Prokaryotic Expression, Preparation Of Monoclonal Antibody Of Part Of The Chicken CD4Molecules

CD4is type Ⅰ transmembrane glycoprotein found on subsets of T cells as well as an important T lymphocyte surface markers. Chicken CD4gene is located chromosome1, its total length is11.5kb including ten exons. Chicken CD4gene encodes a mature protein containing four immuno globulins-like, extracellular region (402AA) Transmembrane region (24AA) and cytoplasm region (33AA). CD4acts as co-receptors for the T cell receptor (TCR) in recognition of antigens associated with MHC Ⅱ molecules. CD4is also a receptor of signal transmission. The cytoplasmic region of CD4through tyrosine kinase pathway is involved in signaling cascades mediated by TCR, triggering a series of immune response. There is highly conserved in the structure and function among chicken and mammalian CD4molecules, it relates to the immune system of chicken. In order to establish a detection method to chicken CD4molecule and study its gene structure and biological function, in this paper prokaryotic expression, preparation and identification of monoclonal antibody of part of the chicken CD4molecule were carried out.First a pair of primers was designed based on the cDNA sequence of chicken CD4gene and multi-clone restriction enzyme sites in prokaryotic expressive vector pET-32a. The gene fragment encoding the first and second Ig domains except signal peptide was amplified by PCR, it was579bp length, which accorded with the sequence in GenBank Y12012. Then the segment was inserted into vector pET-32a and reconstructed the recombinant vector, named pET-32a-CD4. The vector pET-32a-CD4was transformed into BL21(DE3). After the expression condition were optimized, the CD4segment was induced in a lot of expression. The inclusion bodies were purified after cell lysis. Fusion protein His-CD4was obtained from purification procedure with Ni-NTA resin after the dissolution and renaturation in vitro. The molecular weight of His-CD4was about42KD.Secondly the eight week old Balb/c mice were immunized with the purified fusion protein His-CD4. After tour time immunizations, a titer of the srum antibody in mice had more1:10000, the spleen cells were collected and fused with myeloma cells (SP2/0). After more time subcloning and detection of antibody in cell supernatant, a hybridoma cell lines secreting monoclonal antibodie (McAb) against His-CD4were produced, named3E12. The McAb showed specific reaction to His-CD4in western blot. The ascitic fluid was produced by celiac injection of hybridoma cells into BALB/c mouse. The titre of ascitic fluids of the McAb was1:105in indirect ELISA, and the immuno globulin class of McAb belonged to IgG2b subtype with identification with diagnostic kit.In conclusion, in this study the gene fragment of chicken CD4was cloned, then recombinant plasmid pET-32a-CD4was reconstructured and the prokaryotic expression of fusion protein His-CD4carried out. With fusion one hybridoma cell line secreting McAb against His-CD4was obtained. The subtype and titre of ascitic fluids of the McAb was identified. All these provide a experimental material for the study of the biological function of chicken CD4.