Opin Biol Ther,2005;5(1):111-124
5 Muyldermans S,Baral T N,Retamozzo V C,et al.Camelid immunoglobulins and nanobody technology.Vet Immunol Immunopathol,2009;128(1-3):178-183
6 Cui M.Past and recent progress of molecular imaging probes forβ-amyloid plaques in the brain.Curr Med Chem,2014;21(1):82-112
7 Campuzano S,Salema V,Moreno G M,et al.Disposable amperometric magnetoimmunosensors using nanobodies as biorecognition element:Determination of fibrinogen in plasma.Biosens Bioelectron,2013;52:255-260
8孔庆明,姚亚波,陈睿,等.纳米抗体及其在诊断检测中的研究进展.生物工程学报,2014;30(9):1351-1361Kong Qingming,Yao Yabo,Chen Rui,et al.Progress in nanobody and its application in diagnosis.Chin J Biotech,2014;30(9):1351-1361
9 Hassanzadeh G G,Devoogdt N,De Pauw P,et al.Nanobodies and their potential applications.Nanomedicine(Lond),2013;8(6):1013-1026
10 Capon D J,Chamow S M,Mordenti J,et al.Designing CD4 immunoadhesins for AIDS therapy.Nature,1989;337(6207):525-531
11 Chamow S M,Ashkenazi A.Immunoadhesins:principles and applications.Trends Biotechnogy,1996;14(2):52-60
12牛舜,张芳琳,李璞媛,等.人整合素β3亚基毕赤酵母表达载体的构建及鉴定.科学技术与工程,2007;7(15):3660-3664Niu Shun,Zhang Fanglin,Li Puyuan,et al.Construction and identification of human integrinβ3in pichia expression system.Science Technology and Engineering,2007;7(15):3660-3664
13 Thomas P,Smart T G.HEK293 cell line:A vehicle for the expression of recombinant proteins.Journal of Pharmacological&Toxicological Methods,2005;51(3):187-200
14 Tate C G,Haase J,Baker C,et al.Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter.Biochimica et Biophysica Acta,2003;1610(1):141-153
15 Chollangi S,Parker R,Singh N,et al.Development of robust antibody purification by optimizing protein-A chromatography in combination with precipitation methodologies.Biotechnol Bioeng,2015;112(11):2292-2304
16 Bager R,Johansen J S,Jensen J K,et al.Protein conformational change delayed by steric hindrance from an N-linked glycan.J Mol Biol,2013;425(16):2867-2877
2王志忠,张婷,王钦媛,等.癌胚抗原对直结肠偶发高代谢灶良恶性判断.科学技术与工程,2016;16(22):162-164Wang Zhizhong,Zhang Ting,Wang Qinyuan,et al.The application value of serum CEA in incidental focal hypermetabolic colorectal lesions.Science Technology and Engineering,2016;16(22):162-164
3 Holliger P,Hudson P J.Engineered antibody fragments and the rise of single domains.Nat Biotechnol,2005;23(9):1126-1136
4 Revets H,De Baetselier P,Muyldermans S.Nanobodies as novel agents for cancer therapy.Expert Opin Biol Ther,2005;5(1):111-124
5 Muyldermans S,Baral T N,Retamozzo V C,et al.Camelid immunoglobulins and nanobody technology.Vet Immunol Immunopathol,2009;128(1-3):178-183
6 Cui M.Past and recent progress of molecular imaging probes forβ-amyloid plaques in the brain.Curr Med Chem,2014;21(1):82-112
7 Campuzano S,Salema V,Moreno G M,et al.Disposable amperometric magnetoimmunosensors using nanobodies as biorecognition element:Determination of fibrinogen in plasma.Biosens Bioelectron,2013;52:255-260
8孔庆明,姚亚波,陈睿,等.纳米抗体及其在诊断检测中的研究进展.生物工程学报,2014;30(9):1351-1361Kong Qingming,Yao Yabo,Chen Rui,et al.Progress in nanobody and its application in diagnosis.Chin J Biotech,2014;30(9):1351-1361
9 Hassanzadeh G G,Devoogdt N,De Pauw P,et al.Nanobodies and their potential applications.Nanomedicine(Lond),2013;8(6):1013-1026
10 Capon D J,Chamow S M,Mordenti J,et al.Designing CD4 immunoadhesins for AIDS therapy.Nature,1989;337(6207):525-531
11 Chamow S M,Ashkenazi A.Immunoadhesins:principles and applications.Trends Biotechnogy,1996;14(2):52-60
12牛舜,张芳琳,李璞媛,等.人整合素β3亚基毕赤酵母表达载体的构建及鉴定.科学技术与工程,2007;7(15):3660-3664Niu Shun,Zhang Fanglin,Li Puyuan,et al.Construction and identification of human integrinβ3in pichia expression system.Science Technology and Engineering,2007;7(15):3660-3664
13 Thomas P,Smart T G.HEK293 cell line:A vehicle for the expression of recombinant proteins.Journal of Pharmacological&Toxicological Methods,2005;51(3):187-200
14 Tate C G,Haase J,Baker C,et al.Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter.Biochimica et Biophysica Acta,2003;1610(1):141-153
15 Chollangi S,Parker R,Singh N,et al.Development of robust antibody purification by optimizing protein-A chromatography in combination with precipitation methodologies.Biotechnol Bioeng,2015;112(11):2292-2304
16 Bager R,Johansen J S,Jensen J K,et al.Protein conformational change delayed by steric hindrance from an N-linked glycan.J Mol Biol,2013;425(16):2867-2877