C57BL/6NcGAS基因敲除鼠的构建及对猪伪狂犬病病毒增殖的影响  

Construction of C57BL/6NcGAS knockout mice and effect of cGAS knockout on proliferation of porcine pseudorabies virus

作　　者：李丽蕴 梁东阁 于海深 郭江涛 曾磊[1] LI Liyun;LIANG Dongge;YU Haishen;GUO Jiangtao;ZENG Lei(Key Laboratory of Animal Biochemistry and Nutrition,Ministry of Agriculture and Rural Affairs/Henan Key Laboratory of Animal Growth and Development Regulation,College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[1]河南农业大学动物医学院,农业农村部动物生化与营养重点实验室/河南省动物生长发育调控重点实验室,河南郑州450046

出　　处：《中国兽医学报》2023年第10期2162-2170,共9页Chinese Journal of Veterinary Science

基　　金：河北农业大学博士启动基金资助项目(30501221)。

摘　　要：胞质DNA感受器环鸟苷酸-腺苷酸合成酶(cyclic GMP-AMP synthase,cGAS)能够识别胞质中的双链DNA(double stranded DNA,dsDNA),从而激活Ⅰ型干扰素信号通路抵御外界病原微生物的感染。为研究cGAS基因对猪伪狂犬病病毒(PRV)增殖的影响,本试验首先利用CRISPR/Cas9基因编辑技术构建cGAS基因敲除小鼠,通过H&E染色检测C57BL/6N小鼠和C57BL/6N-cGAS^(-/-)小鼠肺脏和脑组织形态差异及感染PRV后肺脏组织中炎性浸润的影响;Q-PCR检测C57BL/6N小鼠和C57BL/6N-cGAS^(-/-)小鼠感染PRV后对PRV-gB、PRV-TK、IFN-β、ISG15和ISG20基因mRNA表达水平的差异;Western blot及免疫组化检测C57BL/6N小鼠和C57BL/6N-cGAS^(-/-)小鼠感染PRV后PRV-gB和PRV-gE蛋白表达水平;最后利用病毒PFU法检测cGAS敲除鼠对PRV子代病毒滴度的影响。Western blot检测结果显示在C57BL/6N小鼠肺脏和脑组织中cGAS基因敲除成功,同时H&E染色结果显示,cGAS基因敲除对其肺脏和脑组织形态无影响,但感染PRV后C57BL/6N-cGAS^(-/-)小鼠组织中的炎性浸润明显高于C57BL/6N;小鼠存活率试验结果显示,感染PRV后,C57BL/6N-cGAS^(-/-)与C57BL/6N小鼠相比,存活率显著降低。Q-PCR及Western blot检测显示C57BL/6N-cGAS^(-/-)小鼠可以促进PRV-gB和PRV-gE mRNA转录和蛋白表达;PFU测定结果显示,感染PRV后,C57BL/6N-cGAS^(-/-)小鼠体内的病毒滴度显著高于C57BL/6N小鼠。另外,Q-PCR结果显示该基因敲除小鼠可以抑制PRV感染引起的IFN-β、ISG15和ISG20基因mRNA表达上调。以上结果表明cGAS抑制PRV在小鼠体内的增殖。Cyclic GMP-AMP synthase(cGAS),a cytosolic DNA receptor,can recognize double stranded DNA(dsDNA)in cytosol,thereby activating type I interferon signaling pathway to resist the infection of external pathogenic microorganisms.To study the effect of cGAS gene on PRV replication,in this study,CRISPR/Cas9 gene editing technology was used to construct cGAS gene knockout mice.H&E staining was used to detect the morphological differences of lung and brain tissue between C57BL/6N mice and C57BL/6N-cGAS^(-/-)mice,and the effect of inflammatory infiltration in lung tissue after PRV infection.The mRNA expression levels of PRV-gB,PRV-TK,IFN-β,ISG15,and ISG20 genes were detected by Q-PCR between C57BL/6N mice and C57BL/6N-cGAS^(-/-)mice infected with PRV.The protein expressions of PRV-gB and PRV-gE in C57BL/6N mice and C57BL/6N-cGAS^(-/-)mice infected with PRV were detected by Western blot and immunohistochemistry.Finally,the effect of cGA S knockout mice on the titer of PRV progeny virus was detected by viral PFU assay.Western blot showed that cGAS gene knockdown was successful in the lung and brain tissue of C57BL/6N mice,and H&E staining showed that cGAS gene knockdown had no effect on the morphology of lung and brain tissue.However,the inflammatory infiltration of C57BL/6N-cGAS^(-/-)mice after PRV infection was significantly higher than that of C57BL/6N.The results of mouse survival test showed that the survival rate of C57BL/6NcGAS^(-/-)mice was significantly lower than that of C57BL/6N mice after PRV infection.Q-PCR and Western blot analysis showed that C57BL/6N-cGAS^(-/-)mice could promote the mRNA transcription and protein expression of PRV-gB and PRV-gE.The results of PFU showed that the virus titer in C57BL/6N-cGAS^(-/-)mice was significantly higher than that in C57BL/6N mice after PRV infection.In addition,Q-PCR results showed that the knockout mice could inhibit the up-regulation of IFN-β,ISG15 and ISG20 mRNA expression induced by PRV infection.These results indicate that cGAS inhibits PRV proliferation in mice.

关 键 词：环鸟苷酸-腺苷酸合成酶(cGAS) 伪狂犬病病毒(PRV) 天然免疫 小鼠 

分 类 号：S852.65[农业科学—基础兽医学]