抗CEACAM5纳米抗体在大肠杆菌中的高效周质表达
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摘要
纳米抗体具有相对分子质量小、稳定性高、特异性强以及能够穿过血脑屏障等众多优势,而且其能够通过大肠杆菌、酵母等简单微生物大量表达。大肠杆菌周质表达纳米抗体是目前纳米抗体制备主要的方法之一,但是纳米抗体的表达水平还相对较低。以pET22b和pMES4为载体构建了两种重组表达质粒,并且比较了他们在不同宿主菌中表达纳米抗体的情况。结果表明,以pMES4为载体的大肠杆菌能够在周质中可溶性表达纳米抗体,而以pET22b为载体的大肠杆菌表达的纳米抗体无法分泌到周质空间中。随后,通过IPTG浓度优化及5 L发酵罐过程,控制实现了纳米抗体在大肠杆菌周质中的表达量达到308. 32 mg/L。研究提供了一种高效表达纳米抗体的方法,为纳米抗体的规模化制备奠定了技术基础。
Nanobodies offer multiple advantages over conventional antibodies in terms of size,stability,solubility,immunogenicity,and production costs,with improved tumor uptake and blood clearance. Additionally,the recombinant expression of nanobodies is robust in various expression systems,such as Escherichia coli,Saccharomyces cerevisiae and Pichia pastoris. Periplasmic expression of nanobody was one of main nanobody production ways in E.coli. However,level of nanobody expression was low in E. coli periplasm. In the study,two recombinant expression vectors,pET22b-2D5 and pMES4-2D5,were constructed for nanobody expression in E. coli periplasm. The results indicated that nanobody 2D5 could be efficiently expression in E. coli periplasm with pMES4-2D5 vector.With optimizations,a high nanobody yield of 308. 2 mg/L was achieved in a 5 L-fermentor. The results of this study provide an efficient way to produce nanobody,and laid a technical foundation for scale-production of nanobodies.
Nanobodies offer multiple advantages over conventional antibodies in terms of size,stability,solubility,immunogenicity,and production costs,with improved tumor uptake and blood clearance. Additionally,the recombinant expression of nanobodies is robust in various expression systems,such as Escherichia coli,Saccharomyces cerevisiae and Pichia pastoris. Periplasmic expression of nanobody was one of main nanobody production ways in E.coli. However,level of nanobody expression was low in E. coli periplasm. In the study,two recombinant expression vectors,pET22b-2D5 and pMES4-2D5,were constructed for nanobody expression in E. coli periplasm. The results indicated that nanobody 2D5 could be efficiently expression in E. coli periplasm with pMES4-2D5 vector.With optimizations,a high nanobody yield of 308. 2 mg/L was achieved in a 5 L-fermentor. The results of this study provide an efficient way to produce nanobody,and laid a technical foundation for scale-production of nanobodies.
引文
1 Jemal A,Bray F,Center M M,et al. Global cancer statistics[J].Ca a Cancer Journal for Clinicians,2015,61(2):69-90
2 Rees M,Tekkis P P,Welsh F K,et al. Evaluation of long-term survival after hepatic resection for metastatic colorectal cancer:A multifactorial model of 929 patients[J]. Annals of Surgery,2008,247(1):125-135
3 Gold P,Freedman S O. Specific carcinoembryonic antigens of the human digestive system[J]. Journal of Experimental Medicine,1965,122(3):467-481
4 Balint J P,Gabitzsch E S,Rice A,et al. Extended evaluation of a phase 1/2 trial on dosing,safety,immunogenicity,and overall survival after immunizations with an advanced-generation CEA vaccine in late-stage colorectal cancer[J]. Cancer Immunology Immunotherapy,2015,64(8):977-987
5 Bilusic M,Heery C R,Arlen P M,et al. Phase I trial of a recombinant yeast-CEA vaccine(GI-6207)in adults with metastatic CEA-expressing carcinoma[J]. Cancer Immunology Immunotherapy,2014,63(3):225-234
6 Marcela D C,Alberto C,Luigi A,et al. Phase 1 studies of the safety and immunogenicity of electroporated HER2/CEA DNA vaccine followed by adenoviral boost immunization in patients with solid tumors[J]. Journal of Translational Medicine,2013,11(1):62
7 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[J].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[J]. Science Technology and Engineering,2016,16(22):162-164
8 王秀红,周素芳.基因工程抗体研究进展[J].生物技术通讯,2007,18(2):304-306Wang Xiuhong,Zhou Sufang. Advances in research on genetically engineered antibody[J]. Letters in Biotechnology,2007,18(2):304-306
9 胡春艳,刘全忠.单克隆抗体制备技术及应用的研究进展[J].安徽预防医学杂志,2008,(2):116-118Hu Chunyan,Liu Quanzhong. Recent advances in the generation and application of the monoclonal antibody[J]. Anhui Journal of Preventive Medicine,2008,(2):116-118
10 孙思凡,张部昌,靳彦文.治疗性单克隆抗体研究进展[J].生物技术通讯,2009,20(2):258-262Sun Sifan,Zhang Buchang,Jin Yanwen. Advances on therapeutic monoclonal antibody[J]. Letters in Biotechnology,2009,20(2):258-262
