Preparation Of Recombinant Human BFGF, Anti-bFGF Polyclonal Antibody And Their Primary Application In Tumor Detection

Basic fibroblast growth factor(bFGF)is one of the most important angiogenesis factor in tumor growth and aggressiveness.In this study,a plasmid has been constructed to direct the synthesis of recombinant human bFGF(rhbFGF) in E.coli as a fusion protein containing a 6xHis-tag at the N-terminus under the control of T7 promoter.The rhbFGF was synthesized in large amounts(up to 30% of the total bacterial protein)through optimization and acumulated in both inclusion bodies and soluble form upon induction with IPTG.. The rhbFGF was purified based on its 6xHis-tag by affinity chromatography using Nickel-Agarose Column(purity>90%) and refolded by dialysis. Two rabbits were immunized with the purified recombinant protein to produce polyclonal anti-human bFGF antibody. The titer was checked by indirect ELISA and the final titer was 1:819200 after the 4th immunization.The polyclonal antibody was purified by Protein G affinity chromatography,and combinated with commercial anti-bFGF monoclonal antibody to establish a sanwich ELISA for the detection of bFGF expression level.We had detected 50 tumor patients'and 50 healthy controls'serum bFGF by the established sandwich ELISA.The detection rate in tumor patients was 68% , which was significantly higher than those in the healthy controls(10%),indicated that serum bFGF had a close correlation with the tumor genesis and development.Using the recombinant DNA approach, we had produced the rhbFGF and anti-bFGF antibody which were used in the sandwich EILSA for the detection of serum bFGF.,it may be benefical to the development of commercial bFGF ELISA kit in China.