论文部分内容阅读
目的筛选Ⅰ型登革病毒(Dengue virus,DV)中国株D06063人胚肺二倍体细胞KMB17的适应株,经噬斑纯化后,分析其生物学特性。方法将Ⅰ型DV中国株D06063在C6/36细胞上进行扩增,采用微量滴定法测定病毒的感染性滴度;RT-PCR法鉴定DV型别;病毒连续以4.0 MOI的量感染KMB17细胞,传代至病毒完全适应在细胞内扩增,再连续传10代,筛选出KMB17细胞适应株,并进行3轮噬斑纯化;免疫荧光法检测纯化病毒,电镜观察病毒颗粒的形态。结果在C6/36细胞上扩增的Ⅰ型DV D06063株的滴度为6.0 lg CCID50/ml,经RT-PCR可扩增出511 bp的DV特异基因和482 bp的Ⅰ型DV型特异性基因;病毒感染KMB17细胞后,可产生明显的细胞病变,至第10代,病毒的感染性滴度达峰值,为6.75 lg CCID50/ml;纯化的病毒经免疫荧光检测呈阳性,电镜观察显示病毒形态正常。结论成功筛选出传代稳定性好、病毒扩增量高的Ⅰ型DVKMB17细胞适应株,纯化的病毒株保持了原始毒株的基本生物学特性。
Objective To screen the adapted strain of D606063 human embryo lung diploid cell type Ⅰ strain Dengue virus (DV), and analyze its biological characteristics after plaque purification. Methods Type Ⅰ DV China strain D06063 was amplified on C6 / 36 cells. The infectious titer was determined by microtitration method. The DV type was identified by RT-PCR. The virus was infected with KMB17 cells at an MOI of 4.0. Passaged to the virus completely adapted to expansion in the cell, and then continuously transmitted 10 generations, screened KMB17 cell adapted strains, and three rounds of plaque purification; immunofluorescence assay of purified virus, electron microscopy morphology of the virus particles. Results The titer of type Ⅰ DV D06063 strain amplified on C6 / 36 cells was 6.0 lg CCID50 / ml, and the 511 bp DV specific gene and 482 bp type I DV type specific gene were amplified by RT-PCR ; Virus infected KMB17 cells, can produce significant cytopathic, to the tenth generation, the virus infectious titer reached the peak, 6.75 lg CCID50 / ml; purified virus was positive by immunofluorescence, electron microscopy showed that the virus morphology normal. Conclusion The type Ⅰ DVKMB17 cell adapted strains with good stability of passage and high amplification of virus were successfully screened. The purified virus strains maintained the basic biological characteristics of the original strain.