牛副流感3型NP融合蛋白原核表达及间接ELISA检测方法的建立  

Prokaryotic expression of NP protein of Bovine Parainfluenza Virus Type 3 and development of an indirect ELISA

作　　者：王苗苗 张玲玲[1] 张峣[1] 冉旭华[1] 闻晓波[1] Wang Miaomiao, Zhang Lingling, Zhang Yao, Ran Xuhua, Wen Xiaobo(College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Heilongjiang Daqing 16331)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《现代畜牧兽医》2018年第3期1-7,共7页Modern Journal of Animal Husbandry and Veterinary Medicine

基　　金：黑龙江省农垦总局攻关课题(HNK125B-11-08A)

摘　　要：为原核表达牛副流感病毒3型(Bovine parainfluenza virus type 3,BPIV3)NX49株截短NP融合蛋白并以此建立间接ELISA检测方法,本研究采用RT-PCR方法扩增了BPIV3截短NP蛋白基因序列并克隆至p ET-28a中进行诱导表达,纯化后产物进行Western blot分析。以重组蛋白作为包被抗原,建立并优化检测BPIV3抗体的间接ELISA方法,并对该方法的特异性、敏感性、重复性进行验证。结果成功表达出截短的NP重组蛋白,该重组蛋白具有良好的反应原性。间接ELISA优化结果如下:抗原包被浓度为4μg/m L,37℃包被1 h后4℃过夜,待检血清以1:80倍稀释37℃孵育1 h,5%脱脂乳37℃封闭1 h。二抗以1:5 000倍稀释37℃孵育1 h,TMB显色时间为15 min。以该蛋白为包被抗原建立的间接ELISA检测方法其特异性为97.4%,敏感性为95.3%,批内、批间检测结果变异系数均小于10%。本试验成功建立检测BPIV3抗体的间接ELISA方法,该方法具有较强的特异性、良好的重复性及敏感性,为开展牛呼吸道疾病综合征的流行病学调查、实验室诊断奠定了基础。To express the truncated NP fusion protein of bovine parainfluenza virus type 3 （BPIV3）, NX49 strain and establish an indirect ELISA detection method. The truncated NT fragment of BPIV3 was amplified by RT-PCR and subcloned into pET-28a vector. The purified recombinant protein was analyzed by western blot and it was used as diagnostic antigen to establish an indirect ELISA assay. Then the specificity, sensitivity and reproducibility were tested. The results demonstrated that the recombinant protein NP-N-C was expressed successfully. The optimal working param- eters were determined as follows： the coating concentration of antigen was 4 μg/mL, incubated at 37 ℃ for l h and then at 4 ℃ for 12 h, the primary sera dilution was set as 1：80 and incubated for i h at 37 ℃ and 5% skim milk was chosen as blocking reagents and incubated at 37 ℃ for l h. The best dilution of IgG/HRP was 1：5000 and was incubated at 37 ℃ for 1 h. The specificity and sensitivity of the indirect ELISA was 97.4% and 95.3%, respectively. Furthermore, both inter-assay and intra-assay the variation coefficients were lower than 10%. An indirect ELISA assay based on the recombinant protein NP-N-C was established in this study, which demonstrated high specificity, excellent repeatability and sensitivity and indicated a potential of the epidemiological investigation and laboratory diagnosis for Bovine respiratory disease complex associated with BPIV3.

关 键 词：牛副流感病毒3型 NP基因 原核表达 间接ELSIA 

分 类 号：S852.659.5[农业科学—基础兽医学]