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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  2016, Vol. 36 Issue (5): 74-80    DOI: 10.13523/j.cb.20160511
研究报告     
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
陈华新1,2, 武静1,2,3, 赵瑾1,2, 姜鹏1,2
1. 中国科学院海洋研究所 实验海洋生物学重点实验室 青岛 266071;
2. 青岛海洋科学与技术国家实验室海洋生物学与生物技术功能实验室 青岛 266071;
3. 中国科学院大学 北京 100049
Expression and Characterization of Fusion Protein of Single-chain Variable Fragment of Alpha Fetoprotein and Allophycocyanin Alpha Subunit
CHEN Hua-xin1,2, WU Jing1,2,3, ZHAO Jin1,2, JIANG Peng1,2
1. Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China;
2. Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China;
3. University of Chinese Academy of Sciences, Beijing 100049, China
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摘要：

目的:构建多基因表达载体,在大肠杆菌中同时表达AFP单链抗体(scFv)和蓝藻别藻蓝蛋白α亚基脱辅基蛋白(apcA)组成的融合蛋白(scFv-apcA)、藻胆蛋白裂合酶(cpcS)及藻红蛋白生物合成酶(Ho1和pebS),获得共价结合藻红胆素的融合蛋白(scFv-apcA-PEB)。方法:利用融合PCR将scFv和apcA基因连接起来,形成scFv-apcA融合基因,并将该融合基因与cpcS克隆到表达载体pCDFDuet-1中;将Ho1和pebS基因克隆到表达载体pRSFDuet-1中。将两种载体共转化到大肠杆菌中,IPTG诱导重组蛋白表达,经亲和层析获得重组蛋白,通过光谱学分析和抗体竞争性抑制法,测定重组蛋白的生物学活性。结果:成功表达融合蛋白scFv-apcA-PEB,分子质量约为45kDa,与理论值相符,其最大吸收峰为549.5nm,最大荧光发射峰为560nm,竞争抑制ELISA法初步鉴定活性,竞争抑制率达到48%。结论:利用大肠杆菌表达系统,获得了同时具有荧光特性和免疫学活性的重组蛋白。
关键词： 融合蛋白;  单链抗体;  甲胎蛋白;  别藻蓝蛋白    
Abstract:

Objective:By using multiple gene expressing plasmids, fusion protein of single-chain variable fragment of alpha fetoprotein and allophycocyanin alpha subunit, phycobiliprotein lyase (cpcS), and enzymes (Ho1 and pebS) for conversion of heme to phycoerythrobilin were co-expressed in E. coli, with an aim to prepare fluorescent fusion protein which is covalently bound with phycoerythrobilin. Method:fragments of alpha fetoprotein and allophycocyanin alpha subunit were fused by fusion PCR. Then the fused gene, together with cpcS gene, were ligated into vector pCDFDuet-1. Ho1 and pebS genes were ligated into another vector pRSFDuet-1. The two vectors were then transformed into E. coli. Recombinant proteinwere expressed efficiently in E. coli by addition of IPTG and purified by affinity chromatography.The activities of the recombinant protein were evaluated by spectral analysis and competitive ELISA.Results:The recombinant protein, with a molecular weight approximately to 45.0kDa, has an absorbance maximum at 549.5nm and an emission maximum at 660nm. Competitive ELISA results showed that inhibition rate reached 48% when the concentration ratio of scFv and parent monoclonal antibodies was 16:1.Conclusion:A recombinant protein, which was fluorescent and immunologic active, was successfully expressed in E. coli.
Key words: Fusion protein    Single-chain variable fragment antibody    Alpha fetoprotein    Allophycocyanin
收稿日期: 2015-11-09 出版日期: 2016-01-26
ZTFLH:  Q51  
基金资助:

国家"863"计划(2014AA093505,2014AA093501),国家海洋公益性行业科研专项经费项目(201205027-2),国家自然科学基金(41276164)资助项目
通讯作者: 姜鹏     E-mail: jiangpeng@qdio.ac.cn
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引用本文:

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.

CHEN Hua-xin, WU Jing, ZHAO Jin, JIANG Peng. Expression and Characterization of Fusion Protein of Single-chain Variable Fragment of Alpha Fetoprotein and Allophycocyanin Alpha Subunit. China Biotechnology, 2016, 36(5): 74-80.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160511    或    https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I5/74

[1] MacColl R. Cyanobacterial phycobilisomes. Journal of Structure Biology,1998,124(2-3):311-334.
[2] Sekar S, Chandramohan M. Phycobiliproteins as a commodity: trends in applied research, patents and commercialization. Journal of Applied Phycology, 2008, 20: 113-136.
[3] 吴萍,顾铭,戚艺华,等.藻胆蛋白荧光探针及其标记.生命科学研究,2001,5(2): 109-113. Wu P, Gu M, Qi Y H, et al. Phycobiliprotein fluorescence probes and the labeling technique. Life Science Research, 2001, 5(2), 109-113.
[4] Chen H X, Lin H Z, Li F C, et al. Biosynthesis of a stable allophycocyanin beta subunit in metabolically engineered Escherichia coli. Journal of Bioscience and Bioengineering, 2013, 115(5):485-489.
[5] Liu S F, Chen Y J, Lu Y D, et al. Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli. Photosynth Research, 2010, 105(2): 135-142.
[6] Biswas A, Vasquez Y M, Dragomani T M, et al. Biosynthesis of cyanobacterial phycobiliproteins in Escherichia coli: chromophorylation efficiency and specificity of all bilin lyases from Synechococcu ssp. strain PCC 7002. Applied and Environmental Microbiology, 2010, 76(9): 2729-2739.
[7] Alvey R M, Biswas A, Schluchter W M. Attachment of noncognate chromophores to cpcA of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 by heterologous expression in Escherichia coli. Biochemistry, 2011, 50(22): 4890-4902.
[8] 秦丽莉,张春明.单链抗体研究进展及其在医学中的应用.国外医学(放射医学核医学分册), 2005, 29(6): 255-257. Qin L L, Zhang C M. The research progress and medicine application of the ScFv antibody. International Journal of Radiation Medicine and Nuclear Medicine, 2005, 29(6): 255-257.
[9] Zhao K H, Su P, Tu J M, et al. Phycobilin:cystein-84 biliprotein lyase, a nearuniversal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins. Proceedings of the National Academy of Sciences, 2007, 104(36): 14300-14305.
[10] Cabrita L D, Bottomley S P. Protein expression and refolding-A practical guide to getting the most out of inclusion bodies. Biotechnology Annual Review, 2004, 10: 31-50.
[11] 牛坚, 张业伟, 刘斌. 抗人AFP单链抗体的制备、鉴定及其免疫学活性的测定.肝胆胰外科杂志,2011, 23(4): 325-328. Niu J, Zhang Y W, Liu B. Construction, identification and determination of immunologic activity of murine anti-AFP ScFv. Journal of Hepatopancreatobiliary Surgery, 2011, 23(4): 325-328.
[12] 姬晓南, 张瑜, 冯丽亚, 等. 抗人甲胎蛋白单链抗体融合蛋白的构建、表达及初步鉴定. 药物生物技术, 2013, 20(6): 471-474. Ji X N, Zhang Y, Feng Y L, et al. Construction,expression and identification of anti-human AFP singledomain antibody fusion protein. Pharmaceutical Biotechnology, 2013, 20(6): 471-474.
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