摘要
金刚烷胺对于流感病毒引起的流感疾病具有较好的疗效，畜牧养殖业中普遍应用，其主要作用机理是通过吸附作用结合于流感病毒上的M2受体蛋白，抑制病毒的复制、脱壳、感染等过程。M2受体蛋白作为金刚烷胺的作用受体，能与金刚烷胺特异性结合，具有开发成为金刚烷胺的特异性生物识别材料的潜力，本论文主要研究了将该蛋白作为金刚烷胺的生物识别材料的可行性。
本论文首先通过仪器检测法对金刚烷胺在鸡肉产品中的残留程度做了检测分析，对金刚烷胺在肉鸡养殖领域的使用情况做了调查；通过分子生物学方法建立了金刚烷胺受体蛋白的表达系统，研究了表达工艺、纯化工艺及复性工艺，评价了重组受体蛋白的亲和活性及其作为生物识别材料用于金刚烷胺残留检测的可行性，为违禁兽药的生物学检测分析提供技术参考，论文的主要成果如下：
1、采用仪器法对肉鸡养殖领域的金刚烷胺使用情况做了调查，结果表明，在肉鸡养殖中对于金刚烷胺等药物的依赖性依然较大，规模化养殖场和小型养殖户中都存在金刚烷胺的滥用问题，而且养殖规模越小，对于禁用兽药的使用越无序。药残检测结果显示，市售鸡肉产品中存在金刚烷胺残留，残留检出率分别为18.4%（19/103）、5.9%（4/68）和11.1%（8/72）不等，金刚烷胺在不同部位的药残含量有所差异，在鸡翅部位最高、鸡腿及肝脏次之，鸡脯肉中未检测到残留。
2、采用分子生物学技术建立了金刚烷胺受体蛋白的表达系统，研究了表达工艺、纯化工艺、复性工艺，评价了受体蛋白的亲和活性。研究结果表明，重组表达系统在37℃、200r/min条件下经3.5h后达到添加IPTG的最佳时间，IPTG诱导浓度为0.1mM，添加IPTG以后在20℃、100r/min条件下继续诱导表达16h，重组蛋白的表达量得到大幅提高，收获率为29.04mg/L。对重组蛋白的亲和活性评价结果表明重组蛋白对金刚烷胺的亲和常数为1.1×105，偶联比为4.2，显示出作为金刚烷胺生物识别材料的潜力。
3、建立了基于生物识别材料的亲和吸附和酶催化信号放大为特点的亲和检测体系，通过方阵法优化了亲和检测体系的反应参数：包被浓度为20μg/mL、金刚烷胺生物识别蛋白的浓度为0.25mg/mL、酶标抗体的稀释浓度为1：1000；评价了该亲和检测体系的特异性，其IC_(50)值为459ng/mL，与同类药物金刚乙胺的交叉反应率为72%，对于其他养殖业常见违禁药物的交叉反应率较低。评价了该检测体系的稳定性，检测回收率在74-103%之间，批内变异系数5.0-19.0%，批间变异系数6.5-15.5%。
本研究确认了以生物法对金刚烷胺进行残留检测的可行性，建立了一种制备金刚烷胺生物识别材料的方法以及对金刚烷胺残留进行检测的生物学方法，为生物识别材料的制备、检测体系的性能评估、快速检测试剂盒的商业化开发等提供有效的数据参考。
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 IC_(50)of459ng/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.
引文
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