[1]余艳玲,冯鹏霏,潘传燕,等.尼罗罗非鱼TGF-β1蛋白原核表达及多克隆抗体的制备[J].江苏农业学报,2019,(06):1407-1412.[doi:doi:10.3969/j.issn.1000-4440.2019.06.020]
　YU Yan-ling,FENG Peng-fei,PAN Chuan-yan,et al.Prokaryotic expression and polyclonal antibodies preparation of TGF-β1 protein in Nile tilapia[J].,2019,(06):1407-1412.[doi:doi:10.3969/j.issn.1000-4440.2019.06.020]
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尼罗罗非鱼TGF-β1蛋白原核表达及多克隆抗体的制备()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2019年06期
页码:
1407-1412
栏目:
畜牧兽医·水产养殖
出版日期:
2019-12-31

文章信息/Info

Title:
Prokaryotic expression and polyclonal antibodies preparation of TGF-β1 protein in Nile tilapia
作者:
余艳玲; 冯鹏霏; 潘传燕; 陈晓汉; 林勇; 张永德; 罗洪林
(广西水产科学研究院广西水产遗传育种与健康养殖重点实验室，广西南宁530021)
Author(s):
YU Yan-ling; FENG Peng-fei; PAN Chuan-yan; CHEN Xiao-han; LIN Yong; ZHANG Yong-de; LUO Hong-lin
(Guangxi Key Laboratory for Aquatic Genetic Breeding and Healthy Aquaculture, Guangxi Academy of Fishery Sciences, Nanning 530021, China)
关键词:
罗非鱼; TGF-β1蛋白; 原核表达; 多克隆抗体
Keywords:
tilapia; TGF-β1 protein; prokaryotic expression; polyclonal antibody
分类号:
S965.125
DOI:
doi:10.3969/j.issn.1000-4440.2019.06.020
文献标志码:
A
摘要:
为制备尼罗罗非鱼TGF-β1多克隆抗体，采用同源重组技术构建尼罗罗非鱼pET-B2m-TGF-β1原核表达载体，转入大肠杆菌B21中诱导表达，将重组表达蛋白质纯化后免疫大耳兔制备多克隆抗体，采用Western Blot和ELISA检测抗体的特异性和效价。结果表明，构建的pET-B2m-TGF-β1原核表达载体经诱导表达获得了分子量为5.2×104的重组蛋白质，免疫大耳兔获得效价为1∶2 048 000的抗尼罗罗非鱼TGF-β1多克隆抗血清，该抗体能够特异性地识别原核表达的TGF-β1蛋白。
Abstract:
To prepare the polyclonal antibody against TGF-β1 of Nile tilapia, the TGF-β1 gene of Nile tilapia was transferred into the prokaryotic expression vector pET-B2m using homologous recombination technology, and transferred into Escherichia coli B21 for expression. The expressed TGF-β1 protein was purified and used to immunize rabbits for the preparation of polyclonal antibodies. The specificity and potency of the antibody were detected by Western blot and ELISA. The results showed that the pET-B2m-TGF-β1 prokaryotic expression vector was successfully constructed, and the recombinant protein with the molecular weight of 5.2×104 was obtained by inducing expression. The anti-Nile tilapia TGF-β1 polyclonal antiserum with a potency of 1∶2 048 000 was obtained from the immunized rabbit. The antibody could specifically recognize the prokaryotically expressed TGF-β1 protein.

参考文献/References:

