|
|
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 |
|
|
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.
|
Received: 14 July 2014
Published: 25 September 2014
|
|
|
|
[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.
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
|
Shared |
|
|
|
|
|
Discussed |
|
|
|
|