Preparation And Determination Of CD4 Polyclonal Antibodies Of Tree Shrew

Tree shrews are squirrel-like animals, belonging to the order Scandentia, which have a claimed close affinity to primates. And owing to a variety of unique characteristics for example, small adult body size, easily tamed, short reproductive cycle and life span, low cost of feeding and management, the tree shrew has been proposed as a viable human infectious disease animal model for example HBV, and EV71, in biomedical research. At present, few studies on tree shrews immune system, in particular, immune-related molecules of tree shrews have been done, which limits the further application of tree shrews as an ideal virus infectious animal model. In the process of virus infection, CD4 molecule recognizes antigen-antibody complexes as a T cell receptor, and plays an important role in anti-infection immunity. Meanwhile, the expression of CD4 and the preparation of antibodies in tree shrews have not reported yet. Therefore sequence of CD4 protein is analyzed firstly. And the expression, purification of CD4 protein and preparation of polyclonal antibody have been completed.In this study, the full-length CD4 cDNA encoding sequence with restriction Enzyme sites was cloned from the RNA of tree shrew peripheral blood lymphocytes by RT-PCR. The amplified product was inserted into vector pGEX-5X-1 and pET-30a(+). The constructed recombinant plasmids pGEX-5X-1-CD4 and pET-30a (+)-CD4 transformed to E. coli BL21 and E. coli BL21 (DE3) respectively, and induced with IPTG The expressed recombinant proteins GST-CD4 and His-CD4 were purified, and GST-CD4 was used as an immunogen combined with different adjuvants (MF59/Al(OH)3/Freund’s adjuvant) for immunization of BALB/c mice. The serum antibody titer was determined by indirect ELISA, and the enhancement of humoral immune response induced by CD4 protein in different groups was compared.In order to evaluate the binding ability of CD4 polyclonal antibody, Western blot is used to detect the protein CD4 expressed by prokaryotic expression systems and the protein CD4 in vivo (tree shrew, human, monkey and so on).Recombinant plasmids pGEX-5X-1-CD4 and pET-30a (+)-CD4 were constructed correctly. The expressed recombinant proteins GST-CD4 and His-CD4 with relative molecular masses of about 70.0×103 and 53.5×103 respectively, existed in forms of soluble and inclusion bodies. The concentrations of proteins GST-CD4 and His-CD4 were 1mg/ml and 600μg/ml respectively. The antibodies were induced and the antiserum titers were high in three adjuvant groups after immunization. Among which the titer tested by ELISA is highest in Freund’s adjuvant group. The Freund’s adjuvant group (GMT:97420) showed the highest geometric mean of antiserum, followed by Al(OH)3 adjuvant group (GMT:67202) and MF59 adjuvant group (GMT:55128). The Freund’s adjuvant group was significantly higher than proteins group(P< 0.001); The Al(OH)3 adjuvant group was significantly higher than proteins group(P<0.01); The MF59 adjuvant group was higher than proteins group(P<0.05). Mouse antiserum with good specificity was prepared. And it could bind to CD4 protein of tree shrew specifically. There is no immunological cross-reaction between species.In conclusion, the protein CD4 of tree shrew were obtained by this experiment, and then good specific CD4 polyclonal antibodies without immunological cross-reaction between species were also prepared in this experiment. And the research laid a foundation of further study on the preparation of monoclonal antibody against CD4 and CD4 function study and immune related experiments of tree shrews as viral infection models.