山羊STING的克隆及其在OFTu细胞的表达
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摘要
为了解山羊干扰素刺激基因(stimulator of interferon genes,STING)及其编码蛋白的生物学信息,本试验采用RT-PCR法克隆了海南黑山羊STING基因并进行序列及其编码蛋白的分析,构建了pEGFP-N1-STING表达载体并转染至OFTu细胞进行了表达。结果表明STING基因的物种内同源性高、种间同源性低,编码蛋白由378个氨基酸组成,存在4个潜在跨膜区,含有1个信号肽;转染和Western blot试验证实,构建的表达载体可在OFTu细胞中成功表达。本试验为构建STING专性表达细胞系及深入研究其在山羊抗感染免疫的机制奠定了基础。
To investigate the stimulator of interferon genes( STING) of Hainan black goat and the biological information of its encoding protein,the STING gene was cloned by RT-PCR and the sequence and encoded protein were analyzed. The expression vector of pEGFP-N1-STING was constructed and transfected into OFTu cells for expression analysis.Results of nucleotide homology analysis showed that the STING gene were conservative within species while highly variant between species.The STING protein is composed of 378 amino acids with 4 potential transmembrane domains which containe one signal peptide. Transfection and Western blot experiments confirmed that the expression vector could be successfully expressed in OFTu cells. These results provided the basic data for construction of STING-specific expression cells lines and further investigation of its anti-infection mechanism of STING in goat.
To investigate the stimulator of interferon genes( STING) of Hainan black goat and the biological information of its encoding protein,the STING gene was cloned by RT-PCR and the sequence and encoded protein were analyzed. The expression vector of pEGFP-N1-STING was constructed and transfected into OFTu cells for expression analysis.Results of nucleotide homology analysis showed that the STING gene were conservative within species while highly variant between species.The STING protein is composed of 378 amino acids with 4 potential transmembrane domains which containe one signal peptide. Transfection and Western blot experiments confirmed that the expression vector could be successfully expressed in OFTu cells. These results provided the basic data for construction of STING-specific expression cells lines and further investigation of its anti-infection mechanism of STING in goat.
引文
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[4]ISHIKAWA H,MA Z,BARBER G N.STING regulates intracellular DNA-mediated,type I interferondependent innate immunity[J].Nature,2009,461(7265):788-792.
[5]FENG H,LIU H,KONG R,et al.Expression profiles of carp IRF-3/-7correlate with the up-regulation of RIG-I/MAVS/TRAF3/TBK1,four pivotal molecules in RIG-I signaling pathway[J].Fish Shellfish Immunol,2011,30(4/5):1159-1169.
[6]DALRYMPLE N A,CIMICA V,MACKOW E R.Dengue virus NS proteins inhibit RIG-I/MAVS signaling by blocking TBK1/IRF3 phosphorylation:Dengue virus serotype 1NS4AIs a unique interferonregulating virulence determinant[J].MBio,2015,6(3):e515-e553.
[7]CHRISTENSEN M H,JENSEN S B,MIETTINEN J J,et al.HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced typeⅠIFN expression[J].EMBO J,2016,35(13):1385-1399.
[8]RAN Y,SHU H B,WANG Y Y.MITA/STING:a central and multifaceted mediator in innate immune response[J].Cytokine Growth Factor Rev,2014,25(6):631-639.
[9]KONNO H,BARBER G N.The STING controlled cytosolic-DNA activated innate immune pathway and microbial disease[J].Microbes Infect,2014,16(12):998-1001.
[10]LIANG D,XIAO-FENG H,GUAN-JUN D,et al.Activated STING enhances Tregs infiltration in the HPV-related carcinogenesis of tongue squamous cells via the c-jun/CCL22signal[J].Biochim Biophys Acta,2015,1852(11):2494-2503.
[11]MA Z,DAMANIA B.The cGAS-STING defense pathway and its counteraction by viruses[J].Cell Host Microbe,2016,19(2):150-158.
[12]POLTORAK A,KURMYSHKINA O,VOLKOVA T.Stimulator of interferon genes(STING):A“new chapter”in virus-associated cancer research.Lessons from wild-derived mouse models of innate immunity[J].Cytokine Growth Factor Rev,2016,29:83-91.
[13]TROTARD M,TSOPOULIDIS N,TIBRONI N,et al.Sensing of HIV-1infection in Tzm-bl cells with reconstituted expression of STING[J].J Virol,2015,90(4):2064-2076.
[14]汪园园,童 双,李 威,等.羊胚胎鼻甲细胞的原代培养及其在羊口疮病毒研究中的应用[J].中国兽医科学,2013,43(5):470-475.
