检测传染性法氏囊病病毒VP4蛋白抗体试纸条的研制及应用

Study and Application on an Immunochromatographic Method for Detection the VP4 Protein Antibody of Infectious Bursal Disease Virus

采用RT-PCR技术从滨州分离株中扩增出传染性法氏囊病病毒(infectious bursal disease virus,IBDV)VP4基因,将VP4基因插入到pGEX-4T-1载体上构建pGEX-4T-1-VP4,诱导表达并用谷胱甘肽-S-转移酶(GST)亲和层析柱纯化得到纯化的重组VP4蛋白。以兔抗鸡IgG为胶体金标记物,以重组IBDV VP4蛋白和羊抗兔IgG为硝酸纤维素膜检测线和质控线的包被物,制备一种能检测IBDV VP4蛋白抗体的胶体金试纸条。结果表明,该试纸条检测IBDV强毒(IBDV BC6/85)免疫的血清检测线显红色,为阳性反应;检测IBDV弱毒(IBDV NB)免疫的血清、新城疫病毒(Newcastle disease virus,NDV)标准阳性血清、禽流感H5和H9标准阳性血清、传染性支气管炎标准阳性血清及0.85%生理盐水检测线不显红色,为阴性反应。该试纸条与建立的ELISA方法相比,敏感度低2个滴度;检测320份临床血清,试纸条与ELISA的符合率达99.38%。提示,该试纸条使用方便、操作简单,10min内可以用肉眼判断结果,可为区分IBDV的强弱毒提供参考数据,具有较大的应用价值。

The VP4 gene was acquired by RT-PCR from Binzhou isolate of infectious bursal disease virus, the VP4 expres- sion plasmid was constructed by inserting the target fragment into pGEX-4T-1 vectors. The expression VP4 proteins were ac- quired by inducing and purifying. An immunochromatograpic strip was developed for the detection VP4 protein antibody of in- fectious bursal disease virus. Rabbit anti-chicken IgG was labled with colloidal gold as a detection reagent, and recombinant VP4 protein of IBDV was blotted on the test line while goat anti-rabbit IgG was used on the control line of the nitrocellulose membrane. The results of specificity showed that the strip was positive in the detection of reference sera against IBDV BC6/85 virulent and negative to the detection of standard positive serum of Newcastle disease virus, anvian influenza virus H5 and H9 subtypes, infectious bronchitis virus were negative, 0.85 % isotonic Na chloride, reference sera against IBDV BC6/85 virulent. Compared with ELISA, the sensitivity of the gold-immunochromatographie assay （OICA） was low two titer. The agreement rate between the two tests was 99.38~//oo. The detection time of VP4 IgO against IBDV by the GICA was less than 10 min. The GICA test strip was a reliable and useful tool for the on-site surveillance of IBDV.