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牛副流感病毒3型HN基因原核表达及间接ELISA方法建立
引用本文:周玉龙,任亚超,朱战波,侯喜林,王密,耿晶,朴范泽,李森.牛副流感病毒3型HN基因原核表达及间接ELISA方法建立[J].病毒学报,2012,28(1):23-28.
作者姓名:周玉龙  任亚超  朱战波  侯喜林  王密  耿晶  朴范泽  李森
作者单位:黑龙江八一农垦大学动物科技学院,大庆,163319;哈尔滨医科大学大庆校区,大庆,163319
基金项目:黑龙江省科技厅项目(GA09B301-2);哈尔滨医科大学伍连德青年基金项目:WLD-QN1111;黑龙江省卫生厅项目2009-259
摘    要:用构建的HJ-1株牛副流感病毒3型HN基因原核表达载体pQE30-HN,在E.coli XL1Blue中表达了重组HN蛋白,并用电洗脱方法从电泳凝胶中纯化了该重组蛋白作为包被抗原,建立了检测该病毒抗体的ELISA方法。建立的ELISA最佳工作条件分别是:抗原浓度为6μg/mL,血清稀释度为1∶50,封闭液为5%脱脂奶粉,封闭条件为37℃60min,酶标抗体工作浓度为1∶10 000;阳性临界值为OD450≥0.30。该方法与病毒中和试验符合率高达96%,板内和板间重复性试验变异系数均小于10%,重复性较好。该方法与牛冠状病毒、传染性鼻气管炎病毒和呼吸道合包体病毒阳性血清无交叉反应。应用该方法检测黑龙江省部分地区323份奶牛血清样本,对牛副流感病毒3型抗体血清阳性率为57.89%。该ELISA方法的成功建立为进一步研发ELISA试剂盒提供了技术基础。

关 键 词:牛副流感病毒3型  HN蛋白  原核表达  ELISA

Prokaryotic expression of HN gene of bovine parainfluenza virus type 3 and the establishment of indirect ELISA method
Zhou Yu-Long,Ren Ya-Chao,Zhu Zhan-Bo,Hou Xi-Lin,Wang Mi,Geng Jing,Piao Fan-Ze,Li Sen.Prokaryotic expression of HN gene of bovine parainfluenza virus type 3 and the establishment of indirect ELISA method[J].Chinese Journal of Virology,2012,28(1):23-28.
Authors:Zhou Yu-Long  Ren Ya-Chao  Zhu Zhan-Bo  Hou Xi-Lin  Wang Mi  Geng Jing  Piao Fan-Ze  Li Sen
Institution:Animal Technology College, Heilongjiang Bayi Agricultural University, Daqing 163319, China.
Abstract:The prokaryotic expression plasmid pQE30-HN of hemagglutinin-neuraminidase (HN) protein gene of bovine parainfluenza virus type 3 (BPIV3) strain HJ-1 was expressed by IPTG induction in E. coli XL1Blue. The recombinant HN protein(rHN) was purified by electroeluting method, and used as coated antigen. An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody valence of BPIV3. The best working conditions of ELISA were as follows: the antigen concentration was 6 microg/mL; the serum dilution was 1:50; the blocking reagent was 5% skimmed milk; the blocking time was 60 min at 37 degrees C; the second antibody concentration was 1:10 000; The cut-off value was 0.30. The method revealed a good specificity, no cross-reaction to the positive sera of BCV, IBRV or BRSV was observed. We applied the method to detect 323 serum samples of dairy cow in Heilongjiang Province, the seropositivity rate of BPIV3 was about 58%. The indirect ELISA established provided a technological basis for the development of ELISA kit.
Keywords:Bovine parainfluenza virus type 3  Hemagglutinin-neuraminidase protein  Prokaryotic expression  ELISA
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