环状RNA的体外构建及表达  

Construction and expression of circular RNA in vitro

作　　者：唐琪 刘宏博 张哲罡 张家友 曹慧 杨晓明 TANG Qi;LIU Hongbo;ZHANG Zhegang;ZHANG Jiayou;CAO Hui;YANG Xiaoming(Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430207,Hubei Province,China;不详)

机构地区：[1]武汉生物制品研究所有限责任公司,湖北武汉430207 [2]国家联合疫苗工程技术研究中心,湖北武汉430207 [3]北京生物制品研究所有限责任公司质量保证部,北京100029 [4]中国生物技术股份有限公司,北京100029

出　　处：《中国生物制品学杂志》2024年第1期32-36,共5页Chinese Journal of Biologicals

基　　金：湖北省重点研发计划(2023BCB028)。

摘　　要：目的利用真核生物mRNA成熟过程中内含子外显子置换(permuted intron exon,PIE)策略,构建可在体外环化(in vitro cyclization,IVC)并具有翻译功能的RNA,转染HEK-293T细胞后进行表达。方法选取具备Ⅰ型催化内含子(groupⅠcatalytic intron)的5’和3’环化臂、柯萨奇病毒B3(Coxsackievirus B3,CVB3)的内部核糖体进入位点(internal ribosome entry site,IRES)以及目的基因序列构建模板质粒,通过PCR法获得线性化质粒模板,经体外转录(in vitro transcription,IVT)合成线性RNA,依次添加环化试剂完成IVC得到环状RNA(circular RNA,circRNA)。采用琼脂糖凝胶电泳及核糖核酸酶R(ribonuclease R,RNase R)消化的方式确认RNA环化。将分别携带增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)、萤火虫荧光素酶(firefly luciferase,Fluc)及流感病毒血凝素(hemagglutinin,HA)(IVR-180)的circRNA转染HEK-293T细胞,逐一验证其体外表达情况。结果RNA环化后电泳主条带变小,且出现环化产生的小片段,经RNase R消化,仅环化后RNA条带仍有部分保留。转染EGFP-circRNA的HEK-293T细胞荧光显微镜下可见明显绿色荧光;转染Fluc-circRNA的HEK-293T细胞Fluc表达值平均为未环化RNA的20倍以上,且随Fluc-circRNA转染剂量的升高,荧光数值(relative light unit,RLU)呈上升趋势;Western blot分析显示,转染HAcircRNA的HEK-293T细胞成功表达了HA蛋白。结论在体外成功环化了线性RNA并完成不同蛋白的表达,为新型流感疫苗及mRNA疫苗的研究奠定了基础。Objective To construct encoding RNA that can be cyclized in vitro by using the permuted intron exon(PIE)strategy in the maturation process of eukaryotic mRNA,and transfect it into HEK-293T cells for expression.Methods The sequences of 5'and 3'cyclic arms with groupⅠcatalytic intron,the internal ribosome entry sites(IRES)of Coxsackievirus B3(CVB3)and the target gene were selected to construct the template plasmid.Linearization plasmid template obtained by PCR was used to synthesize linear RNA through in vitro transcription(IVT),which then started in vitro cyclization(IVC)by the addition of cyclization reagents to obtain circular RNA(circRNA).RNA cyclization was confirmed by agarose gel electrophoresis and ribonuclease R(RNase R)digestion.HEK-293T cells were transfected with circRNAs respectively carrying enhanced green fluorescent protein(EGFP),firefly luciferase(Fluc),and influenza virus hemagglutinin(HA)IVR-180 genes,to verify their expression with in vitro.Results With RNA cyclization,the main band of agarose gel electrophoresis became smaller and small fragments appeared.After RNase R digestion,only some circRNA bands remained.HEK-293T cells transfected with EGFP-circRNA showed significant green fluorescence under the fluorescence microscope.The Fluc expression values of HEK-293T cells transfected with Fluc-circRNA were on average 20 times higher than non cyclized RNA,and the relative light unit(RLU)scaled up with the increase of Fluc-circRNA transfection dose.Western blot analysis showed that HA protein was successfully expressed in HEK-293T cells transfected with HA-circRNA.Conclusion In this study,linear RNA was successfully cyclized in vitro and different proteins were expressed,which lays a foundation of the research of new influenza vaccines and mRNA vaccines.

关 键 词：环状RNA 体外环化 绿色荧光蛋白 萤火虫荧光素酶 流感病毒血凝素 

分 类 号：Q71[生物学—分子生物学]