摘要
目的包装表达轮状病毒VP7基因的重组腺病毒，并检测其免疫活性。方法RT—PCR扩增病毒结构蛋白VP7基因，定向克隆于腺病毒穿梭质粒pAdtrack—CMV中，在细菌中与缺陷型腺病毒基因组pAdeasy-1进行同源重组，并用RT-PCR和Western blot检测。将包装好的腺病毒免疫小鼠，应用间接ELISA检测小鼠血清中特异性轮状病毒IgG抗体。结果酶切和测序鉴定证实成功构建携带VP7基因的重组腺病毒表达载体，并在293细胞中成功包装病毒；RT—PCR和Western blot均能特异检测VP7基因的表达；重组腺病毒免疫小鼠后可诱导针对轮状病毒的特异性免疫。结论重组腺病毒的成功包装，为新型轮状病毒基因疫苗的研制提供了一种可行的途径。
Objective To package a recombinant adenovirus expressing human rotavirus VP7 gene and detect its immune activity. Methods The VP7 gene was amplificated using reverse-transcription PCR, and subcloned into the shuttle vector pAdtrack-CMV. Homologous recombination was performed in the bacteria between recombinant vector and adenoviral backbone plasmid pAdEasy-1, then detected the VP7 expression by RT-PCR and Western blot. The packaged adenovirus immunized mice, and detected serum rotavirns IgG antibodies by indirect ELISA. Results Restriction endonuclease digestion test and sequencing showed that the recombinant adenovector containing the rotavirus VP7 gene. It can be successfully packaged in 293 cells. The VP7 gene expression can be specific detected by RT-PCR and Western blot analysis. Recombinant adenovirus immunized mice could induce specific immunity against rotavirns. Conclusions The successful packaging of the recombinant adenovirus may be potential candidate for rotavirus DNA vaccine.
出处
《国际病毒学杂志》
2012年第6期241-244,共4页
International Journal of Virology
基金
国家自然科学基金青年科学基金项目（30700139）
