Preparation and Determination of CD4 Polyclonal Antibodies of Tree Shrew

doi:10.3969/j.issn.1674-5817.2016.04.001

Abstract

Abstract: Objective To express tree shrew (Tupaia belangeri) CD4 protein in prokaryotic cells, purify the expressed product and prepare its polyclonal antibodies,and determinate CD4 protein in vivo. Methods CD4 gene was cloned and inserted into express vectors. And then recombinant plasmids pET30a (+)-CD4 and pGEX-5X-1-CD4 were constructed. The expressed recombinant proteins His-CD4 and GST-CD4 were expressed and purified. The polyclonal antibodies of CD4 protein were prepared with three different adjuvants including Al (OH)₃, MF59 and Freund’s. The serum antibody specificity was determined by Western blot. Results The expressed recombinant proteins His-CD4 and GST-CD4, with relative molecular masses of about 53.5×10³ and 70.0×10³ respectively. And the concentrations of proteins His-CD4 and GST-CD4 were 600 μg/ml and 1mg/ml respectively. Three groups of adjuvants all can induce antibodies. The comparison results of Geometric Mean Titer (GMT) is that the Freund’s adjuvant group (GMT: 97 420) showed the highest geometric mean of antiserum, followed by Al (OH)₃ adjuvant group (GMT: 67 202) and MF59 adjuvant group (GMT: 55128). The Freund’s adjuvant group was significantly higher than the original protein group (P<0.001). The Al(OH)₃ adjuvant group was significantly higher than the original protein group (P<0.01). The MF59 adjuvant group was higher than the original protein group (P<0.05). Conclusions Mouse antiserum with high specificity was prepared,and it could bind to CD4 protein of tree shrew in vivo, which laid a foundation of further study on the preparation of monoclonal antibody against CD4 and the research of CD4 function and immune related experiments of tree shrews as viral infection models.

Key words: Tree shrew, CD4 Protein, Expression, Purification, Polyclonal antibody

CLC Number:

Q95-33

LU Kong-jie, XU Jing-wen, ZHANG Xue-mei, WU Zhong-xiang, REN Fang-fang, MA Na, GONG Wei, YAN Li-wei, ZHU Wen-bing, DONG Shao-zhong. Preparation and Determination of CD4 Polyclonal Antibodies of Tree Shrew[J]. Laboratory Animal and Comparative Medicine, 2016, 36(4): 243-249.

References

[1] 彭燕章, 叶智彰, 邹如金, 等. 树鼩生物学[M]. 昆明: 云南科技出版社, 1991:1-7.
[2] 殷安国, 匡德宣, 李晓飞,等. 树鼩模型在人类病毒性疾病研究中的应用进展[J]. 中国实验动物学报, 2014, 22(2):86-89.
[3] 陈瑾, 代解杰, 孙晓梅. 树鼩肝炎动物模型的研究进展[J]. 中国比较医学杂志, 2008, 18(2):59-62.
[4] 曹雪涛, 何维, 熊思东, 等. 医学免疫学[M]. 北京: 人民卫生出版社, 2015:200-201.
[5] Wang JH, Yan YW, Garrett TP, et al.Atomic structure of a fragment of human CD4 containing two immunoglobulin-like domains.[J]. Nature, 1990, 348(6300):411-418.
[6] Yan L, Ling W, Xu H, et al.The activation and dynamics of cytokine expression by CD4⁺, T cells and AIDS progression in HIV-1-infected Chinese individuals[J]. Microbial Pathogenesis, 2012, 53(5-6):189-197.
[7] Barboza L, Salmen S, Peterson DL, et al.Altered T cell costimulation during chronic hepatitis B infection[J]. Cell Immunol, 2009, 257(1-2):61-68.
[8] Houman H, Hamzaoui A, Ghorbal IB, et al.Abnormal expression of chemokine receptors in Beh?et's disease: relationship to intracellular Th1/Th2 cytokines and to clinical manifestations[J]. J of Autoimmun, 2004, 23(3):267-273.
[9] Belz GT, Wodarz D, Diaz G, et al.Compromised influenza virus-specific CD8(+)-T-cell memory in CD4(+)-T-cell-deficient mice[J]. J of Virol, 2002, 76(23):12388-12393.
[10] Garcíafruitós E, Vázquez E, Díezgil C, et al.Bacterial inclusion bodies: making gold from waste[J]. Trends Biotechnol, 2012, 30(2):65-70.
[11] Ventura S, Villaverde A.Protein quality in bacterial inclusion bodies[J]. Trends Biotechnol, 2006, 24(4):179-185.
[12] Shi T, Zhang L, Li Z, et al.Expression, purification and renaturation of truncated human integrin β1 from inclusion bodies of Escherichia coli[J]. Protein Expr Purif, 2014, 107(4):13-19.

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