11 Hamers-Casterman C,Atarhouch T,Muyldermans S,et al. Naturally occurring antibodies devoid of light chains[J]. Nature,1993,363(6428):446-448
12 Cortez-Retamozo V,Backmann N,Senter P D,et al. Efficient cancer therapy with a nanobody-based conjugate[J]. Cancer Research,2004,64(8):2853-2857
13 Coppieters K,Dreier T,Silence K,et al. Formatted anti-tumor necrosis factorαVHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collageninduced arthritis[J]. Arthritis&Rheumatology,2010,54(6):1856-1866
14 Ghahroudi M,Desmyter A,Wyns L,et al. Selection and identification of single domain antibody fragments from camel heavy-chain antibodies[J]. FEBS Letters,1997,414(3):521-526
15 Frenken L G,Vanderlinden R H,Hermans P W,et al. Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae[J]. Journal of Biotechnology,2000,78(1):11-21
16 Maggi M,Scotti C. Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies[J].Data in Brief,2017,12(C):132-137
17 Ta D T,Redeker E S,Billen B,et al. An efficient protocol towards site-specifically clickable nanobodies in high yield:Cytoplasmic expression in escherichia coli combined with intein-mediated protein ligation[J]. Protein Engineering Design&Selection Peds,2015,28(10):351-363
2 Rees M,Tekkis P P,Welsh F K,et al. Evaluation of long-term survival after hepatic resection for metastatic colorectal cancer:A multifactorial model of 929 patients[J]. Annals of Surgery,2008,247(1):125-135
3 Gold P,Freedman S O. Specific carcinoembryonic antigens of the human digestive system[J]. Journal of Experimental Medicine,1965,122(3):467-481
4 Balint J P,Gabitzsch E S,Rice A,et al. Extended evaluation of a phase 1/2 trial on dosing,safety,immunogenicity,and overall survival after immunizations with an advanced-generation CEA vaccine in late-stage colorectal cancer[J]. Cancer Immunology Immunotherapy,2015,64(8):977-987
5 Bilusic M,Heery C R,Arlen P M,et al. Phase I trial of a recombinant yeast-CEA vaccine(GI-6207)in adults with metastatic CEA-expressing carcinoma[J]. Cancer Immunology Immunotherapy,2014,63(3):225-234
6 Marcela D C,Alberto C,Luigi A,et al. Phase 1 studies of the safety and immunogenicity of electroporated HER2/CEA DNA vaccine followed by adenoviral boost immunization in patients with solid tumors[J]. Journal of Translational Medicine,2013,11(1):62
7 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[J].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[J]. Science Technology and Engineering,2016,16(22):162-164
8 王秀红,周素芳.基因工程抗体研究进展[J].生物技术通讯,2007,18(2):304-306Wang Xiuhong,Zhou Sufang. Advances in research on genetically engineered antibody[J]. Letters in Biotechnology,2007,18(2):304-306
9 胡春艳,刘全忠.单克隆抗体制备技术及应用的研究进展[J].安徽预防医学杂志,2008,(2):116-118Hu Chunyan,Liu Quanzhong. Recent advances in the generation and application of the monoclonal antibody[J]. Anhui Journal of Preventive Medicine,2008,(2):116-118
10 孙思凡,张部昌,靳彦文.治疗性单克隆抗体研究进展[J].生物技术通讯,2009,20(2):258-262Sun Sifan,Zhang Buchang,Jin Yanwen. Advances on therapeutic monoclonal antibody[J]. Letters in Biotechnology,2009,20(2):258-262
11 Hamers-Casterman C,Atarhouch T,Muyldermans S,et al. Naturally occurring antibodies devoid of light chains[J]. Nature,1993,363(6428):446-448
12 Cortez-Retamozo V,Backmann N,Senter P D,et al. Efficient cancer therapy with a nanobody-based conjugate[J]. Cancer Research,2004,64(8):2853-2857
13 Coppieters K,Dreier T,Silence K,et al. Formatted anti-tumor necrosis factorαVHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collageninduced arthritis[J]. Arthritis&Rheumatology,2010,54(6):1856-1866
14 Ghahroudi M,Desmyter A,Wyns L,et al. Selection and identification of single domain antibody fragments from camel heavy-chain antibodies[J]. FEBS Letters,1997,414(3):521-526
15 Frenken L G,Vanderlinden R H,Hermans P W,et al. Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae[J]. Journal of Biotechnology,2000,78(1):11-21
16 Maggi M,Scotti C. Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies[J].Data in Brief,2017,12(C):132-137
17 Ta D T,Redeker E S,Billen B,et al. An efficient protocol towards site-specifically clickable nanobodies in high yield:Cytoplasmic expression in escherichia coli combined with intein-mediated protein ligation[J]. Protein Engineering Design&Selection Peds,2015,28(10):351-363