CAJ | 학술논문

目的研究乙脑病毒变异的特性以及变异株性状与E蛋白表达的关系.方法通过应用乙脑病毒的野生株以及人肝癌细胞KN73建立持续感染模型.采用胰蛋白酶消化技术每周进行细胞传代.经反复冻融收集持续感染细胞内病毒.采用BHK细胞空斑实验方法进行病毒滴定.采用间接免疫荧光方法检测E和NS3蛋白抗原.采用Western印迹杂交检测E和NS3蛋白的表达.结果感染早期(24小时-36小时)野生株感染的KN73细胞的培养液中病毒滴度为106 PFU/ml.在感染后期(3年)滴度为103-4 PFU/ml.病毒重叠感染实验发现在急性重叠感染野生株的持续感染KN73细胞中培养液的病毒滴度比在同一时相急性感染的正常KN73细胞培养液的滴度要低得多.间接免疫荧光检测表明,在持续感染的KN73细胞中病毒抗原的存在低于急性感染的KN73细胞.Western印迹杂交检测表明,E、NS3蛋白的分子量分别为53KD和73KD.在持续感染的KN73细胞中NS3蛋白的表达很稳定,但E蛋白的表达却明显受抑.结论从持续感染的KN73细胞中获得的病毒具有缺陷干扰病毒的一些特性且参与持续感染,其毒力和增殖均低于亲本病毒.这些变异可能与E蛋白表达减少有关.
목적연구을뇌병독변이적특성이급변이주성상여E단백표체적관계.방법통과응용을뇌병독적야생주이급인간암세포KN73건립지속감염모형.채용이단백매소화기술매주진행세포전대.경반복동융수집지속감염세포내병독.채용BHK세포공반실험방법진행병독적정.채용간접면역형광방법검측E화NS3단백항원.채용Western인적잡교검측E화NS3단백적표체.결과감염조기(24소시-36소시)야생주감염적KN73세포적배양액중병독적도위106 PFU/ml.재감염후기(3년)적도위103-4 PFU/ml.병독중첩감염실험발현재급성중첩감염야생주적지속감염KN73세포중배양액적병독적도비재동일시상급성감염적정상KN73세포배양액적적도요저득다.간접면역형광검측표명,재지속감염적KN73세포중병독항원적존재저우급성감염적KN73세포.Western인적잡교검측표명,E、NS3단백적분자량분별위53KD화73KD.재지속감염적KN73세포중NS3단백적표체흔은정,단E단백적표체각명현수억.결론종지속감염적KN73세포중획득적병독구유결함간우병독적일사특성차삼여지속감염,기독력화증식균저우친본병독.저사변이가능여E단백표체감소유관.
Objective To study the character of mutants originating from Japanese encephalitis viruses and the relationship between the characterization of mutant strains and E protein expression.Methods Persistent infection was established with standard strains of Japanese encephalitis viruse, known as parental viruse, in a human hepatoma cell line, KN73. Cells were subcultured weekly using trypsinization techniques. Cell-associated viruses of persistently infected cells were collected by a freeze and thaw method. Virus titers were examined by plaque method using baby hamster kidney (BHK) cells. Indirect immunofluorescence assays were used to examine E and NS3 protein antigens. Wester*# n blot analysis was used to test expression of E and NS3 proteins. Results In the early phase (24-36 h) post-infection, virus titer in culture fluid from KN73 cells infected with parental viruses were 106 PFU/ml. They were 103-4 PFU/ml in the late phase (3 years) post-infection. The titer of cell-associated viruse was 102-3 PFU/ml. A virus super-infection assay found that virus titers in culture fluid from persistently infected KN73 cells acutely super- infected with parental viruses were much lower than that of culture fluids in acutely infected normal KN73 at the same phase. Indirect immunoflurescence assay revealed that the quantity of viral antigens in persistently infected KN73 cells was lower than that in acutely infected KN73 cells with parental viruses. Western blot analyses indicated that the molecular weights of E and NS3 proteins were 53 kD and 73 kD, respectively. Expression of NS3 protein in persistently infected KN73 cells was stable but expression of E protein was markedly suppressed. Conclusions The virulence and reproduction of viruses obtained from persistently infected KN73 cells, which have some features of DI viruses and were involved in persistent infection, was lower than that of parental viruses. These mutants may have be related to the decrease in E protein expression.