猪轮状病毒DN30209株的驯化培养  

Domestication of porcine rotavirus(PRV) strain DN30209

作　　者：赵高伟[1] 段凤娟[1] 任玉东[1] 佟玲[1] 王婧婧[1] 曹丽艳[1] 任晓峰[1] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2013年第4期384-389,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31270187);教育部新世纪优秀人才项目(NCET-10-0144);教育部高等学校博士学科点专项科研基金项目(博导类)(20122325110019);黑龙江省长江学者后备人才计划项目;哈尔滨科技局创新人才研究资金项目(RC2012XK002003)

摘　　要：为获得一株猪轮状病毒弱毒疫苗的候选株,用恒河猴肾细胞(MA-104)培养之前分离的猪轮状病毒DN30209株(PRV DN30209),并对预处理病毒的胰酶的浓度及时间、维持液(DMEM)中胰酶浓度和时间等条件进行优化,在最佳培养条件下对病毒进行连续传代,检测各代次病毒的TCID50,达到最高且稳定时,用该代次的病毒免疫PRV阴性BALB/c小鼠,于免疫后不同时间检测血清特异性中和抗体效价。结果,经20μg/mL胰酶37℃孵育40min,吸附90min,在含2μg/mL胰酶的DMEM中,病毒能在MA-104稳定增殖。经60代培养病毒TCID50由最初的103.40/mL增至106.00/mL,传至第60～63代时病毒TCID50维持在106.00/mL左右,并趋于稳定。选第60代病毒免疫小鼠,2周后小鼠血清中和效价为1∶91,4周后达到1∶128。结果表明,PRV DN30209株通过传代驯化培养可以作为弱毒疫苗候选株。To obtain an attenuated vaccine candidate strain of porcine rotavirus, a porcine rotavirus DN30209 strain（PRV DN30209） recently isolated in our laboratory was cultured with rhesus monkey kidney cells（MA-104）. The optimized trypsin concentration and conditions were used. The virus was passaged under the best conditions and the TCID50 of each passage was determined. PRV negative BALB/c mice were immunized when the TCID50 of PRV DN30209 reached the highest level. Serum specific neutralizing anti body titer was detected at different time points. The results showed that the virus could stably proliferate in MAq04 cells under conditions of incubation for 40 rain at a final trypsin concentration of 20μg/mL,and then absorption for 90 rain at 2 μg/mL trypsin in DMEM medium. The virus TCIDs0 increased from 103^3.40/mL to 106^6.00/mL and the virus TCIDs0 maintained around 106^6.00/mL when virus was at the passages of 60--63（P60--63）. The serum neutralizing titers was 1 ： 91 on week 2 post-immunization and reached 1 ： 128 on week 4 post-immunization when the mice were immunized with the P60 PRV. The present study provides reference for development of PRV vaccines using PRV DN30209 candidate strain.

关 键 词：轮状病毒 培养特性 疫苗株 

分 类 号：S852.651[农业科学—基础兽医学]