猪cGAS基因的克隆与原核表达
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摘要
【目的】环磷酸鸟苷-腺苷酸合成酶(cyclic guanosine monophosphate-adenosine monophosphate synthase,c GAS)是近期在哺乳动物细胞中发现的一种新型核酸转移酶,能够识别胞质DNA,催化ATP和GTP生成第二信使c GAMP,继而通过STING依赖的方式活化转录因子IRF3,启动机体固有免疫。通过构建含猪c GAS基因的重组质粒p Bb B3a-His6-Nus A-c GAS,进行原核表达,得到c GAS蛋白,为进行体外催化合成c GAMP及探讨其在天然免疫过程中的作用奠定基础。【方法】以猪脾脏c DNA为模板克隆猪c GAS的蛋白编码区(open reading frame,ORF),用非酶连接技术将此基因克隆至丙酸诱导型原核表达载体p Bb B3a-His6-Nus A-LIC中。菌液PCR进行阳性克隆鉴定并测序。将测序鉴定正确的克隆菌液提取质粒,转化至E.coli BL21(DE3)中。当细菌生长到对数期时,丙酸钠诱导表达His6-Nus A-c GAS融合蛋白,用20 mmol·L~(-1)丙酸钠在20℃,180 r/min分别诱导0、2、4、6、8、10 h,以确定最佳诱导时间;然后,分别用0、5、10、15、20、25、30、35、40、45 mmol·L~(-1)的丙酸钠在20℃,180 r/min诱导6 h,以确定最佳诱导丙酸钠诱导浓度;另外,分别在20℃,30℃,37℃条件下用20 mmol·L~(-1)丙酸钠,180 r/min培养6 h,以确定最佳诱导温度。筛选最佳诱导条件,并用SDS-PAGE和Western blotting进行鉴定。【结果】(1)本试验成功克隆了猪c GAS基因,其ORF长度为1 494 bp;(2)构建了c GAS丙酸诱导型原核表达载体p Bb B3a-His6-Nus A-c GAS;(3)His6-Nus A-c GAS融合蛋白在37℃,添加20 mmol·L~(-1)丙酸钠,诱导6 h时表达量最高。(4)His6-Nus A-c GAS融合蛋白在裂解菌液的上清和沉淀中均有表达,相对分子质量为111.87 k D。【结论】运用大肠杆菌表达系统成功表达了c GAS融合蛋白,本试验为体外表达c GAS融合蛋白提供技术方法。
【Objective】c GAS(cyclic guanosine monophosphate-adenosine monophosphate synthase), as a new type of nucleic transferase was found in mammalian cells recently. It can identify cytoplasmic DNA and catalyse ATP and GTP to generate the second messenger c GAMP, c GAMP binds to STING by dependent way, leading to activate transcription factor IRF3. Then the inherent immunity was starting. This experiment was conducted to construct a prokaryotic expression plasmid, p Bb B3a-His6-Nus Ac GAS, containing porcine c GAS gene. The gene expression was induced in E. coli, and the c GAS protein was obtained. Then a foundation of the research on the synthesis of c GAMP in vitro and its function in innate immune progress was made.【Method】 The open reading frame(ORF) of c GAS was amplified from the c DNA of porcine spleen, and cloned it into propionate inducible plasmid p Bb B3a-His6-Nus A-LIC by ligation-independent cloning(LIC) technology. Identification of individual clone was performed by bacteria liquid PCR followed by DNA sequencing. Plasmids were extracted from the confirmed bacteria and transformed into E.coli BL21(DE3). When the bacteria grew to logarithmic phase, sodium propionate was used to induce the expression of His6-Nus A- c GAS fusion protein, 20 mmol·L~(-1) sodium propionate at 20℃, 180 r/min were induced by 2 h, 4 h, 6 h, 8 h, 10 h and 0 h, then the optimal induction time was determined. Sodium propionate at 0, 5, 10, 15, 20, 25, 30, 35, 40 and 45 mmol·L~(-1), respectively, and at 20℃, 180 r/min induced for 6 h to determine the best induction concentration of sodium propionate. Under the conditions of 20, 30 and 37℃, 20 mmol·L~(-1) sodium propionate was used at 180 r/min to cultivate for 6 h to determine the best temperature induction. The His6-Nus A-c GAS fusion protein was induced by sodium propionate and identified by SDS-PAGE and Western Blot. 【Result】(1) Porcine c GAS gene'ORF, which is 1 494 bp in length, was successfully cloned;(2) The propionate inducible plasmid p Bb B3a-His6-Nus A-c GAS was constructed;(3) At 37℃, 20 mmol·L~(-1) sodium propionate to induce for 6 h, the His6-Nus A-c GAS fusion protein's expression amount was the highest.(4) His6-Nus A-c GAS fusion protein was efficiently expressed in soluble form with a molecular weight of about 111.87 k D.【Conclusion】 These results indicate that c GAS fusion protein was successfully expressed in E.coli BL21(DE3) and this will provide technology and methods for c GAS's fusion protein expression in vitro.
