新型狂犬病病毒中和抗体检测方法的建立  

Development of a novel method for detection of rabies virus neutralizing antibody

作　　者：薛向红[1,2,3] 郑学星[2,3] 盖微微[2,3] 李岭[1,2,3] 马金柱[2,3] 冯娜[2,3] 王铁成[2,3] 黄耕[2,3] 赵永坤[2,3] 杨松涛[2,3] 夏咸柱[1,2,3] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]军事医学科学院军事兽医研究所,吉林长春130062 [3]吉林省人兽共患病预防与控制重点实验室,吉林长春130062

出　　处：《中国生物制品学杂志》2013年第8期1165-1169,1174,共6页Chinese Journal of Biologicals

基　　金：公益性行业(农业)科研专项(201103032);"十一五"国家科技支撑计划重点项目(2010BAD04B03)

摘　　要：目的建立一种安全、快速、经济的新型狂犬病病毒中和抗体的检测方法。方法在狂犬病病毒弱毒株HEP-Flury基因组Ψ区插入增强型绿色荧光蛋白(enhanced green fluorescent protein eGFP)基因,利用反向遗传技术,拯救重组病毒rHEP-eGFP,分别采用荧光显微镜观察、直接免疫荧光染色鉴定及电镜观察等方法对rHEP-eGFP病毒进行鉴定;分别绘制rHEP-eGFP和亲本毒株HEP-Flury的生长动力学曲线;将rHEP-eGFP在BHK-21细胞中连续传代9次,测定各代次rHEP-eGFP的滴度,荧光显微镜下观察eGFP的表达;采用TCID50法检测狂犬病病毒标准攻击毒株CVS-11和rHEP-eGFP的毒力;以rHEP-eGFP病毒为抗原,建立新型荧光抗体病毒中和试验(fluorescent antibodyvirus neutralization,FAVN)-eGFP,对25份犬血清的抗体效价进行测定,并与标准FAVN检测结果进行比较。结果经荧光显微镜观察、直接免疫荧光染色鉴定和电镜观察表明,成功拯救出rHEP-eGFP病毒;重组病毒rHEP-eGFP与亲本病毒HEP-Flury的生长特性相似;rHEP-eGFP连续传代9次,均能稳定表达eGFP;CVS-11的毒力为108TCID50/ml,rHEP-eGFP的毒力为107.3TCID50/ml;FAVN-eGFP与FAVN测定25份犬血清抗体效价的结果具有良好的一致性。结论建立的新型狂犬病病毒中和抗体检测方法(FAVN-eGFP)准确度高,特异性好。Objective To develop a safe,rapid and economic method for detection of rabies virus neutralizing antibody.Methods Recombinant plasmid pHEP-eGFP was constructed by inserting enhanced green fluorescent protein（eGFP） gene into the Ψ region of genome of attenuated rabies virus HEP-Flury strain,based on which recombinant rabies virus rHEP-eGFP was rescued by reverse genetic technique,and identified by fluorescent microscopy,direct immunofluorescent staining and electron microscopy.The growth dynamic curves of rHEP-eGFP and its parental strain HEP-Flury were plotted.The rHEP-eGFP was subcultured for nine passages in BHK-21 cells.The titers of various passages were determined,and the expression of eGFP was observed by fluorescent microscopy.The virulence of rabies virus standard challenging strain CVS-11 and rHEP-eGFP was determined by TCID50 method.A novel fluorescent antibody virus neutralization（FAVN）-eGFP assay was developed using rHEP-eGFP as antigen,by which 25 dog serum samples were detected,and the results were compared with those by FAVN assay.Results Fluorescent microscopy,direct immunofluorescent staining and electron microscopy proved that rHEP-eGFP was successfully rescued,of which the characters of growth were similar to those of its parental strain HEP-Flury.After rHEP-eGFP was subcultured for 9 passages,eGFP was expressed stably.The virulence of CVS-11 and rHEP-eGFP were 108 and 107.3 TCID50 / ml respectively.The detection results of 25 dog serum samples by FAVN-eGFP and FAVN were in agreement.Conclusion The developed FAVN-eGFP method showed high accuracy and specificity.

关 键 词：狂犬病病毒 HEP-Flury株 绿色荧光蛋白 荧光抗体病毒中和试验 血清效价 

分 类 号：R373.9[医药卫生—病原生物学] R392.33[医药卫生—基础医学]