适合猪圆环病毒2生长的PK15均质细胞株的构建  

Construction of homogeneous PK15 cell line suitable for cultivation of porcine circovirus 2

作　　者：罗维[1] 赵墩[1] 蒋大良[1] 余兴龙[1] 

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128

出　　处：《湖南农业大学学报（自然科学版）》2013年第4期404-408,共5页Journal of Hunan Agricultural University(Natural Sciences)

基　　金：国家自然科学基金(30571390)

摘　　要：为筛选出PCV2培养滴度最高的PK15细胞克隆株,采用稀释法从PK15混合细胞中分离单细胞克隆株,获得的细胞克隆用于接毒试验,即用同步接种法将猪圆环病毒2(PCV2)病毒细胞混合液以1∶100的比例接种单克隆细胞,接毒后经PCR与定量PCR检测得出A10细胞克隆培养PCV2,获得的病毒含量最高。将A10细胞进行第2轮细胞克隆,之后用同步接种法和异步接种法将PCV2感染A10细胞,并检测A10细胞接毒后的PCV2 DNA含量,2种接毒方法获得的最高PCV2基因组的拷贝数分别为3.0×109、6.2×109/mL,高于用PK15混合细胞培养PCV2所获得的最高PCV2基因组拷贝数(107/mL)。综合以上结果,构建的PK15均质细胞克隆株比PK15混合细胞更适合PCV2的培养。To screen single cell clone line for cultivation of PCV2 with the highest titer, dilution method was used to isolated single clone lines from PK15 heterogeneous cells. The obtained single cell clones were subjected to PCV2 infection through synchronized inoculation at a ratio of I ： 100. PCR and quantitative PCR analysis showed the yield of PCV2 in cell clone A10 was the highest after PCV2 infection. Then second cell clone was conducted on A10 cell to ensure the homogeneous character of A10, followed by PCV2 inoculation test through synchronized and non-synchronized inoculations. The result of quantitative PCR indicated the maximum PCV2 genomic copy numbers obtained with synchronized and non-synchronized inoculations were 3.0×10^9/mL and 6.2×10^9/mL, respectively, which were higher than the maximum PCV2 genomic copy numbers with PK15 heterogeneous cells. These results suggest the constructed homogeneous PKI 5 cell line is more suitable to cultivate PCV2 compared to PK15 heterogeneous cells.

关 键 词：猪圆环病毒2(PCV2) PK15细胞 细胞克隆 定量PCR 同步接毒 异步接毒 

分 类 号：S852.659[农业科学—基础兽医学]