摘要
为建立检测肠道黏膜新城疫病毒(NDV)特异性sIgA抗体的ELISA方法,通过原核表达并纯化的重组NDV HN蛋白作为包被抗原,构建检测肠道黏膜NDV特异性sIgA抗体的间接ELISA方法,并利用该方法检测各肠段黏膜新城疫特异性sIgA抗体含量,探讨了NDV黏膜免疫抗体水平的变化规律。结果显示,成功表达并纯化了HN蛋白,确定的最佳ELISA方案为:重组NDVHN蛋白包被质量浓度为0.5μg/mL,100μL/孔,封闭液为50 mg/mL脱脂奶粉溶液,黏膜洗脱液稀释度为1∶40,作用时间为1.5 h,二抗作用时间为1 h,稀释度为1∶100000,显色时间为10 min。ELISA重复性试验的批内变异系数在2.63%~7.68%之间,批间变异系数在1.43%~7.63%之间。本方法与包被全NDV建立的ELISA方法的符合率达到91.30%。使用本方法检测口服免疫含HN抗原重组乳杆菌(BLCC2-0394)的鸡十二指肠、空肠、回肠和盲肠黏膜中NDV特异性sIgA抗体水平,结果显示,免疫后第19天NDV特异性sIgA抗体显著高于空白对照组(P<0.01),第23天抗体水平达到最高,第26天出现下降趋势。结果表明,所建立的以重组HN蛋白为包被抗原的ELISA检测方法可用于肠道黏膜NDV特异性抗体的检测。
In order to construct an indirect ELISA method for detecting the Newcastle disease virus(NDV)-specific secretory IgA(sIgA)antibody of the intestinal mucosa,NDV HN protein was expressed by prokaryotes and used as the coating antigen to establish indirect ELISA detection method.The method was used to detect the NDV specific sIgA intestinal mucosa in each segment,and the change rule of mucosal anti-NDV antibody level was discussed.The results showed that the HN protein was successfully expressed and purified,and the optimal ELISA reaction conditions were as follows:the recombinant HN protein was coated with 0.5μg/mL,100μL per well,the blocking solution was 50 mg/mL skimmed milk powder solution,the mucosal elution was diluted at 1∶40 and incubated for 1.5 h,the second antibody was diluted at 1∶100000 and reacted for 1 h,and finally the coloring time was 10 min.The repeatability results showed that ELISA had good repeatability with intra-assay coefficient of variation ranging from2.63%to 7.68%and inter-assay coefficients of variation ranging from 1.43%to 7.63%.The coincidence rate between this method and the method,which established with coating whole NDV,reached 91.30%.This method was used to detect the NDV specific sIgA antibody level in the duodenum,jejunum,ileum and cecum mucosa of chickens immunized with recombinant lactic acid bacteria containing HN antigen(BLCC2-0394).The results showed that the NDV specific sIgA antibody level was significantly higher than that of the blank control group on the 19th day after immunization(P<0.01),the antibody level reached the highest on the 23rd day,and showed a downward trend on the 26th day.In conclusion,the ELISA method established with HN protein as antigen could be used to detect the specific antibodies of NDV intestinal mucosa.
作者
陈甜甜
侯玉凤
苏晓彤
孙明杰
张秋婷
谢全喜
谷巍
CHEN Tian-tian;HOU Yu-feng;SU Xiao-tong;SUN Ming-jie;ZHANG Qiu-ting;XIE Quan-xi;GU Wei(Shandong Key Laboratory of Animal Microecological Preparations/Shandong Baolai-Leelai Bioengineering Co.Ltd.,Tai'an 271000,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2023年第1期49-56,共8页
Chinese Veterinary Science
基金
国家重点研发计划项目(2017YFD0501000)。
