摘要
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 investigate the adaptability of Dengue-Ⅳ virus LD34 strain,isolated in China,in KMB17 cells and the biological characteristics of adapted strain.Methods Dengue-Ⅳ LD34 strain was propagated in C6 / 36 cells and determined for infectivity,then inoculated to KMB17 cells at a MOI of 2.0 for subculture,based on which the adapted strain was screened,and the culture condition was optimized.The adapted strain was subcultured in KMB17 cells for 10 passages,then determined for infectious titer by microtitrimetry,and for antigenicity by IFA,from which the specific gene was amplified by RT-PCR.Results The optimal MOI and serum concentration in medium for culture of the screened adapted strain were 0.4 and 5% respectively.The adapted strain caused obvious CPE of KMB17 cells,and reached a titer of 7.75 CCID50 / ml after subculture for 10 passages.The virus of passage 10 was positive for antigenicity,from which a Dengue virus-specific gene fragment at a length of 511 bp and a Dengue virus-Ⅳ-specific gene fragment at a length of 393 bp were amplified.Conclusion A KMB17 cells-adapted Dengue-Ⅳ virus LD34 strain was obtained,which showed the primary biological characteristics of original strain and good antigenicity.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第9期1135-1138,共4页
Chinese Journal of Biologicals
基金
云南省科技厅社会发展科技计划项目(2011CA016)
中国医学科学院病原生物学研究所中央级公益性科研院所科研业务费项目(2009IPB102)
中国医学科学院医学生物学研究所引进人才项目(IMB2009RC01)
云南省自然科学基金项目(2009ZC187M)
关键词
登革病毒
人胚肺二倍体细胞
适应性
生物学特性
Dengue virus
Human embryonic lung diploid cells
Adaptability
Biological characteristics