[1]MASSAGU J. TGF-β signal transduction[J]. Annual Review of Biochemistry, 1998, 67: 753-791.
[2]STEPHEN T L, RUTKOWSKI M R, ALLEGREZZA M J, et al. Transforming growth factor β-mediated suppression of antitumor T cells requires FoxP1 transcription factor expression[J]. Immunity, 2014, 41(3): 427-439.
[3]BARCELLOS-HOFF M H, CUCINOTTA F A. New tricks for an old fox: impact of TGFβ on the DNA damage response and genomic stability[J]. Science Signaling, 2014, 7(341): re5.
[4]HAN A, ZHAO H, LI J, et al. ALK5-mediated transforming growth factor β signaling in neural crest cells controls craniofacial muscle development via tissue-tissue interactions[J]. Molecular and Cellular Biology, 2014, 34(16): 3120-3131.
[5]FENG Y, YUAN F, GUO H, et al. TGF-β1 enhances SDF-1-induced migration and tube formation of choroid-retinal endothelial cells by up-regulating CXCR4 and CXCR7 expression[J]. Molecular and Cellular Biochemistry, 2014, 397(1/2): 131-138.
[6]WIENER Z, BAND A M, KALLIO P, et al. Oncogenic mutations in intestinal adenomas regulate Bim-mediated apoptosis induced by TGF-β[J]. Proceedings of the National Academy of Sciences, 2014, 111(21): 2229-2236.
[7]MUOZ-ESPN D, CAAMERO M, MARAVER A, et al. Programmed cell senescence during mammalian embryonic development[J]. Cell, 2013, 155(5): 1104-1118.
[8]GRAFE I, YANG T, ALEXANDER S, et al. Excessive transforming growth factor-β signaling is a common mechanism in osteogenesis imperfecta[J]. Nature Medicine, 2014, 20(6): 670-675.
[9]WANG J, WANG Y, WANG Y, et al. Transforming growth factor β-regulated microRNA-29a promotes angiogenesis through targeting the phosphatase and tensin homolog in endothelium[J]. Journal of Biological Chemistry, 2013, 288(15): 10418-10426.
[10]PARK J G, LEE D H, MOON Y S, et al. Reversine increases the plasticity of lineage-committed preadipocytes to osteogenesis by inhibiting adipogenesis through induction of TGF-β pathway in vitro. [J]. Biochemical and Biophysical Research Communications, 2014, 446(1): 30-36.
[11]SULAIMAN W, NGUYEN D H. Transforming growth factor beta 1, a cytokine with regenerative functions[J]. Neural Regeneration Research, 2016, 11(10): 1549-1552.
[12]WANG Y W, LIOU N H, CHERNG J H, et al. siRNA-targeting transforming growth factor-β type I receptor reduces wound scarring and extracellular matrix deposition of scar tissue[J]. Journal of Investigative Dermatology, 2014, 134(7): 2016-2025.
[13]MUSTOE T A, PIERCE G F, THOMASON A, et al. Accelerated healing of incisional wounds in rats induced by transforming growth factor-beta[J]. Science, 1987, 237(4820): 1333-1336.
[14]GORDON K J, BLOBE G C. Role of transforming growth factor-β superfamily signaling pathways in human disease[J]. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 2008, 1782(4): 197-228.
[15]CHANG A S, HATHAWAY C K, SMITHIES O, et al. Transforming growth factor-β1 and diabetic nephropathy[J]. American Journal of Physiology-Renal Physiology, 2015, 310(8): 689-696.
[16]ATTISANO L, WRANA J L. Signal transduction by the TGF-β superfamily[J]. Science, 2002, 296(5573): 1646-1647.
[17]DE CAESTECKER M. The transforming growth factor-β superfamily of receptors[J]. Cytokine & Growth Factor Reviews, 2004, 15(1): 1-11.
[18]MARIE J C, LIGGITT D, RUDENSKY A Y. Cellular mechanisms of fatal early-onset autoimmunity in mice with the T cell-specific targeting of transforming growth factor-β receptor[J]. Immunity, 2006, 25(3): 441-454.
[19]YANG M, ZHOU H. Grass carp transforming growth factor-β1 (TGF-β1): Molecular cloning, tissue distribution and immunobiological activity in teleost peripheral blood lymphocytes[J]. Molecular Immunology, 2008, 45(6): 1792-1798.
[20]YANG M, WANG X, CHEN D, et al. TGF-β1 exerts opposing effects on grass carp leukocytes: implication in teleost immunity, receptor signaling and potential self-regulatory mechanisms[J]. PLoS ONE, 2012, 7(4): e35011.
[21]WEI H, YANG M, ZHAO T, et al. Functional expression and characterization of grass carp IL-10: an essential mediator of TGF-β1 immune regulation in peripheral blood lymphocytes[J]. Molecular Immunology, 2013, 53(4): 313-320.
[22]WANG X, YANG X, WEN C, et al. Grass carp TGF-β1 impairs IL-1β signaling in the inflammatory responses: evidence for the potential of TGF-β1 to antagonize inflammation in fish[J]. Developmental & Comparative Immunology, 2016, 59: 121-127.
[23]LI L P, WANG R, LIANG W W, et al. Development of live attenuated Streptococcus agalactiae vaccine for tilapia via continuous passage in vitro[J]. Fish & Shellfish Immunology, 2015, 45(2): 955-963.
[24]ZHAN X, MA T, WU J, et al. Cloning and primary immunological study of TGF-β1 and its receptors TβR I/TβR II in tilapia (Oreochromis niloticus)[J]. Developmental & Comparative Immunology, 2015, 51(1): 134-140.
[25]张永德,林勇,冯鹏霏,等. 尼罗罗非鱼Lck多克隆抗体的制备及鉴定[J]. 南方农业学报, 2018(11): 2304-2310.
[26]PAUKNER R, STAUDIGL P, CHOOSRI W, et al. Expression, purification, and characterization of galactose oxidase of Fusarium sambucinum in E. coli[J]. Protein Expression and Purification, 2015, 108: 73-79.
[27]FERRER-MIRALLES N, SACCARDO P, CORCHERO J L, et al. General introduction: recombinant protein production and purification of insoluble proteins[M]. New York: Humana Press, 2015: 1-24.
[28]LILIE H, SCHWARZ E, RUDOLPH R. Advances in refolding of proteins produced in E. coli[J]. Current Opinion in Biotechnology, 1998, 9(5): 497-501.
[29]SRENSEN H P, MORTENSEN K K. Advanced genetic strategies for recombinant protein expression in Escherichia coli[J]. Journal of Biotechnology, 2005, 115(2): 113-128.
[30]VILLAVERDE A, CORCHERO J L, SERAS-FRANZOSO J, et al. Functional protein aggregates: just the tip of the iceberg[J]. Nanomedicine, 2015, 10(18): 2881-2891.
[31]CARRIO M, CUBARSI R, VILLAVERDE A. Fine architecture of bacterial inclusion bodies[J]. FEBS Letters, 2000, 471(1): 7-11.
[32]MITRAKI A, FANE B, HAASE-PETTINGELL C, et al. Global suppression of protein folding defects and inclusion body formation[J]. Science, 1991, 253(5015): 54-58.

备注/Memo

备注/Memo:
收稿日期：2019-03-22 基金项目：国家自然科学基金面上项目(31760765、31372553)；广西自然科学基金项目(2015GXNSFAA139068) 作者简介：余艳玲（1976-），女，陕西咸阳人，工程师，主要从事水产动物分子生物学研究。(E-mail)yaeling@126.com 通讯作者：罗洪林, (E-mail)541365548@qq.com
更新日期/Last Update: 2020-01-09