CN113088522B - 一种日本鳗鲡转录因子c-Rel基因启动子及其应用 - Google Patents
一种日本鳗鲡转录因子c-Rel基因启动子及其应用 Download PDFInfo
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Abstract
本发明涉及一种日本鳗鲡转录因子c‑Rel基因启动子及其应用。通过c‑Rel基因开放阅读框第一外显子序列比对分析日本鳗鲡基因组,对分析预测的c‑Rel基因5’侧翼区序列进行降落PCR方法克隆获得日本鳗鲡转录因子c‑Rel基因启动子序列,并成功构建了c‑Rel基因启动子pGL3‑c‑Rel‑pro荧光素酶重组载体。实验证明c‑Rel基因启动子可被LPS和嗜水气单胞菌诱导激活,并且发现重要信号通路炎症调控因子Caspase‑1能够显著上调c‑Rel基因启动子的荧光素酶活性。本发明可为研究日本鳗鲡转录因子c‑Rel基因的表达调控机制以及重要鱼类炎症相关NF‑κB信号通路网络调控机制的研究提供良好的实验系统,同时也可以利用该启动子构建表达载体后高效表达外源基因,或将该启动子应用于转基因鱼的构建,具有重要的理论和实际意义。
Description
技术领域
本发明涉及基因工程技术领域,尤其涉及日本鳗鲡转录因子c-Rel基因启动子及其应用。
背景技术
细胞核转录因子kappaB(NF-κB)是细胞中重要的转录调控因子,通过对多种抗菌肽、细胞因子、生长因子、粘附因子以及免疫相关酶的基因表达调控,参与机体炎症反应和免疫调节,并在细胞增殖、分化、凋亡等方面发挥重要作用。
NF-κB家族有5个成员,分别为NF-κB1(p50)、NF-κB2(p52)、Rel-A(p65)、Rel-B和c-Rel。NF-κB/Rel家族蛋白的共同特征是在N端都包含有一个RHD结构域(Rel-homologydomain)。RHD结构域与蛋白的二聚化、DNA结合以及与抑制蛋白IκB(inhibitor of NF-κB)互作等功能相关(Zhang,Q.,M.J.Lenardo and D.Baltimore(2017)."30Years of NF-κB:ABlossoming of Relevance to Human Pathobiology."Cell168(1):37-57.)。当细胞受到TNFα、IL-1β等细胞因子、细菌脂多糖LPS、病毒蛋白、细菌和病毒特有的DNA或RNA、过氧化酶、蛋白激酶、紫外线及X射线刺激后,经过不同信号通路的信号传导最终激活并引起不同NF-κB/Rel蛋白之间的同源二聚化或异源二聚化,进入细胞核对免疫相关基因表达进行调控(Wan,F.and M.J.Lenardo(2010)."The nuclear signaling of NF-KB:currentknowledge,new insights,and future perspectives."cell Research(1):24-33.)。
c-Rel是转录因子NF-κB家族的重要成员之一,通过参与调控T细胞IL-2、Foxp3等多种免疫功能密切相关细胞因子(Isomura,I.,S.Palmer,R.J.Grumont,K.Bunting,G.Hoyne,N.Wilkinson,A.Banerjee,A.Proietto,R.Gugasyan and L.Wu(2010)."c-Rel isrequired for the development of thymic Foxp3+CD4 regulatory T cells."Journalof Experimental Medicine 207(4):899-899.;F.,R.J.Grumont,A.Strasser,D.Metcalf,R.Li,D.Tarlinton and S.Gerondakis(1995)."Mice lacking the c-relproto-oncogene exhibit defects in lymphocyte proliferation,humoral immunity,and interleukin-2expression."Genes&;development 9(16):1965-1977.)以及巨噬细胞和树突状细胞中IL-21、IL-23的转录表达(Chen,G.,K.Hardy,K.Bunting,S.Daley,L.Ma and M.F.Shannon(2010)."Regulation ofthe IL-21gene by the NF-κBtranscription factorc-Rel."Journal ofimmunology(Baltimore,Md.:1950)185(4):2350-2359.;Visekruna,A.,A.Volkov and U.Steinhoff(2012)."A key role for NF-κBtranscription factor c-Rel in T-lymphocyte-differentiation and effectorfunctions."Clinical&;developmental immunology 2012:239368.),从而在机体免疫调节方面发挥关键作用。此外,有研究表明c-Rel可以诱导哺乳动物干扰素调节因子4(IRF-4)在淋巴细胞表达水平的提高(Grumont,R.J.and S.Gerondakis(2000)."Rel InducesInterferon Regulatory Factor 4(IRF-4)Expression in Lymphocytes:Modulation ofInterferon-Regulated Gene Expression by Rel/Nuclear FactorκB."JournalofExperimental Medicine 191(8):1281-1292.)