摘要
【目的】阐明干扰素基因刺激因子(STING)在猪抗病原微生物感染中的作用机制,为猪传染性胃肠炎、流行性腹泻和猪伪狂犬病等病毒性疾病的科学防控提供参考依据。【方法】基于CRISPR/Cas9技术,在STING基因第4、第8外显子中寻找高分靶点并设计sgRNA序列,将退火的sgRNA与酶切的LentiCRISPRv2载体用T4 DNA连接酶连接以获得LentiCRISPRv2-STING-sgRNA慢病毒载体(STING-sgRNA);以不同的STING-sgRNA慢病毒载体组合及包装质粒psPAX2和包膜质粒pMD2.G共同转染293T细胞,得到含sgRNA的慢病毒再转染3D4/21细胞;经嘌呤霉素筛选和有限稀释法获得单克隆细胞株,通过PCR、测序及Western blotting鉴定STING基因的敲除效果;并采用实时荧光定量PCR验证STING基因敲除对I型干扰素表达的影响。【结果】以不同的STING-sgRNA慢病毒载体组合与HA-STING过表达载体共同转染293T细胞,均能在细胞内对STING真核表达载体产生编辑效果,且以STING-sgRNA(1+5)慢病毒载体组合的编辑效率最高。以编辑效率最高的STING-sgRNA(1+5)慢病毒载体组合及包装质粒psPAX2和包膜质粒pMD2.G共同转染293T细胞包装出慢病毒,再用慢病毒感染3D4/21细胞,结果获得1株STING基因大片段(4989 bp)缺失的3D4/21细胞株,Western blotting检测未发现STING蛋白,说明STING基因敲除3D4/21细胞(3D4/21-STING-/-)构建成功。与野生型3D4/21细胞相比,在转染副猪嗜血杆菌DNA刺激下,3D4/21-STING-/-细胞中的IFN-β基因转录水平显著降低(P<0.05)。【结论】采用CRISPR/Cas9技术能成功大片段敲除3D4/21细胞中的STING基因,而导致STING基因功能丧失;STING基因敲除会导致细胞在病原微生物DNA刺激时Ⅰ型干扰素转录障碍,也提示STING基因可能是猪抗病原微生物感染的关键因子。
【Objective】To elucidate the mechanism of interferon gene stimulating factor(STING)in the anti-pathogenic microbial infection of pigs,so as to further provide a reference for the scientific prevention and control of viral diseases such as porcine transmissible gastroenteritis,epidemic diarrhea and porcine pseudorabies.【Method】High-scored targets were found in exons 4 and 8 of STING gene and corresponding sgRNA sequences were designed based on CRISPR/Cas9 technology.The annealed sgRNAs were linked with the enzyme digested LentiCRISPRV2 carrier with T4 DNA ligase to obtain LentiCRISPRV2-STING-sgRNA lentivirus carrier(STING-sgRNA);Different combinations of STING sgRNA lentivirus carriers,packaging plasmid psPAX2 and envelope plasmid pMD2.G were transfected into 293T cells to obtain lentivirus containing sgRNA and then transduced into 3D4/21 cells.Monoclonal cell lines were obtained by puromycin screening and limited dilution method.The knockout efficiencies of the STING gene were identified by PCR amplification,Sequencing and Western blotting;The effect of STING gene knockout on the expression of typeⅠinterferon was verified by real-time fluorescent quantitative PCR.【Result】When 293T cells were transfected with different combinations of STING-sgRNA lentivirus carrier and HA-STING over expression vector,the editing effect of STING eukaryotic expression carrier could be detected in cells,and the combination of STING-sgRNA(1+5)lentivirus carrier showed the supreme editing efficiency.Thus,the STING-sgRNA(1+5)lentivirus carrier combined with the packaging plasmid psPAX2and the envelope plasmid p MD2.G were transfected 293T cells to package lentivirus,and then infected 3D4/21 cells with lentivirus.The results showed that a 3D4/21 cell line with a large deletion of the STING gene(4989 bp)was obtained.The STING protein was not observed by Western blotting,indicating that the STING gene knockout 3D4/21 cells(3D4/21-STING-/-)were successfully constructed.The transcription level of IFN-βin 3D4/21-STING-/-cells de
作者
黄秋艳
杨烨城
张昆丽
孟繁明
李剑豪
朱向星
王塑天
唐冬生
HUANG Qiu-yan;YANG Ye-cheng;ZHANG Kun-li;MENG Fan-ming;LI Jian-hao;ZHU Xiang-xing;WANG Su-tian;TANG Dong-sheng(School of Life Science and Engineering,Foshan University/Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding/Guangdong Provincial Research Center of Gene Editing Engineering Technology,Foshan,Guangdong 528225,China;Institute of Animal Science,Guangdong Academy of Agricultural Sciences/State Key Laboratory of Livestock and Poultry Breeding/Guangdong Key Laboratory of Animal Breeding and Nutrition,Guangzhou,Guangdong 510610,China;Institute of Animal Health,Guangdong Agricultural Science/Key Laboratory of Livestock Disease Prevention of Guangdong Province/Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province,Ministry of Agriculture and Rural Affairs,Guangzhou,Guangdong 510640,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2022年第4期891-898,共8页
Journal of Southern Agriculture
基金
国家自然科学基金项目(32002153,32002298)
广州市基础与应用基础研究项目(202102020177)
广东省农业科学院科技创新战略专项(R2019YJ-YB2004)。
