两种培养条件下重组杆状病毒表达小反刍兽疫病毒N蛋白的丰度  

Abundance of PPRV N Protein Expressed by Recombinant Baculovirus System under Two Culture Conditions

作　　者：师亚玲 韩佃刚 董俊 叶玲玲 李凌枫 陈朝林 信吉阁[1] 艾军 Shi Yaling;Han Diangang;Dong Jun;Ye Lingling;Li Lingfeng;Chen Chaolin;Xin Jige;Ai Jun(Yunnan Agricultural University,Kunming,Yunnan 650201,China;Technology Center of Kunming Customs,Kunming,Yunnan 650200,China)

机构地区：[1]云南农业大学,云南昆明650201 [2]昆明海关技术中心,云南昆明650200

出　　处：《中国动物检疫》2022年第2期97-102,共6页China Animal Health Inspection

基　　金：云南省2014年度农业科技成果转化项目;海关总署科研项目(2019HK032)。

摘　　要：为了在重组杆状病毒上获得高丰度表达的小反刍兽疫病毒(PPRV)N蛋白,利用细胞贴壁培养和悬浮培养法培养重组杆状病毒Vbacmin-PPRV-N 120 h,用显微镜观察不同时间点细胞形态变化,随后在培养24、48、72、96、120 h时分别收集培养液,以ELISA方法检测PPRV N蛋白表达量,并用SPSS软件对数据进行统计分析。显微镜下观察发现,在感染重组杆状病毒后,两种方式培养的细胞随着时间增加,均由形态大小均匀、圆形、透亮、折光性好,变为大小不均匀、折光性差,并且出现了很多细胞碎片。根据ELISA检测结果,摇瓶悬浮培养条件下PPRV N蛋白的表达量显著高于贴壁培养,摇瓶悬浮培养的昆虫细胞蛋白表达量在96 h达到最高峰,120 h蛋白表达量开始下降,贴壁培养的昆虫细胞蛋白表达量在120 h达到最高峰。研究表明,用昆虫杆状病毒系统大规模表达PPRV N蛋白时更适合用摇瓶悬浮培养方法。本研究为优化昆虫细胞表达PRRV N蛋白的培养条件提供了参考。In order to obtain high abundance of peste petit ruminant virus(PPRV)N protein expressed by recombinant baculovirus system,the recombinant baculovirus,Vbacmin-PPRV-N,was cultured by cell adherent culture and shake flask culture for 120 hours,and the morphological changes of the cells were observed by microscope,then the culture supernatant was collected at 24,48,72,96 and 120 hours,respectively,to detect the expression amount of PPRV N protein by ELISA,and the data obtained were summarized and analyzed by SPSS software. It was found that,through microscopic observation,after infection with recombinant baculovirus,the cells cultured by the two methods changed from equal size,round,transparency and good refractivity to unequal size,poor refractivity with many cell fragments. Based on the results of ELISA,the expression amount of PPRV N protein in shake flask culture was significantly higher than that in adherent culture. The protein expression of insect cells cultured in shake flask reached the peak at 96 h and began to decrease at 120 h,while that in adherent culture reached the peak at 120 h.It was concluded that the shake flask culture was appropriate for expression on a large scale when PPRV N protein was expressed by recombinant baculovirus system. A reference was thereby provided for optimizing the culture conditions by recombinant baculovirus system to express PPRV N protein.

关 键 词：小反刍兽疫病毒N蛋白 Sf9昆虫细胞 贴壁培养 悬浮培养 蛋白表达 

分 类 号：S852.65[农业科学—基础兽医学]