绵羊肌肉生长抑制素MSTN原核表达及其纳米抗体文库的构建与鉴定

doi:10.13523/j.cb.20140913

中国生物工程杂志

2014, Vol. 34

Issue (9): 87-93 DOI: 10.13523/j.cb.20140913

技术与方法

绵羊肌肉生长抑制素MSTN原核表达及其纳米抗体文库的构建与鉴定

李尤简¹, 张国奇², 郭吉星², 陈新凯¹, 豆晓霞¹, 陈创夫², 盛金良¹

1. 石河子大学动物科技学院;
2. 新疆地方与民族高发病教育部重点实验室石河子 832003

Expression of Ovine Myostatin Gene and Construction and Identification of Nanobody Library Against Recombinant MSTN

LI You-jian¹, ZHANG guo-qi², Gou ji-xing², CHEN xin-kai¹, DOU Xiao-xia¹, CHEN Chuang-fu², SHENG Jin-liang¹

1. College of Animal Science and Technology;
2. Key Laboratory of Xinjiang Endemic and Ethnic Disease, Shihezi University, Shihezi 832003, China

全文: PDF(795 KB) HTML

摘要：

克隆绵羊肌肉生长抑制素（myostatin，MSTN）基因并在大肠杆菌中诱导表达，纯化重组蛋白免疫健康的双峰驼（Bactrian camel），分离其外周血淋巴细胞提取总RNA，利用RT-PCR扩增骆驼重链抗体IgG2、IgG3的可变区（VHH）基因片段，将VHH片段与pCANTAB5E连接后电转入大肠杆菌TG1构建纳米抗体文库。结果显示，纳米抗体文库容量为9.5×10⁵，挑取部分克隆进行测序分析，所获得的纳米抗体文库具有良好的多态性，为进一步筛选绵羊MSTN的高特异性纳米抗体片段奠定了基础。

关键词： 绵羊; 肌肉生长抑制素; 纳米抗体; 噬菌体展示文库

Abstract:

The ovine myostatin gene was subcloned into the pET-32a(+),and transformed into E.coli BL21.The MSTN recombinant fusion protein was purified and the healthy Bactrian camel was selected for immunization with the MSTN protein.Total RNA was extracted from peripheral lymphocytes for the amplification of VHH fragments by RT-PCR.Primers designed according to the single domain gene sequences of the antibodies and the variable camel IgG2, IgG3 genes were amplified by Nested PCR.PCR products were then purified and inserted into phage mid vector pCANTAB5 E. The VHH antibody gene library was obtained by electroporating recombinant pCANTAB5E VHH vectors into E. coli TG 1 cells.The first set of antibody gene library was composed with 9.5×10⁵ individual colones after cloning VHH genes and the library we possessed high diversity and good capacity,which provides a platform for panning VHH camilid antibody aimed at ovine MSTN.

Key words: Ovine Myostatin Nanobody Phage display library

收稿日期: 2014-07-14 出版日期: 2014-09-25

ZTFLH:

Q78

基金资助:

国际科技合作项目（2013DFR30970）；兵团博士基金（ZD2007JC02）资助项目

通讯作者: 盛金良 E-mail: ccf-xb@163.com;sjlshz@126.com

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引用本文:

李尤简, 张国奇, 郭吉星, 陈新凯, 豆晓霞, 陈创夫, 盛金良. 绵羊肌肉生长抑制素MSTN原核表达及其纳米抗体文库的构建与鉴定[J]. 中国生物工程杂志, 2014, 34(9): 87-93.

LI You-jian, ZHANG guo-qi, Gou ji-xing, CHEN xin-kai, DOU Xiao-xia, CHEN Chuang-fu, SHENG Jin-liang. Expression of Ovine Myostatin Gene and Construction and Identification of Nanobody Library Against Recombinant MSTN. China Biotechnology, 2014, 34(9): 87-93.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140913 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I9/87

[1] McPherron A C,Lawler A M,Lee S J,et al. Regulation of skeletal muscle mass in mice by a new TGF-β superfamily member. Nature, 1997, 387:83-90.

[2] Hamers-Casterman C, Atarhouch T, Muydermans S, et al. Naturally occurring antibodies devoid of light chains. Nature, 1993, 363(6428): 446-448.

[3] 涂追,许杨,刘夏,等.驼源天然单域重链抗体库的构建与鉴定.中国生物工程杂志,2011,31(4):31-36. Tu Z, Xu Y, Liu X,et al.Construction and biopanning of camelid naive single-domain antibody phage display library.China Biotechnology,2011,31(4):31-36.

[4] David R. Maass, Jorge Sepulveda, Aton Pernthaner, et al.Alpaca(Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies(VHHs).Journal of Immunological Methods,2007,324(1-2):13-25.

[5] Ghahroudi M A, Desmyter A, Wyns L, et al. Seleetion and identification of single domain antibody fragments from camel heavy-chain antibodies. Febs Letters, 1997, 414(3): 521-526.

[6] Ma X Y, Cao Y C, Shu D M, et al. Cloning and expression of swine myostatin gene and its application in animal immunization trial.Sci China Ser C:Life Sciences, 2005, 48(4):368-374.

[7] Kim Y S, Bobbili N K, Paek K S, et al. Production of a monoclonal anti-myostatin antibody and the effects of in ovo administration of the antibody on posthatch broiler growth and muscre mass. Poult Sci, 2006, 85(6):1062-1071.

[8] 郭吉星,张辉,胡圣伟,等.绵羊MSTN多克隆抗体的制备及细胞毒性分析.生物技术,2013,23(2):41-44. Guo J X, Zhang H, Hu SH W, et al.Production of Polyclonal Anti-Myostatin Antibody of Ovine and the Analysis of Cytotoxicity.Biotechnology,2013,23(2):41-44.

[9] Ghahroudi M A, Desmyter A, Wyns L, et al. Selection and identification of single domain antibody fragments from camel heavy chain antibodies.Febs Letters,1997,414(3):521-526.

[10] McCafferty J, Griffi ths A D, Winter G, et a1. Phage antibodies: filamentous phage displaying antibody variable domains. Nature, 1990, 348:552-554.

[11] Sblattero D, Florian F, Bradbury A, et al. Analyzing the peripheral blood antibody repertoire of a celiac disease patient using phage antibody libraries. Hum Antibodies, 2000,9(4):199-205.

[12] Vu K B, Ghahroudi M A, Wyns L, et al. Comparison of llama V-H sequenees from conventional and heavy chain antibodies. Molecular Immunology, 1997, 34(16-17): 1121-1131.

[13] Ghahroudi M A, Desmyter A, Wyns L, et al. Seleetion and identification of single domain antibody fragments from camel heavy-chain antibodies. Febs Letters, 1997, 414(3): 521-526.

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