低血清培养PK-15细胞及其增殖猪圆环病毒2型的研究

Study on PK-15 Cells Cultivated with Low Serum Culture and Culture Proliferation of Porcine Circovirus Type 2

依次采用含60、30、20、10mL/L血清浓度的低血清培养基驯化PK-15细胞,并给驯化好的细胞上接种猪圆环病毒2型(PCV-2),以确定PCV-2在该培养体系下的生长情况。结果用含60、30、20mL/L血清浓度的低血清培养基各进行3代次驯化,PK-15细胞能完全适应且生长状态良好;当血清浓度降至10mL/L时,传代1次细胞无法保持良好状态,细胞出现贴壁较差、生长停滞等现象。各血清浓度培养体系中细胞生长曲线测定结果显示,在细胞培养的0、24、48、72、96h各组细胞密度与常规培养的对照组差别不明显。因此用该低血清培养体系培养PK-15细胞时,血清最低添加量为20mL/L。用该体系培养的PK-15细胞接种PCV-2后,通过荧光抗体染色测定病毒滴度。结果显示,低血清培养的PCV-2病毒滴度为10^(6.5)TCID_(50)/mL,与常规条件培养的PCV-2对照(病毒滴度为10^(6.375 )TCID_(50)/mL)差别不明显,表明该体系可用于PCV-2的增殖。表明研究建立了PK-15细胞低血清培养PCV-2体系,为PCV-2的相关研究奠定了基础。

In this study,the low serum media containing 6% 3% 2%and 1% sera, respectively, were used to culture PK-15 cells and to proliferate the porcine circovirus type 2. The results showed that PK-15 ceils were in good growth state in concentrations of 6%, 3% and 2% sera culture medium, respectively, but when the concentration of the serum was decreased to 1%,the cells were in poor attachment and growth. Meanwhile,the densities of PK-15 cells at 0 h,24 h,48 h and 72 h time point in concentrations of 6% ,3% and 2% serum culture medium was not significantly different with the control. Thereby,the culture media containing 2 ~ serum were selected for cell cultivation and virus yield,and the virus titers were determined by indirect immunofluorescent assay. The results showed that the PCV-2 titer of the low serum culture was 10^6.5TCID50/mL,a little bit higher than that of the control （106＇375TCID50/mL）,but the difference was not significant. In this study,the PK-15 cell culture aging system with low serum culture medium for PCV-2 proliferation was established, which laid the foundation for the research of PCV-2.