大熊猫源犬细小病毒VP2基因的原核表达及间接ELISA检测方法的建立

Prokaryotic Expression of Giant Panda-derived Canine Parvovirus VP2 Gene and Establishment of an Indirect ELISA Detection Method

【目的】建立一种检测大熊猫血清中犬细小病毒(canine parvovirus,CPV)抗体的间接酶联免疫吸附剂测定(enzyme linked immunosorbent assay,ELISA)方法。【方法】以大熊猫源CPV DNA为模板,利用PCR扩增VP2基因,再用原核表达系统对VP2基因进行表达,经纯化后的蛋白作为ELISA包被抗原;制备并纯化兔抗大熊猫IgG,采用辣根过氧化物酶(horseradish peroxidase,HRP)标记作为间接ELISA酶标二抗,通过棋盘法确定抗原包被质量浓度及血清稀释度等条件,并评估建立方法与商业试剂盒检测样品结果的符合率。【结果】原核表达成功获得约70 ku的VP2蛋白,将其作为间接ELISA包被抗原,抗原最佳包被质量浓度为2.0μg/mL,血清最佳稀释度为1∶400,用该方法进行检测血清样品时OD450≥0.258为阳性,反之为阴性。采用建立的ELISA方法检测大熊猫血清样本阳性率为60.0%,高于商业化检测试剂盒检测的阳性率(52.5%),两者总符合率达到87.5%。【结论】本研究首次建立了基于大熊猫源CPV VP2蛋白为抗原、自制HRP标记兔抗大熊猫IgG为酶标二抗的间接ELISA方法,该方法特异性强、灵敏性较高、重复性好,为大熊猫血清中CPV抗体的检测提供了技术支持。

[Purpose]To establish an indirect enzyme linked immunosorbent assay(ELISA)method for detecting canine parvovirus(CPV)antibodies in giant panda serum.[Methods]The VP2 gene was amplified by PCR using CPV DNA derived from giant pandas as a template,then the VP2 gene was expressed using a prokaryotic expression system,and the purified protein was used as an ELISA coated antigen.Rabbit anti-giant panda IgG was prepared and purified,and labeled with horseradish peroxidase(HRP)as an indirect ELISA enzyme-labeled secondary antibody.The conditions such as the mass concentration of coated antigen and dilution of serum were determined by the checkerboard method;and the consistency rate between the established method and the test sample results of commercial kits was evaluated.[Results]Prokaryotic expression successfully obtained about 70 ku of VP2 protein,which was used as an indirect ELISA coated antigen.The optimal mass concentration of coated antigen was 2.0μg/mL,and the optimal dilution of serum was 1∶400.When using this method to detect serum samples OD450≥0.258 was positive,and vice versa was negative.The positive rate of giant panda serum samples detected by the ELISA method established in this study was 60.0%,which was higher than the positive rate of commercial detection kits(52.5%),and the total coincidence rate reached 87.5%.[Conclusion]This study establishs an indirect ELISA method based on the giant panda-derived CPV VP2 protein as the antigen,and homemade HRPlabeled rabbit anti-giant panda IgG as the enzyme-labeled secondary antibody for the first time.This method has strong specificity,high sensitivity and good repeatability,and provides technical support for the detection of CPV antibodies in giant panda serum.