尖吻蝮蛇蛇毒金属蛋白酶cDNA序列抗原位点分析及免疫保护效果观察

Epitope analysis of cDNA sequences of Deinagkistrodon acutus snake venom metalloproteinases and observation of their immune protective effects

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 epitopes of cDNA sequences of Deinagkistrodon acutus snake venom metalloproteinases using bioinformatical method,and to observe the immune protective effect of the new immunogen designed according to the identified epitopes.Methods The cDNA sequences of Deinagkistrodon acutus snake venom metalloproteinases were amplified by PCR.The epitopes of the sequences were analyzed by Jameson-Wolf method and Clustal X software,then the sequences of the screened epitopes were artificially synthesized and linked to the vector pIRESneo.BALB/c mice were immunized by the resultant plasmid at 0,2,and 4 weeks for three times,then the titers of the anti-serum were measured by ELISA.The immune protective effects of the anti-serum were tested by the neutralization of venom hemorrhagic activity and venom attacking test.Results Bioinformatical analysis yielded 6 epitopes（MPA-1-MPA-6）.The ELISA results of anti-serum showed that these epitopes could induce immune reaction in mice,and the anti-serum was detectable even when it was diluted to 1：100.The neutralization test and venom attacking test demonstrated that the anti-serum induced by the epitodes could neutralize the venom and protect the mice from haemorrhage.Conclusion Six epitopes of Deinagkistrodon acutus snake venom metalloproteinases have been obtained successfully using bioinformatical method,and the new immunogen designed based on these epitopes shows a primary immune protective effect.