从大容量噬菌体抗体库中筛选抗Acr蛋白人源单链抗体

中国生物工程杂志

2012, Vol. 32

Issue (09): 22-27

研究报告

从大容量噬菌体抗体库中筛选抗Acr蛋白人源单链抗体

王晓娜¹, 米志强¹, 安小平¹, 李建彬¹, 范华昊¹, 张文慧¹, 张博¹, 黄勇¹, 周丽君², 童贻刚¹

1. 军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室北京 100071;
2. 中国人民解放军海军总医院中心研究室北京 100037

Screening of Human Anti-α-crystallin Protein of Mycobacterium Tuberculosis scFv from Large Phage Library

WANG Xiao-na¹, MI Zhi-qiang¹, AN Xiao-ping¹, LI Jian-bin¹, FAN Hua-hao¹, ZHANG Wen-hui¹, ZHANG Bo¹, HUANG Yong¹, ZHOU Li-jun², TONG Yi-Gang ¹

1. State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China;
2. Navy General Hospital, Beijing 100037, China

全文: PDF(396 KB) HTML

摘要： 目的:从天然的大容量噬菌体抗体库中筛选特异的抗结核分枝杆菌晶体蛋白(alpha-crystallin Acr)的人源抗体。方法: 以结核分枝杆菌 Acr蛋白包被免疫管,通过对噬菌体抗体库进行4轮"吸附-洗脱-扩增"的过程从大容量抗体库中筛选特异性抗结核分枝杆菌 Acr蛋白的抗体,并对可变区序列进行了测序分析。将特异性的噬菌体抗体感染HB2151菌,经IPTG诱导表达,制备了抗结核分枝杆菌 Acr蛋白的可溶性单链抗体;对其序列和抗原结合活性进行分析鉴定。结果:经过4轮筛选,获得了43个与结核分枝杆菌 Acr蛋白结合的阳性克隆,其中29个特异结合的克隆;测序分析有26不同的可变区片段;通过可溶性单链抗体(scFv)表达筛选到14株特异性结合Acr蛋白的可溶性单链抗体克隆;经过基因测序,分析了可变区基因的亚群。成功制备了可溶性单链抗体。Westren blotting分析证实筛选的人源单链抗体能与天然蛋白结合。结论:利用单链大容量抗体库获得抗结核分枝杆菌 Acr蛋白的噬菌体抗体并且成功制备抗结核分枝杆菌 Acr天然蛋白的可溶性单链抗体,为今后的研究和应用奠定基础。

关键词： 结核分枝杆菌; Acr 蛋白; 噬菌体抗体库; 单链抗体

Abstract: Objetive:To obtain the specific human scFv against Mycobacterium tuberculosis Acr protein using phage antibody library technology.Methods: A human large phage antibody library was panned four times with the purified Acr protein.Soluble scFvs were prepared through infection of E.coli HB2151 with the selected phage antibodies clones and induction with IPTG. The antigen binding activity were determined ELISA and Western blotting, and DNA sequences of these clones were analyzed by Sanger sequencing.Results:43 positive clones were obtained after 4 rounds of panning and 29 clones had binding ability to Mycobacterium tuberculosis Acr protein. DNA sequencing of the 29 clones showed 26 different positive clones. 14 specific soluble scFvs were obtained after infection in E.coli HB2151. DNA sequence analysis showed that the variable regions of these scFvs belonged to different subgroups.The Western blotting results suggested that the anti-Acr antibodies had good immuno-reactivity with natural Acr protein.Conclusion:Human anti-Acr scFvs are obtained from a large phage antibody library, which will benefit to the application of anti-Acr of Mycobacterium tuberculosis antibody to clinical research.

Key words: Mycobacterium tuberculosis Acr protein Phage antibody library scFv

收稿日期: 2012-04-23 出版日期: 2012-09-25

ZTFLH:

Q511

通讯作者: 童贻刚 E-mail: tong.yigang@gmail.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	王晓娜
	米志强
	安小平
	李建彬
	范华昊
	张文慧
	张博
	黄勇
	周丽君
	童贻刚

引用本文:

王晓娜, 米志强, 安小平, 李建彬, 范华昊, 张文慧, 张博, 黄勇, 周丽君, 童贻刚. 从大容量噬菌体抗体库中筛选抗Acr蛋白人源单链抗体[J]. 中国生物工程杂志, 2012, 32(09): 22-27.

