家蚕微孢子虫经不同冻存条件处理后对家蚕胚胎细胞的侵染性变化  

Infectivity Variation of Nosema bombycis to Bombyx mori Embryonic Cells Under Different Cryopreservation Treatments

作　　者：马强[1] 党晓群[2] 王营[1] 陈洁[1] 刘方燕 赵丽芳[1] 曹琛福[3] 吕建强[3] 潘国庆[1] 周泽扬[1,2] 

机构地区：[1]家蚕基因组生物学国家重点实验室,西南大学,重庆400716 [2]重庆师范大学生命科学学院,重庆400047 [3]深圳出入境检验检疫局动植物检验检疫中心,广东深圳518045

出　　处：《蚕业科学》2014年第6期1055-1061,共7页ACTA SERICOLOGICA SINICA

基　　金：“十二五”农村领域国家科技计划项目(No.2012AA10-1301);国家自然科学基金重点项目(No.31270138);国家自然科学基金青年基金项目(No.31302037);重庆市教委科学技术研究项目(No.KJ130617);质检总局科技项目(No.2013IK042);重庆市教委基础与前沿研究计划项目(No.cstc2014jcyj A80019)

摘　　要：为了解家蚕微孢子虫保存方法对其侵染性的影响及大量获得细胞感染实验材料,将采用不同冻存液及冻存法处理的家蚕微孢子虫分别接种于家蚕胚胎细胞(Bm E),调查家蚕微孢子虫对细胞的感染率及感染细胞的传代能力。与液氮直接冻存法处理和高温高压灭菌法处理的微孢子虫相比较,用液氮梯度冻存法处理的微孢子虫的粘附率和感染率均显著或极显著提高。采用液氮梯度冻存法以水作冻存液处理的微孢子虫对宿主细胞的感染率为64%,感染细胞能进行连续传代培养;而以含10%二甲基亚砜(DMSO)的胎牛血清作冻存液处理的微孢子虫虽然对宿主细胞的感染率达到80%,但感染细胞在培养7 d后大量破裂死亡。因此,选用以水作冻存液经液氮梯度冻存法处理的家蚕微孢子虫有利于感染Bm E细胞并在细胞内增殖,从而获得大量的细胞感染实验材料。In order to study influence of preservation methods on Nosema bombycis infectivity and to acquire infected cells in large quantity for laboratory use,different cryoprotectants and different cryopreservation methods were employed to preserve Nosema bombycis spores which were then used to inoculate Bombyx mori embryonic cells（ Bm E）. The infection rate of microsporidian spores and subculture generation of infected cells were investigated. Compared to microsporidian spores treated by liquid nitrogen direct cryopreservation and autoclaving,microsporidian spores treated by liquid nitrogen gradient cryopreservation had significantly or extremely significantly higher adherence rate and infection rate. Microsporidian spores treated by liquid nitrogen gradient cryopreservation using water as cryoprotectant had an infection rate of 64% to host cells,and the infected cells could be continuously sub-cul-tured. Although the infectivity of spores preserved using 10% dimethy sulfoxide（ DMSO）-containing fetal bovine serum（ FBS） as cryoprotectant had an infection rate as high as 80%,a great deal of the infected cells ruptured and died at 7d post infection. Therefore,microsporidian spores treated by liquid nitrogen gradient cryopreservation using water as cryoprotectant have high proliferative rate in Bombyx mori embryonic cells,which can be utilized to prepare large quantity of infected cells for laboratory use.

关 键 词：家蚕微孢子虫 侵染性 液氮梯度冻存法 冻存液 家蚕胚胎细胞 增殖 

分 类 号：S884[农业科学—特种经济动物饲养]