1种高效纳米抗体生产方法的建立

doi:10.11843/j.issn.0366-6964.2013.09.022

Abstract

Abstract:

The aim of this study was to establish a simple, efficient and inexpensive strategy for expression and purification of Non-taged nanobodies. Chose nanobodies of porcine circovirus type 2 (PCV2) Cap protein as the target protein, three plasmids with N-terminus fusion tags pHSIE-Nb(His-Sumo-Intein）, pHSIE-intein-Nb（His-Intein）and pET32a-intein-Nb（His-Trx-Intein）were constructed and their effects were compared. The recombinant three plasmids were transformed into E. coil BL21(+) and induced by IPTG respectively. The expression and solubility of target proteins were tested under different conditions, and the favorable tags were selected. Recombinant proteins were purified by Ni-NTA column chromatography, and tags were removed by Intein as self-cleavaged, then target protein were prepared, based on which a double antibody sandwich ELISA method was developed. All these three different tagged nanobodies could be successfully expressed. A series of experiments lead to the finding that the placement of His-Trx-Intein before the Nb is the best strategy in availing the soluble expression of the tagged protein. After on-column cleavage was preceded at pH7.0，being incubated at 25 ℃ for 24 h, the recombinant un-tagged Nb were released. The results of double antibody sandwich ELISA showed that the Nb protein could react specifically with cap protein, and there was no significant difference (P>0.05) between positive control group and nanobody group, which suggested that this method of preparation of nanobodies was successful. An efficient expression and simple purification method of preparation of nanobodies was successfully established, which was efficient soluble expression, and simple purification, and no tagged active nanobodies could be prepared. The study provides a technical support for the further theoretical research and production in nanobodies.

CLC Number:

Q789

HE Sheng-fang, FU Xiang-jing, LIU Yang-kun, MU Guo-hui, LIU Hai-jin,WANG Xing-long, DU En-qi, YANG Zeng-qi. Establishing a Method for Highly Efficient Producing Nanometer Antibodies[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(9): 1487-1493.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2013.09.022

https://www.xmsyxb.com/EN/Y2013/V44/I9/1487

References

［1］杨珂，王冬.纳米抗体及其应用［J］.细胞与分子免疫学杂志，2008，24(4): 425-427.

［2］NGUYEN V K, HAMERS R, WYNS L, et al. Loss of splice consensus signal is responsible for the removal of the entire C(H)1 domain of the functional camel IGG2A heavy-chain antibodies［J］. Mol Immunol, 1999, 36(8): 515-524.

［3］DI GUAN C, LI P, RIGGS P D, et al. Vectorsthat facilitate the expression and purification of foreign peptides inEscherichia coliby fusion to maltose_binding protein［J］.Gene, 1988, 67(1): 21-30.

［4］KIM S J, PARK Y, HONG H J. Antibody engineering for the development of therapy antibodies［J］. Mol Cell, 2005, 20(1): 17-29.

［5］XIA L, WU B, CHENG Y T. Preparation and characterization of recombinant VHH zntibody against H5N1 hemagglutinin from avian influenza virus［J］. Chn J Immunol, 2012, 28(2): 137-141.

［6］PANT N, HULTBERG A, ZHAO Y, et al. Lactobacilli expressing variable domain of llama heavy-chain antibody fragments (lactobodies) confer protection against rotavirus-induced diarrhea［J］. J Infect Dis, 2006, 194(11):1580-1588.

［7］HAMERS-CASTERMAN C, ATARHOUCH T, MUYLDERMANS S, et al. Naturally occurring antibodies devoid of light chains ［J］. Nature, 1993, 363(6428): 446-448.

［8］TERPE K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems［J］. Appl Microbiol Biotechnol, 2003, 60(5): 523-533.

［9］DING F X, YAN H L, MEI Q, et al. Novel Cheap and effective fusion expression system for the production of recombinant proteins ［J］. Appl Microbiol Biotechnol, 2007, 77(2): 483-488.

［10］MOHANTY A K, WIENER M C. Membrane protein expression and production: Effects of polyhistidine Tag length and position［J］. Protein Expr Purif, 2004, 33(2):311-325.

［11］姜媛媛, 尹成凯, 李晋南, 等. 利用SUMO融合系统高效表达可溶性重组蛋白的研究［J］. 东北农业大学学报, 2008, 39(10):57-62.

［12］MALAKHOV M P, MATTERN M R, MALAKHOVA O A, et al. SUMO fusions and SUMO specificprotease for efficientexpression and purification of proteins［J］. J Struct Funct Genomics, 2004, 5(1-2): 75-86.

［13］CHONG S, MERSHA F B, COMB D G, et al. Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element［J］.Gene, 1997, 192(2): 277-281.

［14］刘雷, 尹钧. 硫氧还蛋白的研究［J］.东北农业大学学报, 2003, 34(2)：219-225.

［15］MOUAHEB N, THOMAS D, VERDOUCQ L, et al. In vivo functional discrimination between plant thioredoxins by heterologous expression in the yeast Saccharomyces cerevisiae ［J］. Proc Natl Acad Sci U S A, 1998, 95(6): 3312-3317

［16］宋利,萍黄华. 蛋白质剪切及其应用［J］.生物工程学报，2003，19（2）:249-250.

［17］WANG Z, LI N, WANG Y. Ubiquitin-intein and SUMO2-intein fusion systems for enhanced protein. production and purification［J］. Protein Expr Purif, 2012, 82(1): 174-178.

［18］XING L, WU W, ZHOU B H, et al. Streamlined protein expression and purification using cleavable self-aggregating tags［J］. Microb Cell Fact, 2011, 10:42.

［19］BANKI M R, WOOD D W. Inteins and affinity resin substitutes for protein purification and scale up［J］. Microb Cell Fact, 2005, 4(1): 32.

［20］姚广新，张怡轩，胡双纲. Intein 介导的重组人AR DBD的原核可溶性表达、纯化以及活性鉴定［J］. 中国生物工程杂志, 2011, 31(4): 7-11.

［21］MIRANDA-VIZUETE A, DAMDIMOPOULOS A E, PEDRAJAS J R, et al. Human mitochondrial thioredoxin reductase cDNAclonging,expression and genomic organization［J］. Eur J Biochem, 1999, 261(2): 405-412.

[1]	XU Qian-ru, ZHANG Er-qin, GUO Jun-qing, YANG Ji-fei, LIU Yun-chao, WANG Ai-ping, WANG Li, WAN Bo, WANG Lei, JIANG Da-wei, DENG Rui-guang, ZHANG Gai-ping. Expression and Detection of NDV-F Protein in Rice Endosperm [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(6): 1167-1172.
[2]	HU Xiang-yun,GAO Xiao-long，FU Xiang-jing，LIU Dan-dan，FAN Wen-tao，WANG Yan-ping，DU En-qi，WANG Xing-long，DANG Ru-yi，YANG Zeng-qi. Construction and Characterization of a Naive Camelus Bactrianus Variable Domain of Heavy Chain of Heavy-Chain Antibody（VHH）Yeast Two-Hybrid Library [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(6): 1071-1076.
[3]	WANG Xiang-peng, WEI Rui-fang, XIAO Shu-qi, ZHOU En-min. Generation of a Porcine Alveolar Macrophage Cell Line Stably Expressing CD163 by Lentiviral Vector for the Production of Porcine Reproductive and RespiratorySyndrome Virus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(11): 1797-1804.

Establishing a Method for Highly Efficient Producing Nanometer Antibodies

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 3

Recommended Articles

Metrics

Comments