WANG Xiao-na, MI Zhi-qiang, AN Xiao-ping, LI Jian-bin, FAN Hua-hao, ZHANG Wen-hui, ZHANG Bo, HUANG Yong, ZHOU Li-jun, TONG Yi-Gang. Screening of Human Anti-α-crystallin Protein of Mycobacterium Tuberculosis scFv from Large Phage Library. China Biotechnology, 2012, 32(09): 22-27.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I09/22

[1] Williams A, Reljic R, Naylor I, et al.Passive protection with immunoglobulin A antibodies against tuberculous early infection of the lungs. Immunology, 2004. 111(3): 328-333.
[2] Reljic R, Williams A, Ivanyi J, Mucosal immunotherapy of tuberculosis: is there a value in passive IgA? Tuberculosis (Edinb), 2006, 86(3-4): 179-190.
[3] López Y, Yero D, Falero-Diaz G, et al. Induction of a protective response with an IgA monoclonal antibody against Mycobacterium tuberculosis 16 kDa protein in a model of progressive pulmonary infection. International Journal of Medical Microbiology, 2009, 299(6): 447-452.
[4] Reljic R, Clark S, Williams A, et al. Intranasal IFNγ extends passive IgA antibody protection of mice against Mycobacterium tuberculosis lung infection. Clinical & Experimental Immunology, 2006,143(3): 467-473.
[5] Buccheri S, Reljic R, Caccamo N, et al.IL-4 depletion enhances host resistance and passive IgA protection against tuberculosis infection in BALB/c mice. European journal of immunology, 2007, 37(3): 729-737.
[6] Buccheri S, Reljic R, Caccamo N, et al. Prevention of the post-chemotherapy relapse of tuberculous infection by combined immunotherapy. Tuberculosis, 2009, 89(1): 91-94.
[7] Grosset, J.Mycobacterium tuberculosis in the extracellular compartment: an underestimated adversary. Antimicrobial agents and chemotherapy, 2003, 47(3): 833-836.
[8] Nunn P, Williams B, Floyd K, et al.Tuberculosis control in the era of HIV. Nature Reviews Immunology, 2005, 5(10): 819-826.
[9] Teitelbaum R, Glatman-Freedman A, Chen B, et al. A mAb recognizing a surface antigen of Mycobacterium tuberculosis enhances host survival. Proceedings of the National Academy of Sciences, 1998, 95(26): 15688.
[10] Hamasur B, Haile M, Pawlowski A,et al.A mycobacterial lipoarabinomannan specific monoclonal antibody and its F (ab') 2 fragment prolong survival of mice infected with Mycobacterium tuberculosis. Clinical & Experimental Immunology, 2004, 138(1): 30-38.
[11] Pethe K, Alonso S, Biet F, et al.The heparin-binding haemagglutinin of M. tuberculosis is required for extrapulmonary dissemination. Nature, 2001, 412(6843): 190-194.
[12] Chambers M A, Gavier-Widén D, Glyn Hewinson R. Antibody bound to the surface antigen MPB83 of Mycobacterium bovis enhances survival against high dose and low dose challenge. FEMS Immunology & Medical Microbiology, 2004, 41(2): 93-100.
[13] Roy E, Stavropoulos E, Brennan J, et al. Therapeutic efficacy of high-dose intravenous immunoglobulin in Mycobacterium tuberculosis infection in mice. Infect Immun, 2005, 73(9):. 6101-6109.
[14] Olivares N, Puig A, Aguilar D, et al.Prophylactic effect of administration of human gamma globulins in a mouse model of tuberculosis. Tuberculosis (Edinb), 2009, 89(3): 218-220.
[15] Guirado E, Amat I, Gil O, et al.Passive serum therapy with polyclonal antibodies against Mycobacterium tuberculosis protects against post-chemotherapy relapse of tuberculosis infection in SCID mice. Microbes Infect, 2006, 8(5): 1252-1259.
[16] 张廷芬,师长宏. Hsp16. 3 的生物学特性及其在结核病研究中的应用. 中华结核和呼吸杂志, 2007, 30(6): 456-458. Zhang T F, Shi C H. Biology characteristics of Hsp16.3 and its application in tuberculosis. Chin J Tuberc Respri Dis, 2007,30(6): 456-458.
[17] Boon C, Li R, Qi R, et al. Proteins of Mycobacterium bovis BCG induced in the Wayne dormancy model. J Bacteriol, 2001, 183(8): 2672-2676.
[18] Cunningham A F, Spreadbury C L. Mycobacterial stationary phase induced by low oxygen tension: cell wall thickening and localization of the 16-kilodalton α-crystallin homolog. Journal of bacteriology, 1998, 180(4): 801-808.
[19] Lim A, Eleuterio M, Hutter B et al. Oxygen depletion-induced dormancy in Mycobacterium bovis BCG. J Bacteriol, 1999, 181(7): 2252-2256.

