Prokary Otic Expression Of GAPDH And Preparation Of Its Monoclonal Antibodies

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in glycolysis with high and stably expression in cells which being widely used as a control in the expreriments.It is not only involved in the process of energy metabolism, but also DNA repair, apoptosis, up regulate the expression of proteins and physiological functions. It is actually a multifunctional protein involved in many activities in subcellular level. It is very important to discovery and exploration the any other functions of this enzyme and its application in food industry.In order to obtain hybridoma cell lines steadily secreting monoclonal antibody against GAPDH, the GAPDH fragment was amplified by PCR using the GAPDH cDNA as a template,then cloned to pMD18-T. The recombinant plasmid pMD18-T-GAPDH was transformed into E. coli Top10’. After amplified a large number of this plasmid and digested with restriction enzyme,the target fragment was recycled with Gel Extraction Kit and then cloned to the expression vector pTO-T7.The recombinant plasmid pTO-T7-GAPDH was transferred into Escherichia coli ER2566, the protein was induced by IPTG and identified by SDS-PAGE and western blot, the recombinant strain had ability to express a37kDa protein, and the size of target protein is consistent with our expectation. The recombinant protein was precipitated by ammonium sulfate and purified by affinity chromatography and molecular sieve chromatography. The purity of GAPDH was above to99%. The purified recombinant protein was used as a antigen to inject to BALB/C mice by the way of multiple subcutaneous injection. The immunized mouse spleen cells and myeloma cells SP2/0were fused when the serum titer was about1x106. The fusion and positive rates were81.3%and75.4%respectively after screening of hybridoma cell with medium20%FBS-HAT-1640. The highest positive of hybridoma cells were cloned3times,then the6strains of hybridoma cell lines that stability secreting monoclonal antibodies against GAPDH were got.The monoclonal antibodies were prepared by intraperitoneal injection of the way, then purified by Protein A affinity chromatography good specificity, these antibodies with high sensitivity of anti GAPDH monoclonal antibody and titer of, can be widely used in cell biology and immunology.