犬细小病毒VP2抗体竞争ELISA检测方法的初步建立

本研究旨在制备犬细小病毒(canine parvovirus,CPV)VP2抗体,并建立竞争ELISA检测方法,从而为CPV疫苗免疫效果的检测及血清学调查提供技术支持。参照GenBank中犬细小病毒VP2基因序列设计1对添加EcoR Ⅰ和Xho Ⅰ酶切位点的引物,PCR扩增VP2基因全长序列,将其克隆到pET28a(＋)载体中,构建原核表达载体pET28a-VP2,转化大肠杆菌Rosetta,表达并纯化了重组蛋白。SDS-PAGE及Western blotting分析表明,目的蛋白分子质量大小为67 ku,可被CPV抗血清识别,证明其能与特异性抗体结合,有良好的抗原性。以纯化的重组蛋白免疫家兔制备VP2多抗,采用辣根过氧化物酶进行标记,初步建立了竞争ELISA方法。结果显示,VP2重组蛋白的最佳包被浓度为5 μg/mL,酶标多抗的最佳稀释度为1∶3200,封闭条件为4 ℃过夜,最适的封闭液为10%小牛血清,山羊抗兔IgG-HRP的最佳工作浓度为1∶5000,显色时间为25 min。竞争ELISA试验结果表明,该方法具有较强的稳定性,有望为CPV疫苗免疫效果的判定及血清学调查提供技术手段。

Initial Development of the Competition ELISA Detection Method for Canine Parvovirus based on VP2 Antibody

This study focused on the preparation of canine parvovirus VP2 antibodies,and establishing a competitive ELISA detection method,so as to provide technical support for detection of the effect of CPV vaccine and serological investigations of CPV infection.A pair of primers adding EcoR Ⅰ and Xho Ⅰ restriction site were designed according the gene sequence of canine parvovirus VP2.Full-length VP2 gene was obtained by PCR amplification,then cloned into pET28a(+) vector to construct a prokaryotic expression vector pET28a-VP2,and transformed into E.coli host strain Rosetta.Finally,the VP2 recombinant protein was expressed and purified.Rabbits were immunized with purified VP2 recombinant protein,then VP2 polyclonal antibody was prepared,and labeled with horseradish peroxide enzyme,and detected by indirect ELISA method,finally,established a competitive ELISA method initially.The result of SDS-PAGE showed that the VP2 recombinant protein expressed was correct,and the molecular weight size was 67 ku.The result of Western blotting showed that this protein could be recognized by CPV antiserum,demonstrated that it was specific.Indirect ELISA results showed that best packages concentration of the VP2 recombinant protein was 5 μg/mL,and coated at 4 ℃ overnight,the best dilution of enzyme labeled antibody was 1∶3200,the optimal solution for block was the 10% bovine serum,1∶5000 dilution of the goat anti-rabbit IgG-HRP was the best work concentration,coloration time was 25 min.The results of competitive ELISA test showed that the method was stability.In conclusion,this study provides a technological means for determination the effect of CPV vaccine and serological surveys.