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目的确定呼吸道合胞病毒(respiratory syncytial virus,RSV)培养条件,筛选病毒保护剂配方,并建立检测病毒滴度的微量细胞病变方法。方法分别将Hep2、Vero和293R细胞以0.01 MOI接种RSV,选择病毒培养细胞基质;将RSV分别以0.005和0.02 MOI接种Hep2细胞,在4~9 d收获病毒液,检测病毒滴度,确定最佳MOI及病毒收获时间;将6种配方病毒保护剂分别加至病毒液中,反复冻融7次,检测病毒滴度,筛选最佳配方;将病毒分别在33和37℃条件下滴定,第7、9天判定结果,确定微量细胞病变方法的培养温度和判定时间。结果确定病毒培养细胞基质为Hep2细胞,以0.02 MOI RSV接种后,37℃培养7~9 d收获病毒液;配方1(0.1%人血白蛋白)为最佳病毒保护剂配方;微量细胞病变法的实验条件为37℃滴定,7 d判定结果。结论建立了稳定可靠的呼吸道合胞病毒培养及病毒滴度检测方法。
OBJECTIVE: To determine the culture conditions of respiratory syncytial virus (RSV), screen out the formulation of virus protectant and establish a method of detecting trace of virus cytopathic effect. Methods Hep2, Vero and 293R cells were inoculated with RSV at a MOI of 0.01 and the virus culture medium was selected. Hep2 cells were inoculated with RSV at 0.005 and 0.02 MOI respectively, and the virus solution was harvested from 4 to 9 days. MOI and the harvest time of the virus; 6 kinds of formulated virus protective agent were added to the virus solution, repeated freezing and thawing 7 times, the detection of virus titer, screening the best formula; the virus were titrated at 33 and 37 ℃, 7 , 9 days to determine the results to determine the incubation temperature and determination of trace cytotoxicity time. Results The virus-cultured cells were identified as Hep2 cells. After inoculation with 0.02 MOI RSV, the virus solution was harvested at 37 ℃ for 7-9 days. Formulation 1 (0.1% human serum albumin) was the best virus protectant formulation. The experimental conditions were 37 ℃ titration, 7 d judgment results. Conclusion The stable and reliable culture of respiratory syncytial virus and the detection method of virus titer were established.