摘要
为了构建POR基因稳定敲除(POR^(KO))、回补(PORCO)、过表达(POR^(OE))的LO2细胞株,以便为后续深入开展POR基因在AFB_(1)致毒性机理中的功能研究提供细胞模型。本研究首先利用CRISPR/Cas9技术设计2条靶向POR基因的sgRNA并克隆至lentiCRISPR v2载体,进行慢病毒包装与感染,筛选出LO2 POR^(KO)单克隆细胞后进行测序及Western blot鉴定。其次用同源重组方法构建pcDNA3.1(+)/myc-His B-POR重组质粒,经对POR^(KO)细胞和野生型细胞转染分别构建LO2 PORCO及LO2 POR^(OE)细胞株,以G-418筛选后进行Western blotting鉴定。再用4μmol·L^(-1)及8μmol·L^(-1)AFB_(1)分别对野生型、LO2 POR^(KO)、LO2 PORCO、LO2 POR^(OE)细胞株染毒48 h,观察细胞形态并测定细胞存活率。结果表明,靶向exon5的sgRNA-2可成功敲除POR基因,得到LO2 POR^(KO)细胞株;成功构建表达POR重组蛋白的LO2 PORCO及LO2 POR^(OE)细胞株。对AFB_(1)处理后的各细胞存活率测定结果表明,相较于野生型LO2细胞43.66%±0.58%和27.90%±0.46%的存活率,LO2 POR^(KO)细胞存活率具有极显著性差异,均为100%(P<0.0001);LO2 PORCO细胞存活率仍有显著性差异,分别为57.07%±0.74%和42.54%±0.68%(P<0.05);LO2 POR^(OE)细胞存活率则显著降低,分别为24.58%±0.92%和17.99%±0.81%(P<0.01)。综上所述,本研究成功构建了LO2 POR^(KO)、LO2 PORCO、LO2 POR^(OE)细胞株,并以AFB_(1)染毒处理分别研究了各细胞株的存活率和细胞形态学变化,发现POR基因对AFB_(1)所致细胞毒性的影响巨大。研究结果为后续深入开展POR基因在AFB_(1)致毒性机理中的功能研究奠定了基础。
To construct three LO2 cell lines separately with POR gene stable knockout(POR^(KO)),complementation(POR CO),and overexpression(POR^(OE)),and provide cell models for further in-depth research on the function of the POR gene in the mechanism of AFB_(1)toxicity.Firstly,two sgRNAs targeting the POR gene were designed using CRISPR/Cas9 technology and cloned into the lentiCRISPR v2 vector,and then lentivirus packaging and virus infection were performed.After screening LO2 POR^(KO)monoclonal cells,they were sequenced and identified by Western blot.Secondly,the pcDNA3.1(+)/myc-His B-POR recombinant plasmid was constructed by homologous recombination method,and the LO2 POR CO and LO2 POR^(OE)cell lines were respectively constructed through cell transfection and were identified by Western blot after screening with G-418.After the wild-type LO2,LO2 POR^(KO),LO2 POR CO,and LO2 POR^(OE)cell lines were exposed with 4μmol·L^(-1)and 8μmol·L^(-1)AFB_(1)for 48 h,then the cell morphology was observed,and the cell viability was determined.The results showed that sgRNA-2 could successfully knock out the POR gene to obtain the LO2 POR^(KO)cell line,and the LO2 POR CO and LO2 POR^(OE)cell lines were successfully constructed.After each cell line was exposed to 4μmol·L^(-1)and 8μmol·L^(-1)AFB_(1)for 48 h,compared with the wild-type LO2 cell viability rate of 43.66%±0.58%and 27.90%±0.46%,the cell viability rate of LO2 POR^(KO)cells was extremely significant,both 100%(P<0.0001);the cell viability rate of LO2 POR CO cells was still significantly different,respectively 57.07%±0.74%and 42.54%±0.68%(P<0.05);the cell viability rate of LO2 POR^(OE)cells was significantly lower,respectively 24.58%±0.92%and 17.99%±0.81%(P<0.01).In conclusion,this paper successfully constructed LO2 POR^(KO),LO2 POR CO,and LO2 POR^(OE)cell lines,and studied the cell viability rate and cell morphology changes of normal LO2,LO2 POR^(KO),LO2 POR CO,and LO2 POR^(OE)cell lines with AFB_(1)exposure,and discovered that the POR gene has a huge impact on AFB_(1)-induced cytotoxicity.This paper laid a foundation for the subsequent in-depth research on the function of the POR gene in the mechanism of AFB_(1)toxicity.
作者
王琳
袁建林
缪昌
马玉晗
曹三杰
赵勤
WANG Lin;YUAN Jianlin;MIAO Chang;MA Yuhan;CAO Sanjie;ZHAO Qin(Research Center for Swine Diseases,College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Sichuan Science-Observation Experimental Station of Veterinary Drugs and Veterinary Diagnostic Technique,Ministry of Agriculture and Rural Affairs,Chengdu 611130,China)
出处
《浙江农业学报》
CSCD
北大核心
2024年第2期272-283,共12页
Acta Agriculturae Zhejiangensis
基金
四川省“十四五”川猪重大科技专项(2021ZDZX0010)。
