Preparation Of Monoclone Antibody (McAb) Against GST Tag Protein And Its Activity Identification

In genetic engineering, kinds of fusion expression systems are often used to express foreign proteins. GST expression system, with a high exprssion yield, convienent to purify expression products and prepare antibodies, is widely used. It is very important to detect and purify fusion expression products when foreign genes are expressed by GST expression system. At present, fusion proteins, obtained from GST fusion expression system, are usually purified by the commercial particular affinity chromatography purification systems such as Glutathione Sepharose-4B affinity chromatography columns, with disadvantages of expensive cost and limited useful life, not suitable for large-scale application and so on. In this study, specific monoclonal antibody against GST is developed, which is a preparatory work for the system of expression products' purification in large scale with affinity chromatography and immune magnetic beads based on monoclonal antibody.The results are as follows.1.The development of monoclonal antibody against GSTExpression bacterium BL21(DE3) containing plasmid pGEX is used to express GST protein. We obtained highly purified GST tag proteins after the expression products was purified by Glutathione-Sepharose 4B affinity chromatography columns. Purfied GST protein are used to immune Balb/c mice. Spleen cells from immuned mice were fused with SP2/0 myeloma cells through Conventional cell fusion technology; with the same purified GST protein as screening antigen, positive hybridoma cell lines were cloned by limited dilution method. We attained 2 strains hybridization tumour cells that secreted McAb against GST protein, named 3E and 10G2.The trait identification of monoclonal antibody against GSTWith indirect ELISA method, the ascites' titer of 3E and 10G were both higher than 1×10~6, the subtypes were both IgG1, and the numbers of chromosome are between 80 to 94. the reaction bewteen ascites of 10G strains and GST fusion protein (AIV-NS1, IBDV-VP2, IBV-NP) were very low, and ascites' titer of 3E reached 2×10~5 when it response to these three fusion proteins with indirect ELISA.3.The preliminary application of monoclonal antibodies against GSTThe ascites monoclonal antibody of 3E lines were purified by octanoic acid-ammonium sulfate purification, the titers of purified monoclonal antibody were 1.28×10~5 and 6.4×10~4 when it response to GST protein and IBDV-VP2 protein. And monoclonal antibodies of 3E response to GST protein and IBDV-VP2 protein were relatively little different in affinity. 3E cell lines monoclonal antibody can be used for the GST fusion protein detection, and it can be used as a ligand for purification of GST fusion protein.