稳定表达circLAMP3的C57/B6-L和A549细胞系的构建  

Establishment of C57/B6-L and A549 cell lines stably expressing circLAMP3

作　　者：陈福再 赵聪慧 张晓璇 张春萍 黄佳承 陈吉龙[1] 马树杰 CHEN Fuzai;ZHAO Conghui;ZHANG Xiaoxuan;ZHANG Chunping;HUANG Jiacheng;CHEN Jilong;MA Shujie(Fujian Province Joint Laboratory of Animal Pathogen Prevention and Control of the"Belt and Road",College of Animal Sciences,Fujian Agriculture and Forestry University,Fuzhou 350002,China)

机构地区：[1]福建农林大学动物科学学院福建省“一带一路”畜禽重大疫病防控联合实验室,福建福州350002

出　　处：《中国兽医学报》2024年第9期2010-2016,共7页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划基金资助项目(2021YFD1800205);福建农林大学杰出青年科研人才计划基金资助项目(xjq202018);福建省自然科学基金资助项目(2022J05033)。

摘　　要：旨在构建稳定过表达环状RNA LAMP3(circLAMP3)的C57/B6-L和A549细胞系,为后续深入研究circLAMP3的生物学功能奠定基础。分别提取C57/B6-L和A549细胞总RNA,反转录后扩增circLAMP3全长序列,连接到pLC5-ciR载体上,得到pLC5-Mouse-circLAMP3和pLC5-Human-circLAMP3重组质粒。利用瞬时转染方法在HEK293T细胞上包装慢病毒,将慢病毒分别感染C57/B6-L和A549细胞,经嘌呤霉素筛选得到稳定表达circLAMP3的细胞系。利用荧光显微镜、PCR扩增、荧光定量PCR(qPCR)和Sanger测序对构建的细胞系过表达circLAMP3效果进行验证。结果表明,pLC5-Mouse-circLAMP3和pLC5-Human-circLAMP3过表达质粒构建成功,荧光显微镜下可见构建的C57/B6-L和A549细胞系表达强烈绿色荧光。PCR和qPCR结果可见过表达细胞系circLAMP3表达显著增强,Sanger测序结果表明circLAMP3环化位点正确。本研究成功构建了过表达circLAMP3的C57/B6-L和A549细胞系,为进一步探索circLAMP3在流感病毒复制中的生物学功能提供了生物材料。This study aims to construct C57/B6-L and A549 cell lines that stably overexpress circular RNA LAMP3(circLAMP3),laying the foundation for further research on the biological functions of circLAMP3.Total RNA was extracted and reverse transcripted into cDNA from C57/B6-L and A549 cells to amplify the full-length sequence of circLAMP3.Then,the fragments of circLAMP3 were ligated into pLC5-ciR vector to obtain pLC5-Mouse-circLAMP3 and pLC5-HumancircLAMP3 recombinant plasmids.The lentiviruses expressing circLAMP3 were packaged on transient transfected HEK293T cells.C57/B6-L and A549 cells were infected with lentiviruses to generate cell lines overexpressing circLAMP3 through puromycin screening.To verify the overexpression efficiency of circLAMP3 of cell lines,we performed the fluorescence microscopy,PCR amplification,quantitative PCR(qPCR),and Sanger sequencing experiments.The results indicated that the overexpression plasmids of pLC5-Mouse-circLAMP3 and pLC5-Human-circLAMP3 were successfully constructed.Strong green fluorescence was observed under a fluorescence microscopy.C57/B6-L and A549 cell lines showed a significant increase in the expression of circLAMP3 by PCR and qPCR methods.Sanger sequencing results showed that the junction site of circLAMP3was correct.This study successfully constructed C57/B6-L and A549 cell lines overexpressing circLAMP3,providing biomaterials for further exploration of the biological function of circLAMP3in influenza virus replication.

关 键 词：环状RNA circLAMP3 载体构建 细胞系 

分 类 号：S855.3[农业科学—临床兽医学]