关键词: 树鼩, CD4蛋白, 表达, 纯化, 多克隆抗体

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