摘要
目的 采用人胚肺二倍体细胞(2BS细胞)培养,结合实时荧光定量逆转录PCR(qRT-PCR)检测,开展甲型肝炎病毒(HAV)在2BS细胞核酸增殖培养模型的建立研究。方法 将不同浓度的HAV活病毒感染2BS细胞,分别于感染后0、8、10、14、20d收获HAV感染的细胞,应用已建立的HAV核酸检测和抗原检测方法分别检测HAV病毒核酸含量和抗原含量的动态变化。结果HAV在2BS细胞中可有效复制和扩增,高剂量组以1×10^(4) CCID_(50)/mLHAV感染0d即可检测到HAV核酸,10~20d病毒核酸达到峰值7.607lgcopies/μL;中剂量组以1×10^(2) CCID_(50)/mLHAV感染6d可检测到HAV核酸,6~10d病毒增殖明显(P<0.05),14~20d病毒核酸含量达到峰值7.074lgcopies/μL并趋于稳定;而低剂量组以1CCID_(50)/mLHAV感染后,10d HAV开始增殖,20d病毒核酸含量与中、高剂量组别相近且基本达到峰值;当HAV感染2BS细胞10 d时,病毒核酸检出拷贝数与感染HAV浓度呈良好的线性关系,可根据标准曲线准确反映样品中的HAV活病毒含量。细胞培养结合病毒抗原表达也可有效反映HAV的扩增情况,但较病毒核酸的出现呈明显滞后,且当样品中抗原含量低时,至少需要20d以上才可完全检测到病毒抗原的表达。结论 应用2BS细胞培养和qRT-PCR检测建立了2BS细胞HAV核酸增殖模型,可快速、准确地反映HAV活病毒在细胞中的动态感染和增殖情况,可应用于HAV活病毒的快速定量、病毒特性、药物筛选和疫苗评价等相关研究。
Objective The study aims to research and establish the proliferative culture model of hepatitis A virus’ s nucleic acid in human embryo lung diploid cell(2 BS) by culturing in 2 BS cells and quantifying with quantitative real-time PCR(qRT-PCR). Methods Different concentrations of HAV live virus were used to infect 2 BS cells, and the infected cells were harvested at 0, 8, 10, 14, 20 days after infection. Dynamic changes of RNA and antigen content were detected by using the established methods for nucleic acid and antigen quantification. Results HAV could be replicated and amplified efficiently in 2 BS cells. Viral nucleic acid could be detected at 24 hours after infection, and then reached its peak of 7.607 lg copies/μL from 10 d to 20 d when 2 BS cells were infected with 1×10^(4) CCID50/mL. By using 100 CCID50/mL, viral nucleic acid can be detected at 6 d after infection, and virus amplified apparently(P<0.05) at 6 d to 10 d, and the peak value of 7.074 lg copies/μL appeared to be stable at 14 d to 20 d. Moreover, virus proliferated at 10 d evidently and continued after that with 1 CCID50/mL;then the virus nucleic acid content reached its peak until 20 d, which was similar to that of medium and high dosage groups. When 2 BS cells were infected by 10 d, the infectious concentration had a good linearity relation with RNA copies, and it could accurately reflect the contents of the HAV live virus in samples according to the standard curve. Combinating the cell culture and antigen detection results can effectively reflect the amplification of HAV, but it showed an obvious lagging compared to the appearance of viral nucleic acid. It took at least 20 d to detect the expression of antigen when the antigen content was low. Conclusion The nucleic acid proliferation models of HAV are established by culturing 2 BS cells and RT-PCR detection, which can quickly and accurately reflect the dynamic infection and proliferation of HAV live virus. It can be used in the quantitative and rapid detection, viral characteristics, drug screening, vaccine evaluation and the other related research regarding HAV.
作者
闫旭佳
袁亚迪
夏青娟
徐艳玲
王春雨
徐涵禹
张洁
YAN Xu-jia;YUAN Ya-di;XIA Qing-juan;XU Yan-ling;WANG Chun-yu;XU Han-yu;ZHANG Jie(Second Department of Vaccine,Changchun Institute of Biological Products Co.,Ltd.,Changchun 130012,Jilin Province,China;不详)
出处
《微生物学免疫学进展》
CAS
2021年第3期14-19,共6页
Progress In Microbiology and Immunology
基金
国家“十三五”重点研发计划(2017YFC1600703)。
