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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 passage adaptability of recombinant rotavirus strain LD (9G2) on Vero cells and to select high-titer adaptive strains. Methods Three rotavirus strains, LD9 (G2) (LD9-1, LD9-2, LD9-3) were passaged on Vero cells at 0.05, 0.10, 0.15 and 0.20 MOI, respectively. One Vero cell-adapted strain was identified and its optimum MOI was determined. The cells were passaged continuously on Vero cells for 15 passages. The virus titers and genomic nucleic acid bands were detected. The VP7 gene was amplified by nestRT-PCR. The 8th, 10th and 13th generation strains were selected and inoculated into Vero cells at 0.10MOI to observe the proliferation of the virus. Results Inoculation of Vero cells with 0.10MOI resulted in significant cytopathic effect (CPE) in the selected strain of LD9-2. The virus titer decreased first and then increased with the increase of passage number, and reached the lowest level at the 4th passage. After 9 generations, 6.75lgCCID50 / ml; continuous passage 15 times, the virus genomic nucleic acid band type consistent with the original strain, and can amplify the 502bp VP7 gene. The peak of virus proliferation of LD9-2 strain appeared on the 7th to 8th days, the virus titer was 6.00 ~ 7.00lgCCID50 / ml. Conclusion The recombinant strain of rotavirus LD9 (type G2) was adapted to Vero cells and a candidate vaccine strain with high expression of virus and stable passage was obtained.