轮状病毒基因重配株LD_9(G_2型)Vero细胞适应株的选育及其生物学特性  

Screening and Biological Characteristics of Vero Cell-adapted Reassortant Rotavirus LD_9 Strain(G_2 Type)

作　　者：寇桂英[1] 胡广宏[1] 包红[1] 王名强[1] 陈汉泉[1] 汪洲[1] 周旭[1] 

机构地区：[1]兰州生物制品研究所第二研究室,兰州730046

出　　处：《中国生物制品学杂志》2010年第8期857-860,共4页Chinese Journal of Biologicals

摘　　要：目的探讨轮状病毒基因重配株LD(9G2型)在Vero细胞上的传代适应性,选育高滴度的适应株。方法将3个轮状病毒基因重配株LD9克隆株(G2型)(LD9-1、LD9-2、LD9-3)在Vero细胞上分别以0.05、0.10、0.15和0.20MOI传代,选育出1株Vero细胞适应株,并确定其最适MOI,然后在Vero细胞上连续传代15代,检测病毒滴度及基因组核酸带型,采用nestRT-PCR法扩增其VP7基因。取第8、10、13代适应株病毒,以0.10MOI接种于Vero细胞,观察病毒的增殖动态。结果选育出的LD9-2株以0.10MOI接种Vero细胞可产生明显的细胞病变(CPE),病毒滴度随传代次数的增加先下降后升高,至4代时最低,9代后稳定于6.75lgCCID50/ml左右;连续传代15次,病毒基因组核酸带型均与原始毒株一致,且均能扩增出502bp的VP7基因。LD9-2株病毒增殖高峰期均出现在第7～8天,病毒滴度为6.00～7.00lgCCID50/ml。结论轮状病毒基因重配株LD9株(G2型)经Vero细胞适应性培养,获得了病毒表达量高且能稳定传代的疫苗候选株。Objective To investigate the adaptability of reassortant rotavirus LD9 strain（G2 type）in Vero cells and screen the adapted strain with high titer. Methods Three clones of reassortant rotavirus LD9 strain（G2 type）, i.e. LD9-1, LD9-2 and LD9-3, were inoculated into Vero cells at MOIs of 0. 05, 0. 10, 0. 15 and 0. 20 separately for subculture, from which one Vero cell-adapted strain was screened, and its MOI was optimized. The adapted strain was subcultured in Vero cells for 15 passages then determined for virus titer and nucleic acid band patterns of genome, from which VP7 gene was amplified by nest RT-PCR. The adapted strain of passages 8, 10 and 13 were inoculated into Vero cells at a MOI of 0. 10 and observed for proliferation. Results The screened LD9-2 strain, inoculated into Vero cells at a MOI of 0. 10, caused obvious CPE, of which the titer decreased at first then increased with the increasing passages. The titer of passage 4 was the lowest, while those of more than passage 9 was stable at about 6. 75 lgCCID50 / ml. All the nucleic acid band patterns of the adapted strain of passages 1 ～ 15 were consistent with that of primary strain, from each of which the VP7 gene at a length of 502 bp was amplified. All the proliferation peaks of LD9-2 strain of passages 8, 10 and 13 appeared on days 7 ～ 8 after culture, with titers of 6. 0 ～ 7. 0 lgCCID50 / ml. Conclusion A candidate rotavirus vaccine strain （G2 type）with high titer, which may be stably subcultured, is obtained by adaptive culture of reassortant rotavirus LD9 strain in Vero cells.

关 键 词：轮状病毒疫苗 基因重配株 VERO细胞 生物学特性 

分 类 号：R373.2[医药卫生—病原生物学] R392-33[医药卫生—基础医学]