Study On Biological Recognition Protein To Amantadine Residue In Chicken And Development Of Affinity Detection Method To Amantadine

Amantadine has better treatment and prophylaxis effect against Influenza A virus.The mainly mechanism was regarded as the affinity bind between amantadine and itsacceptor protein, named as M2protein. Amantadine blocks the ion channel andinhibits gene replicate and infect of the virus. As the acceptor, protein M2wouldspecifically bind with amantadine. On the other hand, M2protein has the potentialability to be used as one kind of recognition material to amantadine in vitro. Thepossibility of M2protein as the recognition material to amantadine was evaluated.Three part of experiment in this research was carried out. First, the amantadineresidue in chicken was determined using HPLC-MS/MS and the investigation aboutillegal use of amantadine in chicken breeding farms was carried out by interview indifferent farms. Second, the biological recognition protein to amantadine wasprepared using genetic technique. Third, the affinity features of recombination proteinwas evaluated and the affinity detection system using recombination biologicalrecognition protein was developed followed by the evaluation of stability of thedetection system. The main results were summarized as follows:1, The investigation result indicated that although amantadine had been bannedin animal breeding, it was still been depended badly on the treatment and prophylaxisof virus disease, both in the good-sized farms and small farms. The residue detectionresult demonstrated, the amantadine residue in chicken was detected positive withpositive ratio of18.4%（19/103）,5.9%（4/68）, and11.1%（8/72）, respectively. Differentpart of chicken has different residue; chicken wing had the highest residue ofamantadine compared with chicken drumsticks and chicken livers. No residue wasdetected in chicken breast.2, The recombination vector encoding M2protein was constructed and transformed into E. coli system and the optimization of expression condition wascarried out followed by research of purification and refolding technique. The resultindicated that the E. coli was fit to be induced by IPTG after growing3.5h in liquidmedium with37℃and200r/min. IPTG was adequate with the final concentration of0.1mM. After the addition of IPTG, the culture condition was amended with20℃,100r/min and the cells were cultured for another16h. It was showed, under suchcondition, the M2protein was expressed with biggest yield of29.04mg/L. The resultof refolding experiment showed the denatured protein was refolded effectively in thepresence of arginine with the optimized final concentration of0.6M. The protein’saffinity function was evaluated by two methods, ELISAand Equilibrium DialysisExperiments. The result from the former method indicated the recombination M2protein has the same structure with the native M2protein onAIV virus. The resultfrom the later experiment demonstrated the recombination protein has the bindconstant of1.1×105and the binding stoichiometry of4.2.3, The method of biological recognition system to amantadine was developedbase on affinity features of biological recognition protein and information amplifiedby enzyme catalysis. The optimization of response parameter in recognition system wasdetermined as follows: the coating concentration and recombination concentrationwere20μg/mL and0.25mg/mL, respectively. The dilution factor of antibody conjugatedby HRP was1:1000. The specificity of biological recognition system was evaluatedwith IC₅₀of459ng/mL and cross-reactivity of72%with rimantadine, the other ionchannel inhibitor beside amantadine. However, the biological recognition systemhas no cross-reactivity with other kinds of antivirus drugs, such as moroxydine orribavirin. In this experiment, the stabilities of the biological recognition system wasalso evaluated with with an intra-assay variation of5.0-19.0%and inter-assay of6.5-15.5%. The average recovery rates varied from74%to103%.In brief, the feasibility of using recombination M2protein as biological recognitionmaterial was affirmed in this research. And the preparation method of biologicalrecognition material to amantadine and the evaluation method to the affinity detection systemtoamantadine residue in chicken using biological recognition material was displayed. The whole result in this research was effective to the preparation, evaluation of biological recognitionmaterial and the commercialization kit to amantadine residue.