STAT3纳米抗体的制备与功能鉴定

doi:10.16431/j.cnki.1671-7236.2023.03.026

摘要/Abstract

摘要： 【目的】获得抗信号转导和转录激活因子3(signal transducer and activator of transcription 3,STAT3)蛋白的特异性纳米抗体,通过验证该纳米抗体在免疫组织化学和Western blotting检测中的应用及对黑色素瘤细胞B16生长的作用,对其功能进行鉴定。【方法】通过噬菌体展示淘筛技术从羊驼B16纳米抗体文库中筛选抗STAT3纳米抗体,获得阳性克隆并测序;根据序列信息设计和构建pColdⅠ原核表达质粒,并通过大肠杆菌进行纳米抗体表达;通过镍柱、分子筛和超滤管进行纯化和浓缩;利用免疫组织化学和Western blotting验证纳米抗体是否结合脑组织中STAT3蛋白;采用CCK8法分析该纳米抗体对B16细胞增殖的影响,并用Western blotting检测其对其分子路径相关基因表达的影响。【结果】经过噬菌体展示淘筛及阳性克隆测序后,成功获得1株特异性结合STAT3蛋白的纳米抗体单克隆菌株,命名为STAT3-VHH-10B;将构建的该纳米抗体的pColdⅠ原核表达载体进行诱导表达和纯化,SDS-PAGE结果显示,成功得到大小约为15 ku的抗STAT3纳米抗体;免疫组织化学和Western blotting结果显示,纯化制备的纳米抗体可成功检测到大脑组织中STAT3的表达;细胞增殖检测结果显示,该纳米抗体对B16细胞增殖具有明显的抑制作用,并下调细胞增殖路径的主要基因CycD1、Bcl2、cMyc编码蛋白的表达。【结论】本研究成功筛选到1株抗STAT3蛋白的特异性纳米抗体,且该纳米抗体可用于免疫组织化学和Western blotting法检测组织中STAT3的表达,此外,通过与抗原结合后,抑制主要基因CycD1、Bcl2、cMyc的表达,从而抑制B16细胞增殖。本研究结果将为STAT3及其纳米抗体的功能研究提供新工具。

关键词: 纳米抗体; 信号转导和转录激活因子3(STAT3); 黑色素瘤; B16细胞

Abstract: 【Objective】 This study was aimed to obtain specific nanobody against signal transducer and activator of transcription 3 (STAT3) protein and then validate its use for immunohistochemistry and Western blotting,as well as the function on the cell growth of B16 melanoma cell.【Method】 Anti-STAT3 nanobodies were screened from B16 nanobody library from alpaca by phage display.After the positive clones were obtained and sequenced,one clone was ligated into the prokaryotic expression vector pColdⅠto construct the plasmid which was expressed by E.coli and purified by nickel columns,molecular sieves and ultrafiltration tubes.Immunohistochemistry and Western blotting were used to verify whether anti-STAT3 nanobody bound STAT3 protein in brain tissue and had the effects on the proliferation of B16 cells by CCK8 method and related genes expression on the proliferation pathway by Western blotting.【Result】 After phage display screening and sequencing of positive clones,a monoclonal strain of nanobody specifically binding STAT3 protein was successfully obtained and named as STAT3-VHH-10B.The plasmid of the ligation of STAT3-VHH-10B and pColdⅠwas expressed and then purified,which was verified by SDS-PAGE and showed anti-STAT3 nanobody with the size of about 15 ku.The results of immunohistochemistry and Western blotting showed that the purified anti-STAT3 nanobody could detect STAT3 expression in brain tissue.The results of cell proliferation test showed that anti-STAT3 nanobody could significantly inhibit the proliferation of B16 cells,and down regulate the protein expression of the main genes on the cell proliferation pathway,including CycD1, Bcl2 and cMyc.【Conclusion】 A specific anti-STAT3 nanobody was successfully screened and could be used for immunohistochemistry and Western blotting to detect the expression of STAT3 in tissues.Moreover,it could inhibit the proliferation of B16 cells by inhibiting the expression of main genes CycD1, Bcl2 and cMyc on the basis of binding with STAT3.The results would provide a new tool for the functional research of STAT3 and its nanobody.

