益于猪圆环病毒2型增殖的PK-15细胞系的克隆筛选与应用

Cloning,screening and application of PK-15 cell lines enhancing proliferation of porcine circovirus type 2

通过有限稀释法对被猪圆环病毒2型（PCV2）感染的PK-15细胞进行2轮细胞克隆筛选,得到1株对PCV2高度易感且稳定的同源性PK-15细胞克隆株,以提高PCV2在PK-15细胞系中的感染效价。结果显示,利用间接免疫荧光试验（IFA）从成功获得的7株PK-15细胞克隆株中筛选得到3株荧光反应较强的PK-15细胞克隆株。通过IFA、半定量PCR和Western-blot等方法确定PCV2可以在PK-15细胞克隆株2中高效克隆并表达PCV2 Cap蛋白。QPCR结果表明,PCV2感染该PK-15细胞克隆株后第48小时病毒表达量达到最高。这证实,获得的高敏感性PK-15细胞克隆株2可用于持续产出高效价的PCV2以便于相关疫苗和诊断制剂的研制。

To improve the proliferation capacity of porcine circovirus type 2（PCV2） in the PK-15 cell,PK-15 cells infected with PCV2 were cloned and screened in two rounds by using limited dilution method to obtain a highly sensitive and stable homologous PK-15 clone.The results showed that 3 cell clones with strong fluorescence reaction were successfully screened from 7 PK-15 cell clones by indirect immunofluorescence assay（IFA）.It was determined that PCV2 can be effectively cloned and express PCV2 Cap protein in PK-15 clone 2 by IFA,semi-quantitative PCR and Western-blot.QPCR showed that PCV2 expression level was the highest in PK-15 clone 2 after 48 h.This indicated that the highly sensitive PK-15 clone 2 can be used to produce high titer PCV2 continuously for the development of PCV2 vaccine and diagnostic preparation.