摘要
旨在建立一种灵敏、准确、高通量检测猫杯状病毒(feline calicivirus, FCV)抗体的间接ELISA方法。本研究通过优化表达条件,在大肠杆菌中表达FCV的VP1蛋白,对重组蛋白进行镍离子亲和层析纯化和透析复性,通过SDS-PAGE和Western blot验证重组蛋白;以重组VP1蛋白为包被抗原建立检测FCV抗体的间接ELISA方法,通过方阵滴定法确定抗原最佳包被浓度和抗体最佳稀释度,优化抗原包被条件、样品孵育时间、封闭液种类等条件,并对其性能进行验证。结果:表达的目的蛋白条带清晰、大小正确,与FCV阳性血清具有良好的反应性,建立的间接ELISA方法检测6份FCV阴性血清,猫细小病毒(feline parvovirus, FPV)阳性血清,猫疱疹病毒(feline herpesvirus, FHV)阳性血清均为阴性,对FCV阳性血清的最低检出效价可达1∶10 240,组间与组内变异系数均小于10%,与商品化试剂盒相比符合率达100%。综上,本研究建立的间接ELISA方法具有良好的灵敏度、重复性和符合率,与其他猫常见病毒阳性血清不发生交叉反应,可以用于临床样品的检测,为FCV感染的诊断、防控、疫苗免疫效力评价提供了技术手段。
The aim of this study was to establish an indirect ELISA method for the detection of feline calicivirus(FCV)antibody with good specificity,high sensitivity and high throughput.In this study,the FCV-VP1 was expressed in Escherichia coli by optimizing the expression conditions.The recombinant protein was purified by nickel ion affinity chromatography and was refolded by dialysis.Then,the recombinant protein was verified by SDS-PAGE and Western blot.The recombinant VP1 protein was used as the coating antigen to establish an indirect ELISA method for detecting FCV antibody.Next,the optimal coating concentration of antigen and the optimal dilution of the antibody were determined using the square titration method.Finally,the conditions of antigen coating,sample incubation time and blocking solution were optimized,and its performance was verified.The results showed that the expressed target protein bands were clear and correct in size,and it possessed a good reaction with FCV positive serum.The established indirect ELISA method was used to detect six FCV negative serum samples,feline parvovirus(FPV)positive serum sample,feline herpesvirus type 1(FHV-1)positive serum sample;and they were all negative.The minimum detection titer of FCV positive serum was 1∶10240,and the coefficient of variation between and within groups was less than 10%.Compared with the commercial kit,the coincidence rate was 100%.The present results indicated that the indirect ELISA method had good sensitivity,repeatability and coincidence rate,but it had no cross-reaction with other cat common virus positive serum samples.The method could be used for the detection of clinical samples,which provided a technical means for the diagnosis,prevention and control of FCV and the evaluation of vaccine immune efficacy.
作者
姬晶晶
王彬
何昭群
李嘉豪
单衍可
刘斐
JI Jingjing;WANG Bin;HE Zhaoqun;LI Jiahao;SHAN Yanke;LIU Fei(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)
出处
《畜牧与兽医》
北大核心
2025年第3期87-94,共8页
Animal Husbandry & Veterinary Medicine
基金
南京农业大学新进博士科研启动费项目(804006)。