[15]WANG D,ZHOU L,ZHOU H,et al.Effects of nutrition level of concentrate-based diets on growth performance and carcass characteristics of Hainan black goats[J].Trop Anim Health Prod,2014,46(5):783-788.
[16]LUO W W,LI S,LI C,et al.iRhom2is essential for innate immunity to DNA viruses by mediating trafficking and stability of the adaptor STING[J].Nat Immunol,2016,17(9):1057-1066.
[17]HOLM C K,RAHBEK S H,GAD H H,et al.Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses[J].Nat Commun,2016(7):10680.
[18]DE ALMEIDA L A,CARVALHO N B,OLIVEIRA F S,et al.MyD88and STING signaling pathways are required for IRF3-mediated IFN-beta induction in response to Brucella abortus infection[J].PLoS One,2011,6(8):e23135.
[2]ZHONG B,YANG Y,LI S,et al.The adaptor protein MITA links virus-sensing receptors to IRF3transcription factor activation[J].Immunity,2008,29(4):538-550.
[3]SUN W,LI Y,CHEN L,et al.ERIS,an endoplasmic reticulum IFN stimulator,activates innate immune signaling through dimerization[J].Proc Natl Acad Sci USA,2009,106(21):8653-8658.
[4]ISHIKAWA H,MA Z,BARBER G N.STING regulates intracellular DNA-mediated,type I interferondependent innate immunity[J].Nature,2009,461(7265):788-792.
[5]FENG H,LIU H,KONG R,et al.Expression profiles of carp IRF-3/-7correlate with the up-regulation of RIG-I/MAVS/TRAF3/TBK1,four pivotal molecules in RIG-I signaling pathway[J].Fish Shellfish Immunol,2011,30(4/5):1159-1169.
[6]DALRYMPLE N A,CIMICA V,MACKOW E R.Dengue virus NS proteins inhibit RIG-I/MAVS signaling by blocking TBK1/IRF3 phosphorylation:Dengue virus serotype 1NS4AIs a unique interferonregulating virulence determinant[J].MBio,2015,6(3):e515-e553.
[7]CHRISTENSEN M H,JENSEN S B,MIETTINEN J J,et al.HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced typeⅠIFN expression[J].EMBO J,2016,35(13):1385-1399.
[8]RAN Y,SHU H B,WANG Y Y.MITA/STING:a central and multifaceted mediator in innate immune response[J].Cytokine Growth Factor Rev,2014,25(6):631-639.
[9]KONNO H,BARBER G N.The STING controlled cytosolic-DNA activated innate immune pathway and microbial disease[J].Microbes Infect,2014,16(12):998-1001.
[10]LIANG D,XIAO-FENG H,GUAN-JUN D,et al.Activated STING enhances Tregs infiltration in the HPV-related carcinogenesis of tongue squamous cells via the c-jun/CCL22signal[J].Biochim Biophys Acta,2015,1852(11):2494-2503.
[11]MA Z,DAMANIA B.The cGAS-STING defense pathway and its counteraction by viruses[J].Cell Host Microbe,2016,19(2):150-158.
[12]POLTORAK A,KURMYSHKINA O,VOLKOVA T.Stimulator of interferon genes(STING):A“new chapter”in virus-associated cancer research.Lessons from wild-derived mouse models of innate immunity[J].Cytokine Growth Factor Rev,2016,29:83-91.
[13]TROTARD M,TSOPOULIDIS N,TIBRONI N,et al.Sensing of HIV-1infection in Tzm-bl cells with reconstituted expression of STING[J].J Virol,2015,90(4):2064-2076.
[14]汪园园,童 双,李 威,等.羊胚胎鼻甲细胞的原代培养及其在羊口疮病毒研究中的应用[J].中国兽医科学,2013,43(5):470-475.
[15]WANG D,ZHOU L,ZHOU H,et al.Effects of nutrition level of concentrate-based diets on growth performance and carcass characteristics of Hainan black goats[J].Trop Anim Health Prod,2014,46(5):783-788.
[16]LUO W W,LI S,LI C,et al.iRhom2is essential for innate immunity to DNA viruses by mediating trafficking and stability of the adaptor STING[J].Nat Immunol,2016,17(9):1057-1066.
[17]HOLM C K,RAHBEK S H,GAD H H,et al.Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses[J].Nat Commun,2016(7):10680.
[18]DE ALMEIDA L A,CARVALHO N B,OLIVEIRA F S,et al.MyD88and STING signaling pathways are required for IRF3-mediated IFN-beta induction in response to Brucella abortus infection[J].PLoS One,2011,6(8):e23135.