3种细胞穿透肽对体外转录mRNA的递送效果  

Delivery Effects of Three Cell Penetrating Peptides on Transcribed mRNA In Vitro

作　　者：王军华 罗画叶 张翠玲 李军伟 WANG Jun-hua;LUO Hua-ye;ZHANG Cui-ling;LI Jun-wei(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China)

机构地区：[1]青岛农业大学动物医学院,山东青岛266109

出　　处：《中国兽医杂志》2022年第4期35-39,44,共6页Chinese Journal of Veterinary Medicine

基　　金：青岛农业大学创新训练项目(201910435019)。

摘　　要：为了开发合适的mRNA递送载体,本试验采用体外转录、加帽、加尾修饰及氯化锂(LiCl)纯化的方法获得具有生物活性的绿色荧光蛋白mRNA(mRNA-EGFP),然后用合成的3种细胞穿透肽(RVG、RVG9dR、RVG11dR)进行递送。先通过凝胶阻滞试验确定mRNA与3种细胞穿透肽的最佳包被质量比例;再在293T细胞水平上测试3种细胞穿透肽的细胞毒性、包被mRNA后对RNase A的耐受性,并通过凝胶阻滞试验和细胞荧光试验来测试3种细胞穿透肽对mRNA的包被和递送效力。细胞毒性试验结果显示,3种细胞穿透肽的半抑制浓度(IC_(50))分别是95、90μmol/L和81μmol/L;RNase A保护试验结果显示,3种细胞穿透肽均可保护mRNA不被RNase A降解;凝胶阻滞试验和细胞荧光试验结果显示,3种细胞穿透肽均可成功包被mRNA-EGFP,并把mRNA递送到细胞内进行表达,其中RVG11dR对mRNA的递送效果最好。结果表明,在细胞穿透肽RVG的末端添加一定数量的精氨酸可提高其对mRNA的递送效力。In order to develop a suitable mRNA delivery vector,this study assessed the efficiency of three cell-piercing peptides(RVG,RVG9dR and RVG11dR)for delivering bioactive mRNA-EGFP obtained by in vitro transcription,capping,polyadenylation and LiCl purification.The optimal encapsulation mass ratio of mRNA to three cell-penetrating peptides was determined by gel shift assay.Cytotoxicity and RNase A resistance of three cell-penetrating peptides were tested on 293T cell line.Encapsulation and delivery of mRNA by three cell-penetrating peptides were determined by gel retardation and cytofluorescence assay.Cytotoxicity test showed that the 50%inhibiting concentration(IC_(50))of three cell-penetrating peptides was around 95,90μmol/L and 81μmol/L,respectively.RNase A protection test showed that all three cell-penetrating peptides could protect mRNA-EGFP from RNase A degradation.Gel retardation and cytofluorescence assay showed that all three cell-penetrating peptides could successfully encapsulate mRNA-EGFP and deliver mRNA into cells for expression,and RVG11dR has the best mRNA delivery effect.Thus,addition of arginine at the end of RVG improves its mRNA delivery effect.

关 键 词：细胞穿透肽 狂犬病毒糖蛋白 MRNA 体外转录 递送效果 

分 类 号：S852[农业科学—基础兽医学]