尖吻蝮蛇毒金属蛋白酶多表位抗原肽的原核表达和免疫原性研究

Prokaryotic expression and immunogenicity research of multiepitope peptide of Deinagkistrodon acutus metalloproteinases

目的研究人工合成的尖吻蝮蛇毒金属蛋白酶抗原位点的原核表达蛋白对小鼠的免疫效果。方法将人工合成的尖吻蝮蛇毒金属蛋白酶关键抗原位点序列连接到表达载体pET-28a(+)进行原核表达,溶解的包涵体蛋白免疫Balb/c小鼠,用ELISA法检测抗体水平,并进行出血中和实验和攻毒实验,观察其免疫保护效果。结果成功表达蛇毒金属蛋白酶抗原位点重组蛋白,诱导表达的蛋白免疫小鼠能使小鼠产生中和抗体,ELISA检测抗体表明,抗血清稀释10倍后相对对照组结果为阳性;皮下注射尖吻蝮蛇毒,免疫小鼠的出血面积显著减少;在全数致死剂量的尖吻蝮蛇毒攻毒下,实验组3只小鼠存活2只。结论原核表达的蛇毒金属蛋白酶多表位抗原肽免疫后能够有效刺激小鼠的免疫应答反应,产生中和尖吻蝮蛇毒的抗体。

One of effective treatment for snake envenoming is administration of antivenom prepared from sera of horses or sheep hyperimmunized with individual venom or a range of venoms.But the specificity of the conventional antivenoms is low since snake venoms contain numerous non-toxic components and many low molecular weight venom proteins are highly toxic but weakly immunogenic.To enhance the therapeutic value of antivenom,we prokaryotically expressed a multiepitope peptide of Deinagkistrodon acutus metalloproteinases,and determined the immunogenicity of the expressed recombinant protein.A sequence encoding mutiepitope of Deinagkistrodon acutus metalloproteinases was ligated into pET28a（＋） plasmid and expressed in E.coli BL21.Then the expressed fusion protein was used to immunize Balb/c mice.The titers of the anti-serum were measured by ELISA,and the immune protective effects were determined by neutralization of venom hemorrhagic activity and venom attacking tests.We found that mice immunized by the recombinant protein could produce anti-snake venom serum,and the antibody was detectable by ELISA when diluted to 1∶10;the neutralization of venom hemorrhagic activity test showed that the hemorrhagic area of immunized mice was reduced;two of three immunized mice survived in the venom attacking test.In conclusion,multiepitope peptide of Deinagkistrodon acutus metalloproteinases can stimulate immune response effectively in mice,and induce the generation of anti-snake venom antibody.