摘要
目的制备表达人成熟miR-155的重组腺病毒,检测该免疫调控分子在肺癌细胞A549的表达及其生物学活性。方法以正常人基因组DNA为模板,PCR扩增约1000bp完整的pri-miR-155基因序列,克隆到T载体pTA2上,测序证实后,正向亚克隆入腺病毒穿梭载体pAdTrack-CMV中,构建pAdTrack-CMV/pri-miR-155重组质粒,与pAdEasy-1腺病毒骨架质粒重组并扩增后转染293FT细胞,进行重组腺病毒的包装、扩增,对其进行安全性、滴度的测定。用此腺病毒感染A549细胞,采用RT-PCR法检测成熟miR-155的表达,双报告基因法检测其干扰活性。结果构建的人pAdTrack-CMV/pri-miR-155重组质粒经酶切及测序鉴定正确;用此腺病毒感染A549细胞后,RT-PCR检测成熟miR-155表达较无关序列病毒感染对照组显著增强。腺病毒感染A549细胞后,通过转染后双报告基因检测相对萤光素酶活性较对照组降低,显示此腺病毒感染A549细胞后,可有效表达成熟miR-155,并具有生物学活性。结论成功制备人pri-miR-155重组腺病毒,并在感染肺腺癌细胞A549后高效表达成熟有活性miR-155。
The aim of this study is to prepare a recombinant adenovirus for expression of human mature miR-155, express the recombinant adenovirus in lung cancer A549 cells, and identify its biologic activity. Firstly primary miR-155 coding sequence was amplified by PCR using healthy volunteer＇s gDNA as template, and then cloned into pTA2 T vector and sequenced. Subsequently full-length pri-miR-155 coding DNA was subeloned into pAdTrack-CMV shuttle plasmid. The produc, t was linearized to homologous recombination with pAdEasy-1 vector in BJ5183 bacteria; the positive clone was identified by PCR plus restriction endonuelease digestion; the recomhined adenovirus DNA transfeeted into 293FT cells for packaging and amplifying Ad/pri-miR-155 virus. Then the virus was purified by CsCl density gradient centrifugation. The expression of miR-155 was verified by RT-PCR and EGFP fluorescence in infected A549 cells. According to the miRBase targets database, the target sequence and its mutated target sequence of miR-155 was synthesized and inserted to the pGL3-Control Report luciferase reporter vector to construct pGL3-Control/miR-155wt and pGL3-Control/miR-155mut, which was cotransfected with pRL-CMV into A549 cells , then infected with Ad/pri-miR-155 virus or Ad/FK3 control virus, and the relative lu- ciferase activity was detected. The results of DNA sequencing showed that sequence of pAdTrack-CMV/pri-miR-155 was correct. The specific expression of human mature miR-155 was identified by RT-PCR in A549 cells after infection with Ad/pri-miR-155 virus, but not in the control A549 cells. Dual-Luciferase Reporter Assay showed that mature miR-155 expressed from Ad/pri-miR-155 virus possessed biological activity. The results mean that we successfully prepared the recombinant adenovirus of hsa-pri-miR-155 and effectively expressed its in A549 cells.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2010年第3期246-250,254,共6页
Immunological Journal
基金
重庆市自然科学基金(CSTC,2007BB5077)
中国博士后科学基金(20070420769)资助项目