【Objective】c GAS(cyclic guanosine monophosphate-adenosine monophosphate synthase), as a new type of nucleic transferase was found in mammalian cells recently. It can identify cytoplasmic DNA and catalyse ATP and GTP to generate the second messenger c GAMP, c GAMP binds to STING by dependent way, leading to activate transcription factor IRF3. Then the inherent immunity was starting. This experiment was conducted to construct a prokaryotic expression plasmid, p Bb B3a-His6-Nus Ac GAS, containing porcine c GAS gene. The gene expression was induced in E. coli, and the c GAS protein was obtained. Then a foundation of the research on the synthesis of c GAMP in vitro and its function in innate immune progress was made.【Method】 The open reading frame(ORF) of c GAS was amplified from the c DNA of porcine spleen, and cloned it into propionate inducible plasmid p Bb B3a-His6-Nus A-LIC by ligation-independent cloning(LIC) technology. Identification of individual clone was performed by bacteria liquid PCR followed by DNA sequencing. Plasmids were extracted from the confirmed bacteria and transformed into E.coli BL21(DE3). When the bacteria grew to logarithmic phase, sodium propionate was used to induce the expression of His6-Nus A- c GAS fusion protein, 20 mmol·L~(-1) sodium propionate at 20℃, 180 r/min were induced by 2 h, 4 h, 6 h, 8 h, 10 h and 0 h, then the optimal induction time was determined. Sodium propionate at 0, 5, 10, 15, 20, 25, 30, 35, 40 and 45 mmol·L~(-1), respectively, and at 20℃, 180 r/min induced for 6 h to determine the best induction concentration of sodium propionate. Under the conditions of 20, 30 and 37℃, 20 mmol·L~(-1) sodium propionate was used at 180 r/min to cultivate for 6 h to determine the best temperature induction. The His6-Nus A-c GAS fusion protein was induced by sodium propionate and identified by SDS-PAGE and Western Blot. 【Result】(1) Porcine c GAS gene'ORF, which is 1 494 bp in length, was successfully cloned;(2) The propionate inducible plasmid p Bb B3a-His6-Nus A-c GAS was constructed;(3) At 37℃, 20 mmol·L~(-1) sodium propionate to induce for 6 h, the His6-Nus A-c GAS fusion protein's expression amount was the highest.(4) His6-Nus A-c GAS fusion protein was efficiently expressed in soluble form with a molecular weight of about 111.87 k D.【Conclusion】 These results indicate that c GAS fusion protein was successfully expressed in E.coli BL21(DE3) and this will provide technology and methods for c GAS's fusion protein expression in vitro.
引文
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[11]WU J,SUN L,CHEN X,FENGHE D,HEPING S.Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA.Science,2013,339(6121):826-830.
[12]SUN L,WU J,DU F,CHEN X,CHEN Z J.Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.Science,2013,339(6121):786-791.
[13]LI X,SHU C,YI G,CHATON C,SHELTON C.Cyclic GMP-AMP synthase is activated by double-stranded DNA-induced oligomerization.Immunity,2013,39(6):1019-1031.
[14]GAO D,WU J,WU Y T,FENGHE D,CHUKWUEMIKA A.Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses.Science,2013,341(6148):903-906.
[15]LAM E,STEIN S,FALCK-PEDERSEN E.Adenovirus detection by the c GAS/STING/TBK1 DNA sensing cascade.Journal of Virology,2013,88(2):974-981.
[16]TANAKA Y,CHEN Z J.STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway.Science Signaling,2012,5(214):20.
[17]DING Q,CAO X Z,LU J,HUANG B,LIU Y J,KATO N,SHU H B,ZHONG J.Hepatitis C virus NS4B blocks the interaction of STING and TBK1 to evade host innate immunity.Journal of Hepatology,2013,59(1):52-58.
[18]ASLANIDIS C,DE JONG P J.Ligation-independent cloning of PCR products(LIC-PCR).Nucleic Acids Research,1990,18(20):6069-6074.