Key words: nanobody; signal transducer and activator of transcription 3 (STAT3); melanoma; B16 cells

中图分类号:

S852.4

秦蓉芬, 贾琼, 于雷涛, 李佳敏, 迟志端, 范瑞文. STAT3纳米抗体的制备与功能鉴定[J]. 中国畜牧兽医, 2023, 50(3): 1118-1128.

QIN Rongfen, JIA Qiong, YU Leitao, LI Jiamin, CHI Zhirui, FAN Ruiwen. Preparation and Functional Identification of STAT3 Nanobody[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1118-1128.

参考文献

[1] CHAI E Z,SHANMUGAM M K,ARFUSO F,et al.Targeting transcription factor STAT3 for cancer prevention and therapy[J].Pharmacology & Therapeutics,2016,162:86-97.
[2] GARCIA R,JOVE R.Activation of STAT transcription factors in oncogenic tyrosine kinase signaling[J].Journal of Biomedical Science,1998,5(2):79-85.
[3] SHI S,MA H Y,ZHANG Z G.Clinicopathological and prognostic value of STAT3/p-STAT3 in cervical cancer:A meta and bioinformatics analysis[J].Pathology-Research and Practice,2021,227:153624.
[4] 陈越,季鸣,陈晓光.STAT3与肿瘤关系的研究进展[J].药学学报,2017,52(9):1351-1358. CHEN Y,JI M,CHEN X G.Research progress of relationship between STAT3 and tumor[J].Acta Pharmaceutica Sinica,2017,52(9):1351-1358.(in Chinese)
[5] TOŠI AC＇G I,FRANK D A.STAT3 as a mediator of oncogenic cellular metabolism:Pathogenic and therapeutic implications[J].Neoplasia,2021,23(12):1167-1178.
[6] LEE H,JEONG A J,YE S K.Highlighted STAT3 as a potential drug target for cancer therapy[J]. BMB Reports,2019,52(7):415-423.
[7] 赵辨.中国临床皮肤病学[M].第2版.南京:江苏科技技术出版社,2017. ZHAO B.Clinical Dermatology in China[M].2nd ed. Nanjing:Jiangsu Science and Technology Press,2017.(in Chinese)
[8] GUO W,WANG H,LI C.Signal pathways of melanoma and targeted therapy[J].Signal Transduction and Targeted Therapy,2021,6(1):424.
[9] PALMA S D,MCCONNELL A,VERGANTI S,et al.Review on canine oral melanoma:An undervalued authentic genetic model of human oral melanoma?[J].Veterinary Pathology,2021,58(5):881-889.
[10] 毛爱迪,陈爱军,王萍.恶性黑色素瘤治疗最新研究进展[J].重庆医学,2021,50(20):3581-3585. MAO A D,CHEN A J,WANG P.Recent research progress in the treatment of malignant melanoma[J].Chongqing Medicine,2021,50(20):3581-3585.(in Chinese)
[11] 王永芳,谭谦.皮肤恶性黑色素瘤诊断和外科治疗的研究进展[J].东南大学学报(医学版),2021,40(5):721-725. WANG Y F,TAN Q.Research progress in the diagnosis and surgical treatment of cutaneous malignant melanoma[J].Journal of Southeast University (Medical Science Edition),2021,40(5):721-725.(in Chinese)
[12] HAMERS-CASTERMAN C,ATARHOUCH T,MUYLDERMANS S,et al.Naturally occurring antibodies devoid of light chains[J].Nature,1993,363(6428):446-448.
[13] HARMSEN M M,RUULS R C,NIJMAN I J,et al.Llama heavy-chain V regions consist of at least four distinct subfamilies revealing novel sequence features[J].Molecular Immunology,2000,37(10):579-590.
