Ⅲ型登革病毒D9964株在人胚肺二倍体细胞KMB17上适应株选育纯化及其增殖动力学研究

Adaptability and Purification of Dengue-Ⅲ Virus D9964 Strain in KMB17 Cells and Proliferation Kinetics of Adapted Strain

选育出Ⅲ型登革病毒中国株在人胚肺二倍体细胞KMB17上的适应株并对其生物学特性和增殖动力学进行研究,为研究登革病毒减毒活疫苗奠定基础。Ⅲ型登革病毒中国株D9964经RT-PCR鉴定型别正确后,进行毒种扩增和滴度测定;以4.0MOI接种KMB17细胞,反复传代直至病毒能在细胞中适应和增殖;连续传10代,选育出KMB17细胞适应株,经三轮噬斑纯化筛选适应株,微量滴定法检测10代病毒培养液的感染性滴度;免疫荧光法检测纯化病毒株的抗原性;将病毒纯化株接种KMB17细胞,取第3～7d细胞提取RNA,以Real-time PCR检测病毒适应株在细胞中的增殖动态。结果显示,经适应性培养后Ⅲ型登革病毒中国株D9964能在KMB17细胞中稳定增殖,经噬斑纯化获得了高纯度的细胞适应株,病毒保持了原始毒株良好的抗原性;增殖动力学研究显示第5～6d为病毒在细胞内的增殖高峰期。

To select the adaptive strain of Dengue-Ⅲ virus D9964 strain（China strain） in KMB17 cells, elu- cidate the biological characteristics and proliferation kinetics of adapted strain, and to lay the foundation for the development dengue inactivated vaccine and attenuated live vaccine. Dengue-Ⅲ virus D9964 strain was firstly identified by amplification of the type-specific gene segment of dengue virus by RT-PCR, and the ti- ter was determined. The virus was then subcultured in KMB17 cells with 4.0 MOI till completely adaptive to multiply in cell S. After subculturing in KMB17 cells for 10 consecutive passages, the adapted strain was screened, and purified through plaque. Virus titer of each passage was measured by microtitrimetry, and the antigenicity was detected by IFA. The purified virus RNA extraction of 3- 8 day cultured from KMB17 cells, was performed to detect the proliferation kinetics of adapted strain. The results showed that after continuous subculture, dengue-Ⅲvirus D9964（China） strain could stably proliferate in KMB17 cells, a highly purled virus adapted strain was obtained through plaque purification. Purified strain maintained the good antigenicity with a highest replicating activity during the 5th-6th day.