摘要
旨在获得MRC1基因修饰的PCV2靶细胞并在体外验证该细胞对PCV2的抑制作用,为后续基因编辑猪的生产提供理论基础。本研究:1)首先利用基因工程技术建立了慢病毒介导的高效同源重组载体和CRISPR/Cas9基因靶向系统;2)通过细胞和分子生物学技术筛选了定点整合表达MRC1的PK15细胞系;3)随后通过细胞试验和PCV2的攻毒试验对其抗PCV2的作用进行了相关验证。结果:1)成功构建了含有红色荧光标记基因、嘌呤霉素标记基因、同向LoxP序列和多克隆位点的pLV-LoxP-mCherry-puro-LoxP载体,同时根据不同猪品种间MRC1基因启动子差异序列设计同源臂,成功构建同源重组载体和靶向同源臂的CRISPR/Cas9基因靶向载体;2)通过病毒包装和共转染的方法,利用嘌呤霉素筛选并验证了MRC1启动子基因编辑的PK15细胞株,成功将MRC1基因转录起始位点上游多出的14 bp敲入到PK15细胞中;3)随后对基因编辑的PK15细胞系进行抗病毒验证,MRC1基因编辑的PK15细胞株MRC1的蛋白表达量显著增高(P<0.05),同时显著降低了PCV2的复制(P<0.05)。MRC1启动子编辑的PK15细胞中MRC1蛋白表达量显著增高,该位点可以用于抗PCV2猪的制备。
This study aimed to obtain target cells with MRC1 gene-modified PCV2 and verify the inhibitory effect of the cells on PCV2 in vitro,which would provide a theoretical basis for the subsequent production of gene-edited pigs.In this study:1)Firstly,a lentivirus-mediated high-efficiency homologous recombination vector and a CRISPR/Cas9 gene targeting system were established using genetic engineering technology;2)The gene-edited MRC1 promoter in PK15 cells were screened by cell and molecular biology techniques;3)Then,the anti-PCV2 effect was verified by PCV2 infection tests.The results showed that:1)The pLV-LoxP-mCherry-puro-LoxP vector containing red fluorescent gene,puromycin gene,homologous LoxP sequence and multiple cloning sites was successfully constructed.The homologous arm was designed according to the differential sequences of MRC1 promoter among different pig breeds and the recombinant vector was constructed.Also the CRISPR/Cas9 mediated gene targeting vector were successfully constructed;2)The PK15 cell line edited by MRC1 promoter gene was screened and verified by puromycin through virus packaging and co-transfection.The 14 bp upstream of the transcription start site of MRC1 gene was successfully knocked into PK15 cells;3)The protein expression of MRC1 in PK15 cell line edited by MRC1 gene was significantly increased(P<0.05),and the replication of PCV2 was significantly reduced(P<0.05).The expression of MRC1 protein in PK15 cells edited by MRC1 promoter was significantly increased,which could be used for the preparation of anti-PCV2 pigs.
作者
费晓钰
石超群
刘雪明
苏峰
姜运良
FEI Xiaoyu;SHI Chaoqun;LIU Xueming;SU Feng;JIANG Yunliang(Key Laboratory of Animal Biotechnology and Disease Control and Prevention of Shandong Province,College of Animal Science and Veterinary Medicine,Shandong Agricultural University,Taian 271018,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2023年第3期934-946,共13页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
山东省农业良种工程(2019LZGC019)
山东省重点研发项目(2019GNC106055)。