[19]谈蓉,马立新.一种新的产生天然蛋白的不依赖于连接的克隆载体.湖北大学学报(自然科学版),2009,31(4):415-418.TAN R,MA L X.Novel ligation-independent cloning vectors for production of native proteins.Journal of Hubei University(Natural Science Edition),2009,31(4):415-418.(in Chinese)
[20]刘爽,胡宝成.原核系统可溶性表达策略.生物技术通讯,2005,16(2):172-175.LIU S,HU B C.The strategy of prokaryotic system soluble expression.Letters in Biotechnology,2005,16(2):172-175.(in Chinese)
[21]LEE S K,KEASLING J D.A Salmonella-based,propionate-inducible,expression system for Salmo-nella enterica.Gene,2006,377:6-11.
[2]KEATING S E,BARAN M,BOWIE A G.Cytosolic DNA sensors regulating type I interferon induction.Trends in Immunology,2011,32(12):574-581.
[3]KATO H,TAKAHASI K,FUJITA T.RIG-I-like receptors:cytoplasmic sensors for non-self RNA.Immunological Reviews,2011,243(1):91-98.
[4]ISHIKAWA H,MA Z,BARBER G N.STING regulates intracellular DNA-mediated,type I interferon-dependent innate immunity.Nature,2009,461(7265):788-792.
[5]BOWIE A.The STING in the tail for cytosolic DNA-dependent activation of IRF3.Science Signaling,2012,5(214):9.
[6]ISHIKAWA H,BARBER G N.STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling.Nature,2008,455(7213):674-678.
[7]MARGOLIS R L,STINE O C,MCINNIS M G,RANEN N G,RUBINSZTEIN D C.c DNA cloning of a human homologue of the Caenorhabditis elegans cell fate-determining gene mab-21:expression,chromosomal localization and analysis of a highly polymorphic(CAG)n trinucleotide repeat.Human Molecular Genetics,1996,5(5):607-616.
[8]SCHOGGINS J W,WILSON S J,PANIS M,MURPHY M Y,JONES C T.A diverse range of gene products are effectors of the type I interferon antiviral response.Nature,2011,472(7344):481-485.
[9]CHOW K L,HALL D H,EMMONS S W.The mab-21 gene of Caenorhabditis elegans encodes a novel protein required for choice of alternate cell fates.Development,1995,121(11):3615-3626.
[10]LI X D,WU J,GAO D,WANG H,SUN L,CHEN Z J.Pivotal roles of c GAS-c GAMP signaling in antiviral defense and immune adjuvant effects.Science,2013,341(6152):1390-1394.
[11]WU J,SUN L,CHEN X,FENGHE D,HEPING S.Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA.Science,2013,339(6121):826-830.
[12]SUN L,WU J,DU F,CHEN X,CHEN Z J.Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.Science,2013,339(6121):786-791.
[13]LI X,SHU C,YI G,CHATON C,SHELTON C.Cyclic GMP-AMP synthase is activated by double-stranded DNA-induced oligomerization.Immunity,2013,39(6):1019-1031.
[14]GAO D,WU J,WU Y T,FENGHE D,CHUKWUEMIKA A.Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses.Science,2013,341(6148):903-906.
[15]LAM E,STEIN S,FALCK-PEDERSEN E.Adenovirus detection by the c GAS/STING/TBK1 DNA sensing cascade.Journal of Virology,2013,88(2):974-981.
[16]TANAKA Y,CHEN Z J.STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway.Science Signaling,2012,5(214):20.
[17]DING Q,CAO X Z,LU J,HUANG B,LIU Y J,KATO N,SHU H B,ZHONG J.Hepatitis C virus NS4B blocks the interaction of STING and TBK1 to evade host innate immunity.Journal of Hepatology,2013,59(1):52-58.
[18]ASLANIDIS C,DE JONG P J.Ligation-independent cloning of PCR products(LIC-PCR).Nucleic Acids Research,1990,18(20):6069-6074.
[19]谈蓉,马立新.一种新的产生天然蛋白的不依赖于连接的克隆载体.湖北大学学报(自然科学版),2009,31(4):415-418.TAN R,MA L X.Novel ligation-independent cloning vectors for production of native proteins.Journal of Hubei University(Natural Science Edition),2009,31(4):415-418.(in Chinese)
[20]刘爽,胡宝成.原核系统可溶性表达策略.生物技术通讯,2005,16(2):172-175.LIU S,HU B C.The strategy of prokaryotic system soluble expression.Letters in Biotechnology,2005,16(2):172-175.(in Chinese)
[21]LEE S K,KEASLING J D.A Salmonella-based,propionate-inducible,expression system for Salmo-nella enterica.Gene,2006,377:6-11.