摘要
目的建立重组杆状病毒滴度间接免疫荧光法的检测方法,并对其进行验证。方法构建含有重组质粒pGEX-6p-1-GST-GP64大肠埃希菌BL21(DE3),进行IPTG(isopropyl-β-D-thiogalactoside,IPTG)诱导,表达并纯化杆状病毒截短GP64糖蛋白。配伍佐剂后采用背部多点注射的方式免疫日本大耳白兔,制备兔抗杆状病毒GP64多抗作为免疫荧光结合抗体,在96孔板里贴壁培养Sf9细胞,接种10倍系列稀释的重组杆状病毒共孵育一定时间,80%冷丙酮固定、透化,一抗稀释比例为1∶200、荧光二抗稀释比例为1∶500,荧光显微镜下显色,计数荧光灶数目计算病毒滴度。并对建立的方法进行验证。结果成功制备了GP64兔多抗;检测时Sf9细胞与重组杆状病毒共孵育时间4 d;重组杆状病毒感染细胞可与GP64抗体发生特异的荧光灶反应,未感染细胞对照组未出现特异性荧光灶;组内和组间测定结果的RSD均<5%;同一样品不同人员检测差异无统计学意义(F=1.86,F<F_(表));同一样品梯度稀释后,稀释倍数的对数与病毒滴度呈线性相关(R^(2)=0.997);病毒滴度的回收率在90%~110%之间。结论建立了间接免疫荧光法测定重组杆状病毒滴度的方法,该方法具有良好的专属性、精密度、线性和准确度。
Objective An indrecct immunofluorescence method for detection of the recombinant baculovirus titer was estab-lished and validated.Methods The Escherichia coli BL21(DE3)containing recombinant plasmid pGEX-6p-1-GST-GP64 was induced by IPTG(isopropyl-β-D-thiogalactoside,IPTG)for expression of the truncated GP64 glycoprotein of baculovirus,then the protein was purified and immunized with adjuvant by multiple point injection on the back in Japanese white rabbits.The rabbit anti-GP64 polyclonal antibody was prepared as an immunofluorescence primary antibody to identify sf9 cells infected by baculovirus.Sf9 cells were cultured in 96 well plates,inoculated with 10 folds serially diluted recombinant baculoviruses and incubated for a certain time.Infected cells were fixed and permeabilized with 80%of cold acetone The concentrations of the first and second fluorescent antibodies were optimized at dilutions of 1∶200 and 1∶500.The fluorescent foci were observed and counted using fluorescence microscope to calculate the virus titers.The established method was verified at last.Results GP64 rabbit polyclonal antibody was successfully prepared.Confluent adherent SF9 cells were co-incubated with diluted recombinant viruses for 4 days.The infected SF9 cells by recombinant baculoviruses could be detected with the specific GP64 antibody.In contrast,mock infected cells did not show the specific fluorescent focus.The relative standard deviations of the measurement values intra-group and inter-group were less than 5%.There was no statistically significant difference between the detection results performed by different personnel for the same sample(F=1.86,F<F_(表)).After gradient dilution of the same sample,the logarithm of dilution times is linearly related to the virus titer,and the linear correlation coefficient R^(2)=0.997.The recovery rate of virus titer was between 90%and 110%.Conclusion An indirect immunofluorescence method for measuring the titers of recombinant baculoviruses was established and validated.The method has good specificity,precision,linearity and accuracy.
作者
熊宇
左勇
郭靖
孟胜利
申硕
陈晓琦
XIONG Yu;ZUO Yong;GUO Jing;MENG Sheng-li;SHEN Shuo;CHEN Xiao-qi(Viral Vaccine Research Laboratory,Wuhan Institute of Biological Products,Wuhan 430207,Hubei Province,China)
出处
《微生物学免疫学进展》
CAS
2023年第3期14-20,共7页
Progress In Microbiology and Immunology
基金
国家“重大新药创制”科技重大专项(2015ZX09102021)。
关键词
杆状病毒
GP64糖蛋白
间接免疫荧光方法
荧光灶反应
Recombinant baculovirus
GP64 glycoprotein
Indirect immunofluorescence method
Fluorescence foci reaction
