鸡RIPK2基因慢病毒干扰载体的构建及稳定低表达RIPK2的HD11筛选  

Construction of Gallus gallus RIPK2 Gene Lentiviral Interference Vector and Screening of HD11 with Stably Low Expressing RIPK2

作　　者：孙红艳[1,2] 陈婷宏 吴钰湘 孙长花 李欢[3] SUN Hong-Yan;CHEN Ting-Hong;WU Yu-Xiang;SUN Chang-Hua;LI Huan(College of Animal Science and Technology,Yangzhou University,Yangzhou 225009;Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China,Yangzhou 225009;College of Biological and Chemical Engineering,Yangzhou Polytechnic College,Yangzhou 225012)

机构地区：[1]扬州大学动物科学与技术学院,扬州225009 [2]中国教育部国际农业与农产品安全研究联合实验室,扬州225009 [3]扬州市职业大学生物与化工工程学院,扬州225012

出　　处：《农业生物技术学报》2022年第6期1031-1042,共12页Journal of Agricultural Biotechnology

基　　金：国家自然科学青年基金(31802053);江苏省自然科学青年基金(BK20180907);中国博士后面上基金(2019M661950);江苏省博士后面上基金(137070510);扬州市省自然科学青年基金(YZ2017104);扬州大学大学生科创基金(X20210658);江苏省高校“青蓝工程”资助项目;扬州市第四期“英才培育计划”优秀教育人才资助项目。

摘　　要：前期通过高通量测序发现,受体互作丝氨酸苏氨酸激酶2(receptor interacting serine/threonine kinase 2,RIPK2)介导的NOD/RIPK2信号通路在鸡(Gallus gallus)免疫炎症反应过程中显著性富集。为研究RIPK2在鸡巨噬细胞系(chicken macrophage cell line,HD11)中免疫炎症的功能,本研究采用慢病毒(Lentivirus)介导的RNA干扰技术,建立了稳定干扰RIPK2基因表达的HD11。根据鸡RIPK2基因的序列,设计了3个RNA干扰靶点序列和1个阴性对照序列,连接到pLVshRNA-EGFP(2A)Puro干扰载体,瞬时转染HD11。利用qRT-PCR技术检测各靶点对RIPK2的干扰效率,将干扰效率最佳的重组载体进行慢病毒包裹。利用包裹干扰载体的慢病毒感染HD11,采用qRT-PCR及Western blot检测不同实验组中RIPK2及NOD/RIPK2信号通路下游关键基因:核因子-κB激酶复合物抑制剂(component of inhibitor of nuclear factor kappa B kinase complex,IKKα)、因子-κB激酶亚单位β抑制剂(inhibitor of nuclear factor kappa B kinase subunit beta,IKKβ)、核因子κB亚单位1(nuclear factor kappa B subunit 1,NFκB)和白细胞介素1β(interleukin 1 beta,IL1β)的表达量变化。同时构建了RIPK2过表达载体(pcDNA3.1-RIPK2),并以此载体开展RIPK2基因的拯救实验。构建了3条靶向RIPK2的慢病毒干扰表达载体RIPK2-shRNA1(小发卡RNA1,short hairpin RNA 1)、RIPK2-shRNA2和RIPK2-shRNA3,其中RIPK2-shRNA1和RIPK2-shRNA3重组质粒对RIPK2基因的干扰效率分别为(77.4±0.61)%和(90.21±0.68)%。将RIPK2-shRNA3重组质粒进行慢病毒包裹,获得病毒滴度为2×10^(8)TU/mL;将RIPK2-shRNA3慢病毒转染HD11,干扰组RIPK2 mRNA和蛋白表达水平较对照组显著性降低(P<0.05),NOD/RIPK2信号通路下游关键基因IKKα、IKKβ、NFκB和IL1β的表达量发生了显著下调(P<0.05);干扰组转染RIPK2过表达载体(pcDNA3.1-RIPK2)后RIPK2的表达水平能够恢复。本研究成功构建了靶向鸡RIPK2的shRNA慢病毒表达载体,可有效沉默RIPK2基因在HD11中的表达,并�In a previous study,by high-throughput sequencing,receptor interacting serine/threonine kinase 2(RIPK2)was demonstrated to be highly up-regulated in the immune and inflammatory response of chickens(Gallus gallus).To investigate the function of RIPK2-mediated NOD/RIPK2 signaling pathway in chicken macrophage cell line(HD11)for immunity and inflammation,a chicken HD11 stably interfering with RIPK2gene was established by using Lentivirus-mediated RNA interference approach.According to the sequence of chicken RIPK2 gene,three RNA interference target sequences and one negative control sequence were designed for RIPK2 gene,recombined with the pLVshRNA-EGFP(2A)Puro interference vector,and then transiently transfected into HD11.Interference efficiency of each target on RIPK2 was tested by qRT-PCR and the recombinant vector with high interference efficiency was packaged with Lentivirus to transfect HD11.qRTPCR and Western blot were used to detect the expression changes of RIPK2 and the downstream key genes IKKα(component of inhibitor of nuclear factor kappa B kinase complex),IKKβ(inhibitor of nuclear factor kappa B kinase subunit beta),NFκB(nuclear factor kappa B subunit 1)and IL1β(interleukin 1 beta)of NOD/RIPK2 signaling pathways in different experimental groups.Meanwhile the RIPK2 overexpression vector(pcDNA3.1-RIPK2)was constructed for the rescue experiment of RIPK2 gene.Three recombinant plasmid of RIPK2-shRNA1(small hairpin RNA 1),RIPK2-shRNA2 and RIPK2-shRNA3 were successfully constructed.The recombinant plasmids of RIPK2-shRNA1 and RIPK2-shRNA3 had the most obviously inhibitory effect of(77.4±0.61)%and(90.21±0.68)%,respectively on the RIPK2 gene.The RIPK2-shRNA3 recombinant plasmid was packaged into retrovirus with a virus titer of 2×10^(8)TU/mL.After the RIPK2-shRNA3 Lentivirus was transfected into HD11,the results of q RT-PCR and Western blot showed that RIPK2 mRNA and protein expression level in the interference group were significantly low compared to control group(P<0.05).The expression of downstream k

关 键 词：鸡受体互作丝氨酸苏氨酸激酶2(RIPK2)基因 小发卡RNA(shRNA) 慢病毒载体 鸡巨噬细胞系(HD11) 

分 类 号：S831.2[农业科学—畜牧学]