[1]	李彤彤,宋彩玲,杨凯越,王文静,陈慧宇,刘明. 抗犬细小病毒VP2蛋白单链抗体的制备与中和活性研究 ^*[J]. 中国生物工程杂志, 2020, 40(4): 10-16.
[2]	安明晖,田文,韩晓旭,尚红. 表达HIV单链抗体的重组乳酸杆菌的构建及表型分析 ^*[J]. 中国生物工程杂志, 2019, 39(10): 1-8.
[3]	谭杨,刘胜,罗凤玲,章晓联. 结核分枝杆菌H37Rv刺激巨噬细胞后差异表达lncRNA分析及鉴定 ^*[J]. 中国生物工程杂志, 2018, 38(5): 1-9.
[4]	高鑫,韦攀健,闫卓红,易玲,王小珏,杨斌,张洪涛. 一株针对人EGFR的单链抗体克隆与哺乳细胞表达 ^*[J]. 中国生物工程杂志, 2018, 38(5): 73-78.
[5]	赵荣,陈含宇,黄春,章晓联,潘勤. 靶向B细胞并特异结合其分泌IL-10的重组融合蛋白的构建、表达和初步鉴定 ^*[J]. 中国生物工程杂志, 2018, 38(2): 1-6.
[6]	庞倩,陈晶,王小红,王佳. 基于噬菌体展示技术抗黄曲霉毒素B1单链抗体的筛选及其蛋白结构分析 ^*[J]. 中国生物工程杂志, 2018, 38(12): 41-48.
[7]	王冬冬, 张国利, 岳玉环, 吴广谋, 田园, 刘雨玲, 吉元刚, 王金鹏, 李建, 潘荣荣, 马洪圆. 抗A型产气荚膜梭菌α毒素全人源双价单链抗体的构建、表达及其活性的初步研究[J]. 中国生物工程杂志, 2017, 37(4): 18-25.
[8]	温杰, 宋琳琳, 张莹, 王荷, 何金生, 洪涛. 稳定表达Aβ特异性单链抗体的哺乳动物细胞株构建和功能研究[J]. 中国生物工程杂志, 2017, 37(2): 1-7.
[9]	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.
[10]	代云见, 张勇侠, 何勇智, 丛聪, 张涛, 王明蓉. 技术与方法Anti-IgE单链抗体纯化工艺研究及活性鉴定[J]. 中国生物工程杂志, 2015, 35(12): 51-57.
[11]	陆健, 江佳稀, 刘建平, 王洪海. 结核分枝杆菌抗原重组酿酒酵母免疫诱导小鼠特异性免疫应答[J]. 中国生物工程杂志, 2014, 34(11): 47-53.
[12]	徐丹, 刘敏, 孔菊, 李校堃, 姜潮. CFP10-ESAT-6-MPB64在杆状病毒系统中的表达纯化及免疫原性鉴定[J]. 中国生物工程杂志, 2014, 34(06): 23-30.
[13]	王报贵, 武晓丽, 董素琴, 甘敏, 陈星星, 陈飞, 明星, 徐锋. 抗肠炎沙门氏菌单链抗体制备及其特异性分析[J]. 中国生物工程杂志, 2013, 33(5): 62-67.
[14]	刘霞, 郭庆龙, 王若珺, 王洪海, 裴秀英, 张雪莲. 结核分枝杆菌生物膜形成相关基因的筛选与鉴定[J]. 中国生物工程杂志, 2013, 33(4): 15-21.
[15]	陈继军, 毛晓燕, 乔玉玲, 毕司英. 抗狂犬病病毒单链抗体的筛选及鉴定[J]. 中国生物工程杂志, 2013, 33(11): 27-31.

Viewed

Full text

Abstract

Cited

Shared

Discussed