Prokaryotic Expression,Purification of Human Adapter Protein NRBP and Preparation of Its Polyclonal Antibody

Objective:To prepare anti-NRBP antibody and study the titers and specificity of this antibody.Method:The full-length cDNA or cDNA encoding the first 99 Aa at N terminal of human NRBP was amplified from pEF-NRBP plasmid by PCR and then subcloned into prokaryotic expression vector pET-21a or pGEX-6P-1.The recombinant plasmids were then transformed into E.coli BL21 and induced.The fusion proteins purified were used to immune rabbit.The titers and specificity of the generated antibodies were analyzed by indirect ELISA and Western blot.Result:The human NRBP cDNA was obtained successfully.The recombinant NRBP or NRBP(1-99Aa) can be expressed by IPTG induction,the purified proteins were used to immune rabbit.The result of ELISA was positive,and the titers of antibody from four different immunized rabbits were from 1:5 120 to 1:40 000.Western blot analysis showed that the antibody can detect exogenous and endogenous NRBP protein(about 60 kDa) and had strong ability of immunoprecipitation.Conclusion:The anti-NRBP antibody was approved having good specificity and sensitivity.The polyclonal antibody of NRBP provided an important tool to study the function of NRBP.