The Preparation Of Monoclonal Antibody Against GST-LRF And Purification For GST-LRF Fusion Protein

Luman-recruiting factor (LRF) appears to be a basic-domain leucine zipper (bZIP) transcription factor. LRF functions as a transcription co-factor and is required by Luman in the transactivation process. LRF recruits Luman to a specific subnuclear domain and facilitates formation of the active transcription complex. At present, the physiological function of the LRF is still unclear.Accquiring monoclonal antibodies againest LRF has considerable significance to studying the physiological function of LRF. The optimal condition for GST-LRF Fusion Protein were analyzed. The GST-LRF Fusion Protein was purified by electroelution. The GST-LRF Fusion Protein was used to immune BALB/C mice. Prepared and identified of monoclonal antibodies.The results are as follows:1 The condition for expression of GST-LRF fusion protein in E. coli: GST-LRF Fusion Protein was expressed in the optimal condition of 37℃, 0.1 mmol/L of IPTG and induction for 5h. GST-LRF Fusion Protein was purified by electroelution.2 Indirect ELISA method is established: HRP-labelled goat anti mouse IgG diluted by PBST (pH 7.4, 0.1 mol/L) in l∶1 200 was choosed as secondary antibody, coated antigen 10μg/mL GST-LRF dissolved in carbonate buffer solution (CBS, pH 9.6, 0.05 mol/L), 1.0% gelatin as blocking agent, and 1.0% TMB as a substrate, 2 mol/L H2SO4 as stop buffer.3 Hybridoma clone secreting monoclonal antibodies against GST-LRF were screened by indirect ELISA, and positive clones were subcloned by limited dilution.Three strain was obtioned and was named: A2E4,B2G6,D2F5.4 Three monoclonal antibodied was produced in vivo by the hybridoma against A2E4,B2G6 and D2F5, and identified of monoclonal antibodies. The titer of ascites was 1:10 000. The MCAbs showed a high specificity to GST-LRF.Reative affinity of monoelonal antibody was detected by indirect ELISA. It was 0.773μg/mL,0.800μg/mL and 0.355μg/mL。...