[14] LI C,TANG Z,HU Z,et al.Natural single-domain antibody-nanobody:A novel concept in the antibody field[J].Journal of Biomedical Nanotechnology,2018,14(1):1-19.
[15] SALVADOR J P,VILAPLANA L,MARCO M P.Nanobody:Outstanding features for diagnostic and therapeutic applications[J].Analytical and Bioanalytical Chemistry,2019,411(9):1703-1713.
[16] VERHAAR E R,WOODHAM A W,PLOEGH H L.Nanobodies in cancer[J]. Seminars in Immunology,2021,52:101425.
[17] BAO G,TANG M,ZHAO J,et al.Nanobody:A promising toolkit for molecular imaging and disease therapy[J].EJNMMI Research,2021,11(1):6.
[18] 吕巧,邹超霞,吴永梅,等.STAT3基因对广西黄牛肌肉干细胞成肌诱导分化的调控研究[J].中国畜牧兽医,2021,48(8):2771-2777. LYU Q,ZOU C X,WU Y M,et al.Study on regulation of STAT3 gene on myogenic differentiation of muscle stem cells in Guangxi cattle[J].China Animal Husbandry & Veterinary Medicine,2021,48(8):2771-2777.(in Chinese)
[19] YU H,KORTYLEWSKI M,PARDOLL D.Crosstalk between cancer and immune cells:Role of STAT3 in the tumour microenvironment.[J].Nature Reviews Immunology,2007,7(1):41-51.
[20] MITCHELL L S,COLWELL L J.Comparative analysis of nanobody sequence and structure data[J].Proteins Structure Function & Genetics,2018,86(7):697-706.
[21] ZAVRTANIK U,LUKAN J,LORIS R,et al.Structural basis of epitope recognition by heavy-chain camelid antibodies [J].Journal of Molecular Biology,2018,430(21):4369-4386.
[22] SUN S,DING Z,YANG X, et al.Nanobody:A small antibody with big implications for tumor therapeutic strategy[J].International Journal of Nanomedicine,2021,16:2337-2356.
[23] 李宏芬,单保恩,陈晶,等.抗Stat3多克隆抗体的制备和其在乳腺癌诊断中的应用[J].第四军医大学学报,2006,27(24):2242-2244. LI H F,SHAN B E,CHEN J,et al.Preparation of polyclonal antibody against STAT3 and its application in the diagnosis of breast cancer[J]. Journal of the Fourth Military Medical University,2006,27(24):2242-2244.(in Chinese)
[24] 严昊,冯建远,张子仪,等.纳米抗体的制备与临床应用研究进展[J].中国畜牧兽医,2021,48(2):685-694. YAN H,FENG J Y,ZHANG Z Y,et al.Progress in preparation and clinical application of nanobody[J].China Animal Husbandry & Veterinary Medicine,2021,48(2):685-694.(in Chinese)
[25] MOHASSAB A M,HASSAN H A,ABDELHAMID D,et al.STAT3 transcription factor as target for anti-cancer therapy[J].Pharmacological Reports,2020,72(5):1101-1124.
[26] SIVEEN K S,SIKKA S,SURANA R,et al.Targeting the STAT3 signaling pathway in cancer:Role of synthetic and natural inhibitors[J]. Biochimica et Biophysica Acta,2014,1845(2):136-154.
[27] AL ZAID SIDDIQUEE K,TURKSON J.STAT3 as a target for inducing apoptosis in solid and hematological tumors[J].Cell Research,2008,18(2):254-267.
[28] LIN L,DEANGELIS S,FOUST E,et al.A novel small molecule inhibits STAT3 phosphorylation and DNA binding activity and exhibits potent growth suppressive activity in human cancer cells[J].Molecular Cancer,2010,9(1):217.