转染E1B55K基因提高Hep2细胞包装肠腺病毒Ad41的能力

人F组腺病毒Ad40、Ad41难以在体外培养的细胞中传代,被称为难养腺病毒(Fastidious adenovirus).本研究观察了在Hep2细胞表达Ad41 E1B55K基因对Ad41复制的促进作用.从Ad41阳性粪便标本中用PCR的方法获得E1B55K基因,构建真核表达载体,转染Hep2细胞,筛选单克隆,用RT-PCR检测了E1B55K基因的表达.用引起293细胞完全CPE比较产毒量的方法对所得细胞克隆进行初步筛选,获得一株产毒相对较强的细胞Hep2-E1B#4.与对照细胞Hep2、Hep2-DNA3相比,等量Ad41接种Hep2-E1B#4产生的细胞病变效应(CPE)程度明显加深.用免疫细胞化学的方法测定产毒的感染滴度,等量Ad41接种后,Hep2-E1B#4产生的子代腺病毒滴度大于对照的9倍;半定量PCR测得Hep2-E1B#4子代病毒基因组拷贝数约为对照细胞的4倍.结果说明转染E1B55K基因促进了Ad41在Hep2细胞的复制,获得的Hep2-E1B#4细胞株可用于Ad41的分离、培养和体外扩增.

Improved Replication of Enteric Adenovirus Type 41 in Hep2 Cell Line Expressing E1B55K

Adenovirus type 40 and 41(Ad40,Ad41),which belong to human adenovirus subgroup F,are called fastidious adenoviruses due to their property of poor growth in cultured cell lines in vitro The effect of expression of exogenous E1B55K in Hep2 on Ad41 replication in this cell line was investigated.E1B55K gene was amplified by PCR with DNA extracted from Ad41-positive feces supernatant as template.Eukaryotic expression plasmid(pcDNA3) carrying E1B55K was constructed,purified,and transferred into Hep2 cell.Expression of E1B55K in G418-resistant clones was assayed by RT-PCR,and one clone named as Hep2-E1B#4 could produce more Ad41 progenies when compared with other clones by the method of inducing complete cytopathic effect(CPE) in 293 cells.Infection of equivalent Ad41 caused more significant cytopathic effect(CPE) in Hep2-E1B#4 than that in the control cells of Hep2 or Hep2-DNA3,also suggesting enhanced viral replication in Hep2-E1B#4.The titer of Ad41 was further determined by method of immunocytochemical staining,and semi-quantity PCR was employed to compare the copy number of Ad41 genome DNA.The results showed that the yield of Ad41 in Hep2-E1B#4 was more than 9 times of that in control cells when equal amount of seed viruses were incubated,and the copy number of Ad41 genome increased 4 times in the raw extract from the infected Hep2-E1B#4 when compared with that from control cells.In conclusion,E1B55K gene transfer improved the ability of Hep2 in packaging Ad41,and the Hep2-E1B#4 cell line,which expressed E1B55K constitutively,would be helpful in isolation,cultivation and amplification of Ad41.