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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 establish a safe, rapid and economical detection method of novel rabies virus neutralizing antibody. Methods The gene of enhanced green fluorescent protein eGFP was inserted into the Ψ region of attenuated HEP-Flury strain of rabies virus. Recombinant virus rHEP-eGFP was rescued by reverse genetic technique. The expression of rHEP-eGFP was detected by fluorescence microscopy and direct immunofluorescence staining The growth kinetic curves of rHEP-eGFP and parental strain HEP-Flury were respectively plotted, and the rHEP-eGFP was serially passaged in BHK-21 cells for 9 times to determine the expression of rHEP-eGFP. The rHEP-eGFP titer was observed under a fluorescence microscope to observe the eGFP expression. The TCID50 method was used to detect the virulence of the rabies virus standard challenge strains CVS-11 and rHEP-eGFP. The rHEP-eGFP virus was used as a new fluorescent antibody The antibody titers of 25 canine serums were determined by fluorescent antibody virus neutralization (FAVN) -eGFP and compared with the standard FAVN test results. The results of fluorescence microscopy, direct immunofluorescence staining and electron microscopy showed that the rHEP-eGFP virus was successfully rescued; the recombinant virus rHEP-eGFP had similar growth characteristics with the parental virus HEP-Flury; rHEP-eGFP was passaged 9 times in succession, Stable expression of eGFP; virulence of CVS-11 108TCID50 / ml, rHEP-eGFP virulence was 107.3TCID50 / ml; FAVN-eGFP and FAVN determination of 25 canine serum antibody titer results with good consistency. Conclusion The detection method of new rabies virus neutralizing antibody (FAVN-eGFP) has high accuracy and specificity.