BHK-21细胞稳定测定猪乙型脑炎病毒半数细胞感染量效价（TCID_（50））的条件优化及应用

为建立BHK-21细胞测定乙型脑炎病毒（Japenese encephalitis virus,JEV）病毒效价的方法,对96孔细胞板中不同初始接种量BHK-21细胞在多种维持液中的生长状态进行探讨,发现当BHK-21细胞以1250～2500个/孔接种96孔板培养24 h后,更换含3%新生牛血清的DMEM后在37℃、5%CO2条件下可至少良好维持72～96 h。在此条件下并基于Reed-Muench法测定病毒效价的原理,本文建立了准确测定JEV TCID50的方法,利用该方法对JEV野毒分离株及商品疫苗进行测定,均获得稳定、准确的病毒效价。本实验为JEV的体外培养、病毒定量、疫苗评价等提供了一种准确、稳定、易判定的参考方法。

A RELIABLE METHOD FOR DETERMNATION OF JAPANESE ENCEPHALITIS VIRUS TITERS

For accurately determining the 50% tissue culture infective dose（TCID50） of Japanese encephalitis virus（JEV）,the growth parameters of BHK-21 cells were optimized.These parameters included initial inoculation numbers of BHK-21 cells in 96-well plates and concentrations of new-born calf serum（NCS） supplemented to DMEM medium.After 1250-2500 BHK-21 cells were seeded per well,the plates were incubated for 24 hours at 37℃ in a 5% CO2 incubator.Then,the spent medium was replaced by the maintenance medium containing 3% NCS.The cell monolayers could be maintained for at least 72-96 hours.The Japanese encephalitis viruses（JEV） and live vaccines were inoculated onto BHK-21 cell monolayers and their titers were calculated by the Reed-Muench method.Results indicated that the established technique was a simple,accurate and reliable method for JEV antigen preparation,titration and vaccine quality evaluation.