论文部分内容阅读
目的研究Ⅳ型登革病毒中国株LD34在人胚肺二倍体细胞KMB17上的传代适应性及其生物学特性。方法将Ⅳ型登革病毒中国株LD34在C6/36细胞上扩增,并采用微量细胞病变法测定病毒的感染性滴度。以2.0 MOI的病毒接种KMB17细胞传代培养,筛选KMB17细胞适应株,并进行培养条件的优化。将Ⅳ型登革病毒中国株LD34 KMB17细胞适应株连续传10代,测定病毒的感染性滴度,免疫荧光法检测病毒的抗原性,RT-PCR法扩增登革病毒的特异性基因。结果筛选出的Ⅳ型登革病毒中国株LD34在KMB17细胞上的最佳培养条件为病毒接种MOI 0.4,培养基血清浓度5%;其感染KMB17细胞后可产生明显的细胞病变(CPE),连续传10代,病毒滴度达7.75 CCID50/ml;第10代病毒的抗原性呈阳性;第10代病毒能扩增出511 bp的登革病毒特异性基因和393 bp的Ⅳ型登革病毒特异性基因。结论获得了Ⅳ型登革病毒中国株LD34 KMB17细胞适应株,病毒保持了原始毒株的基本生物学特性,且具有较好的抗原性。
Objective To study the passage adaptability and biological characteristics of dengue virus type Ⅳ dengue virus (HBV) in human embryo lung diploid cell line KMB17. Methods The dengue virus type Ⅳ LD34 was amplified on C6 / 36 cells and the infectious titer of the virus was determined by the method of micro cytopathic assay. KMB17 cells were inoculated with virus at 2.0 MOI and passaged to KMB17 cells for selection. The culture conditions were optimized. The dengue virus type Ⅳ Dengue virus strain LD34 KMB17 was continuously passaged for 10 generations, the infectious titer of the virus was determined, the antigenicity of the virus was detected by immunofluorescence, and the dengue virus-specific gene was amplified by RT-PCR. Results The optimal culture conditions of dengue virus type Ⅳ dengue virus type Ⅳ on KMB17 cells were MOI 0.4 and 5% serum concentration. The cells infected with KMB17 cells could produce significant cytopathic effect (CPE) The passage 10 generation showed a titer of 7.75 CCID50 / ml. The antigenicity of the 10th generation of virus was positive. The 10th generation of virus could amplify 511 bp of dengue virus-specific gene and 393 bp of dengue virus type IV Sexual gene. Conclusion The strain of dengue virus type Ⅳ LD34 KMB17 was obtained. The virus retained the basic biological characteristics of the original strain and had good antigenicity.