Recombinant Expression,Purification,Antibody Production And Application Of Human Adiponectin Gene

With the improvement of people's living standard, the diabetes such as type II diabetes, insulin resistance and hyperinsulinemia are continuously increasing. Research shows that the concentration of plasma adiponectin (ADPN) was negativedly correlated with these diseases, so it is possible to predict the occurrence of such diseases by detecting the levels of human plasma adiponectin, the information can be provided to the patient as soon as possible. Currently, the ELISA is used to to detect human plasma adiponectin levels, but most ELISA test kit in the market are from abroad, but in chinese there are no relevant products.According to ADPN sequence (NM₀04797.2) published in NCBI, a pair of primers was designed in the use of primer5.0. The total RNA was extracted from human adipose tissue, then, the 732bp gene fragment of the whole gene of adiponectin was acquired by RT-PCR. The fragment was cloned into vector plasmid pET-CKS, Then they were transformed into E.coli TOP10F'. The single colonies was identificated by PCR and the plasmid were digested by enzyme and were sequenced, the sequencing results was blasted with NCBI, the recombinant plasmid with the correct reading frame was named pET-CKS/ADPN.The recombinant plasmid pET-CKS/ADPN was transformed into E.coli BL21-gold. SDS-PAGE electrophoresis showed that the recombinant protein was induced expression in the form of inclusion bodies by IPTG. Western blot demonstrated that the fusion protein had immunoreactivity with rabbit anti-ADPN antibody. The best inductive conditions were established:30℃,0.1mM IPTG concentration, induced expression 8h. Because N-terminal of fusion protein contained six histidine, the target protein can be purificated from the bacterial protein by nickel metal affinity chromatography. the target protein was refolded in the method of dialysis, its concentration was relatively high, it is suitable to animal immunity.After the renaturated recombinant proteins was immunized into rabbits, anti-serum titer was detected by indirect ELISA. The titer reached 5×104 or so after the sixth immunization. Then verificated the rabbit anti-serum titers by double immunod-iffusion test, while its titer reached 1:64. Rabbit was killed for anti-serum, anti-ADPN polyclonal antibody of rabbit was purificated by ammonium sulfate precipitation and Ion Exchange Chromatography. The concentration of polyclonal antibody was about 2.3mg/mL.The anti-ADPN monoclonal antibody was used as the coating antibody, the ADPN polyclonal antibody conjugated with horseradish peroxidase by the method of modified sodium iodide was used as labeled antibody. Then the double antibody Sandwich ELISA for the early and specific detection of ADPN was established. Checkerboard titration method was used to establish the best dilution of ADPN monoclonal antibody was 1:1000, the best dilution of antibody marked with HRP was 1:1500. With recombinant ADPN protein the standard curve was drawn, the best range was about 2ng-10ng. Clinical determinated ADPN concentrations of 111 type II diabetes was 6.5±0.33 ng/mL, the ADPN concentrations of 89 non type II diabetes was 9.9±1.08 ng/mL.The recombinant ADPN protein which had immunogenicity was obtained through genetic engineering approaches, as the same time, we also obtained anti-ADPN polyclonal antibody, then the initial establishment of the double-antibody sandwich detective model of ADPN was successful. It was lay the foundation for the further study for ADPN's biochemical nature and physiological functions and ADPN's ELISA test kit.