【作者】 姜涛；

【导师】 殷玉和；

【作者基本信息】 长春工业大学 ， 化学， 2016， 硕士

【摘要】 目前,国内仅有的轮状病毒疫苗是由兰州生物制品研究所有限责任公司生产并于2000年注册批准上市。此疫苗采用lgCCID₅₀和ELISA联合法(lgCCID₅₀–ELISA)检测轮状病毒滴度,但试验已证明采用该方法测定病毒滴度在时间花费和重复性方面有其不足。在本试验中,我们建立了间接免疫荧光法（indirect immunofluoresce assay,IFA）检测兰州生物制品研究所有限责任公司生产的兰州羔羊轮状病毒疫苗的滴度,并对采取两种检测方法所得出的结果进行比较。本文重点对IFA法测定轮状病毒中各条件进行优化,其中包括MA104细胞最佳接入密度、稀释轮状病毒用胰酶的最适浓度和病毒感染MA104细胞的最佳时间等。结果表明:IFA法最佳工作条件为,MA104细胞密度2×104个/孔为最佳,稀释病毒用胰酶浓度为1mg/ml,轮状病毒最佳培养时间为18h,80%的丙酮作为固定液且固定时间为10min,兔抗SA11多克隆抗体为一抗的最适稀释度和作用时间分别为1:800、60min,Alexa-488标记的山羊抗兔抗体为二抗的最适稀释度和作用时间分别为1:500、60min。通过以上条件的摸索,确立了轮状病毒滴度的检测方法。将已知轮状病毒滴度的样品1′106 FCFU/ml经倍比稀释至10 FCFU/ml,应用IFA法检测各稀释倍数的滴度,最后确定应用该方法测定病毒样品的敏感性为10FCFU/ml;采用此IFA法测定经RV感染的MA104细胞在荧光显微镜下呈阳性,而检测狂犬病毒、流感病毒、水痘病毒感染MA104细胞为阴性,证明了IFA法的特异性;取相同批次的兰州生物制品研究所有限责任公司（LLR株）疫苗,使用该法进行9次重复检测试验,计算此批次疫苗病毒滴度的变异系数（CV）为2.3%。经试验证明我们建立轮状病毒的IFA检测方法具有敏感、特异、可重复等优点,此法为轮状病毒定性定量分析和疫苗生产中质量检测提供可靠依据。更多还原

【Abstract】 Currently, the only licensed rotavirus vaccine in China was developed by Lanzhou Institute of Biological Products Co. Ltd. in 2000. The rotavirus vaccine was detected with the log 50% cell culture infectious dose–enzyme-linked immunosorbent assay(lgCCID₅₀–ELISA), but experiments show that this method is limited on the time spent and experimental repeatability in quantifying rotaviral titers. In this experiment, we established an indirect immunofluorescence assay（IFA） to determine the infectious titer of Lanzhou lamb rotavirus（LLR） vaccine produced by Lanzhou Institute of Biological Products Co. Ltd, and we also compared the IFA and lgCCID₅₀–ELISA methods for assaying rotavirus vaccines.This article focus on optimizing the experimental conditions for IFA, including the optimal cell density for MA104 cells, the optimal trypsin concentration required to activate rotavirous, the best time to fluorescently stain the infected MA104 cells and so on. The research we find the optimum conditions of IFA was as follows: the cell density was 2 ′ 104 cells/well; Dilute the sample use virus diluents which comprise 1 μg/ml trypsin; The best time of virus infection for 18h; the 80% cold acetone as the stationary liquid and a fixation time of 10min; The working concentration of a rabbit anti-SA11 polyclonal antibody as the primary antibody and Alexa-488 labeled the goat anti-rabbit antibody as the secondary antibody were 1:800 and 1:500, and the time for bonding were all 60 min. Through exploring the optimum conditions of IFA, the test was established for assaying the titer of RV.Using IFA, it’s easy to detect a sample when the viral titer was diluted from 1 ′ 106 FCFU/ml to 10 FCFU/ml. This result showed that IFA was sufficiently sensitive to detect rotavirus at 10 FCFU/ml; The results were positive in MA104 cells infected with RV and were negative in MA104 cells infected with rabies virus, influenza virus, varicella virus. It showed that the IFA was specificity; To determine the reproducibility of IFA, the experiment using Lanzhou lamb rotavirus vaccine was repeated nine times. The coefficient of variation（CV） for the viral titers of the rotavirus vaccine was 2.3%. In summary, it is evident that this IFA is a sensitive, specific and repeatable method for the detection of RV in MA104 cells. This method provided a reliable basis for the qualitative and quantitative analysis of rotavirus and certifying the production of rotavirus vaccine.更多还原