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目的采用生物信息学方法分析尖吻蝮蛇蛇毒金属蛋白酶cDNA序列的关键抗原位点,并观察根据这些位点设计合成的新型免疫原的免疫保护效果。方法扩增尖吻蝮蛇蛇毒金属蛋白酶cDNA序列,采用Jameson-Wolf方法和ClustalX软件结合的生物信息学方法分析其抗原位点,人工合成筛选的抗原位点序列,并连接到pIRESneo表达载体,3次(0、2、4周)对BALB/c小鼠进行核酸免疫,ELISA法测定免疫后机体抗体水平,出血中和实验和攻毒实验初步观察其免疫保护效果。结果生物信息学方法分析得到6个关键抗原位点(MPA-1～MPA-6);ELISA法检测抗血清结果表明,抗原位点序列诱导小鼠产生的抗血清相对未免疫的小鼠血清稀释100倍后仍能表现出阳性结果;出血中和实验和攻毒实验表明,将抗原位点序列免疫小鼠可以诱导机体产生免疫反应,使体内产生抗体,能有效中和蛇毒,从而对尖吻蝮蛇蛇毒引起的出血有防护作用。结论用生物信息学方法成功获得尖吻蝮蛇蛇毒金属蛋白酶cDNA序列的6个关键抗原位点,针对这些位点设计合成的新型免疫原展示出初步免疫保护效果。
OBJECTIVE: To analyze the key antigenic sites of the cDNA sequence of the metalloproteinase from Agkistrodon snake venom using bioinformatics methods and to observe the immunoprotective effect of novel immunogens designed and synthesized according to these sites. Methods The cDNA sequence of Agkistrodon acutus venom was amplified by using Jameson-Wolf method and ClustalX software, and its antigenic site was analyzed by bioinformatics method. The synthetic antigenic site was synthesized and ligated into pIRESneo expression vector. The BALB / c mice were immunized with nucleic acid in the second (week 0, week 2, week 2, week 4), and the antibody level after immunization was determined by ELISA. RESULTS: Six key antigenic sites (MPA-1 ~ MPA-6) were obtained by bioinformatics analysis. The antiserum assayed by ELISA showed that the antisera induced by the antigenic site sequence were diluted in serum relative to non-immunized mice 100 times, it still showed positive result. The hemorrhage neutralization experiment and challenge experiment showed that immunizing mice with the antigenic site sequence can induce the body to produce immune reaction, produce antibody in the body and effectively neutralize snake venom, Viper snake venom caused by bleeding protective effect. Conclusion Six key antigenic sites of the cDNA sequence of Agkistrodon snake venom were successfully obtained by bioinformatics methods. The new immunogen designed and synthesized based on these sites showed preliminary immunoprotective effect.