The Expression Of HIV P24 Antigen, The Preparation Of Monoclonal Antibody And Preliminary Research On Its Application

A pair of PCR primers with two enzyme digested sites (BamHl and HincKll) were designed according to the sequence of gag gene of HIV-1 reported by Triques, K. The foil length gene fragment(693bp) of HIV p24 was amplified by PCR and inserted into the pRSET vector in order to construct the pRSET-p24 recombined vector. After transforming into E.coil (BL21) , the purified p24 protein with a singal expected band about 24 KD in SDS-PAGE was prepared by metal-ligand affinity chromatography (IMAC). The antigenicity of purified p24 protein was tested by Western-blot. The specificity and activity of purified p24 protein were similar with those of the product obtained from the National Institute for the Control of Pharmaceutical and Biological Products.We immunized Balb/c mice with purified p24 protein and fused the immunized spleen cells with SP2/0 cells. Three hybridoma cell lines (named 24-1 24 24-3) secreting anti-HIV p24 antibody were established by limiting dilution. The monoclonal antibodies (McAbs) were produced in ascites fluid of mice by injecting these hybridomacells and purified by affinity chromatography. The double-antibody sandwiched (AbS) ELISA method detecting HIV p24 antigen was established using purified McAbs. The sensitivity (80pg) of our AbS reagent was a little lower than that (20pg) of Do Pont kit. The specificity of both reagents was 100%. The preliminary experiments are the important base for developing a more sensitive diagnostic reagent kit for detecting HIV p24 antigen.