Expressing Of Rice Allergens RAG2 Gene,Preparation And Application Of Polyclonal Antibodies Against RAG2

Rice is the staple food in Asian countries and some western countries. In cereal protein, rice protein is the best. However, for some people, rice protein can also induce allergic reactions. 14-16 kDa ?-amylase/trypsin inhibitor is the main allergen in rice. This research to obtain the recombinant proteins RAG2 corresponding to 14-16 kDa ?-amylase/trypsin inhibitor was based on t he genetic engineering technology and prokaryotic expression;To prepare rabbit polyclonal antibody against 14-16 kDa ?- amylase/trypsin inhibitor by using recombinant protein; To construct indirect competitive ELISA method for screening rice varieties and obtaining the hypo-allergenic rice varieties.The main methods and conclusions are as follows:1.Using gene synthesis technology to synthesis the gene of 14-16 kDa ?-amylase/trypsin inhibitor RAG2. PSUMO-Mutant plasmid and E.coliBL21(DE3)pLys S were selected as vector and expression system, respectively. Connecting the gene RAG2 to pSUMO-Mutant construct the recombinant plasmid, then introduced them into E.coli ER2925 for amplification in quantity. Plasmids were extracted and digested by restriction enzyme; the result shows that synthetic gene sequence is correct.2. pSUMO-RAG2 recombinant plasmid in the E.coli BL21(DE3) p LysS was induced successfully. Tracking the SUMO-RAG2 fusion protein, it was turned out that it is insoluble intracellular inclusion bodies. In order to get the SUMO-RAG2 fusion protein, denaturation and renaturation of inclusion bodies, then purification the recombinant protein through Ni column.3. Polyclonal anti-14-16 kDa ?-amylase/trypsin inhibitor polyclonal antibody was raised by inoculating rabbits. The titer of polyclonal antibody was up t o 1:320,000.4.Collection 28 r ice varieties from Jiangxi province and extraction the salt soluble proteins. Using SDS-PAGE and based on t he polyclonal antibody against RAG2 western blot method to detect the contents of allergen RAG2 in the 28 varieties. The preliminary result shows that, all of them were resistance to react to anti-RAG2 serum. Finally, using indirect competitive Elisa method measured IC50 of each variety,and obtained low antigenicity rice varieties.