。最近的研究发现通过抑制或敲除小鼠中c-Rel可以特异性损害调节性T细胞的功能,降低小鼠黑色素瘤细胞的生长,该研究结果提示c-Rel可以作为肿瘤免疫靶向治疗的免疫分子(Grinberg-Bleyer,Y.,H.Oh,A.Desrichard,D.M.Bhatt,R.Caron,T.A.Chan,R.M.Schmid,U.Klein,M.S.Hayden andS.Ghosh(2017)."NF-κB c-Rel Is Crucial for the Regulatory T Cell ImmuneCheckpoint in Cancer."Cell 170(6):1096-1108.e1013.)。
目前,虽然在圆口纲东北七鳃鳗中获得了c-Rel基因及其启动子序列,但对于含有众多养殖经济鱼类的硬骨鱼纲c-Rel基因的研究报道十分有限,仅有斑马鱼c-Rel能够诱导IRF-4表达的研究报道,而有关硬骨鱼类c-Rel基因启动子方面的转录调控机制研究尚未见报道(Li,S.,X.Guo,L.-F.Lu,X.-B.Lu,N.Wu and Y.-A.Zhang(2015)."Regulationpatternoffish irf4(the gene encoding IFN regulatory factor 4)by STAT6,c-Rel andIRF4."Developmental&Comparative Immunology 51(1):65-73.)。
当前,功能基因的表达调控已成为分子生物学研究领域的热点,启动子是基因表达调控的重要元件。鉴于鱼类c-Rel在NF-κB信号通路中的重要性,研究日本鳗鲡c-Rel基因表达调控机制将为硬骨鱼类抗细菌和病毒病免疫应答机制的深入研究提供重要的理论依据。
发明内容
本发明的目的在于克服现有技术的不足之处,提供了一种日本鳗鲡转录因子c-Rel基因启动子及其应用,解决了上述背景技术中的问题。
本发明解决其技术问题所采用的技术方案之一是:提供了日本鳗鲡转录因子c-Rel基因启动子,其核苷酸序列如SEQ ID NO:1所示。
本发明解决其技术问题所采用的技术方案之二是:提供了含有上述启动子的表达盒、重组载体、转基因细胞系、重组菌或重组病毒。
优选地,所述表达盒由上述的启动子、由上述启动子启动转录的目的基因和终止子组成。
优选地,所述重组载体为pGL3-basic、pGL2-Basic、pGL4.10、pGLuc。
优选地,所述重组菌为大肠杆菌、枯草杆菌、乳酸菌、酵母菌。
本发明解决其技术问题所采用的技术方案之三是:提供了上述日本鳗鲡转录因子c-Rel基因启动子在构建真核表达载体、鱼类细胞或哺乳动物细胞中高效表达外源基因中的应用。
本发明解决其技术问题所采用的技术方案之四是:提供了上述日本鳗鲡转录因子c-Rel基因启动子在构建转基因鱼中的应用。
本发明具有如下有益效果:
本申请人成功克隆获得了日本鳗鲡NF-κB重要亚基c-Rel基因序列。由于启动子是决定基因表达及其调控的关键因素,为研究鱼类c-Rel基因的表达调控机制,我们通过日本鳗鲡c-Rel开放阅读框序列与基因组序列比对分析获得可能的日本鳗鲡c-Rel基因5′侧翼调控区序列,经过引物设计PCR克隆验证获得日本鳗鲡c-Rel基因启动子序列,分析表明该c-Rel基因启动子存在多个转录因子结合位点,如AP-1、C/EBPalp、SP1、NF-κB、MBP-2、C/EBPβ、c-Jun、RelA、Sox-2、Sox-4、Hb、GLO、MBP-2、RAR-alph、MyoD等,具有复杂的表达调控机制,其中转录因子Sox-2、Sox-4、Hb、GLO、MBP-2、MyoD等为硬骨鱼类所特有,未在圆口纲东北七鳃鳗c-Rel基因启动子序列中出现,显示出硬骨鱼类c-Rel基因启动子具有不同于圆口纲类c-Rel基因的特有表达调控机制。
经报告基因检测实验证明了该日本鳗鲡c-Rel基因启动子具有较强的启动子活性。并且首次证实日本鳗鲡转录因子c-Rel基因启动子活性的可以被革兰氏阴性菌重要表面抗原LPS和水产动物重要致病菌嗜水气单胞菌诱导激活,该研究未在硬骨鱼类、软骨鱼类以及圆口纲东北七鳃鳗等研究报道过,因此可为研究包含众多经济养殖硬骨鱼类转录因子c-Rel基因的表达调控机制以及重要的类炎症相关NF-κB信号通路网络调控机制的研究提供良好的实验系统,在应用方面为利用该启动子构建表达载体高效表达外源基因或将该启动子应用于转基因鱼构建创造条件,具有重要的理论和实际意义。
附图说明
图1为日本鳗鲡c-Rel基因启动子SP1、NF-1等转录因子结合位点示意图
图2为日本鳗鲡c-Rel基因启动子Hb、C/EBPalp等转录因子结合位点示意图
图3为日本鳗鲡c-Rel基因启动子Sox-2、Antp等转录因子结合位点示意图
图4为日本鳗鲡c-Rel基因启动子MBP-2、I d2等转录因子结合位点示意图
图5为日本鳗鲡c-Rel基因启动子RAR-alph、MyoD等转录因子结合位点示意图
图6为采用双荧光素酶报告基因检测系统定量分析日本鳗鲡c-Rel基因启动子的活性图。
其中,横坐标pGL3表示空载体pGL3-Basic转染EPC细胞的荧光素酶相对活性(作为对照组);
pGL3-c-Rel-pro为荧光素酶重组载体pGL3-c-Rel-pro转染EPC细胞的荧光素酶相对活性(作为实验组)。
如图6所示,重组载体pGL3-c-Rel-pro转染EPC细胞中的荧光素酶相对活性为空载体pGL3-Basic转染EPC细胞的11.5倍,说明了日本鳗鲡c-Rel基因启动子可以较好地启动荧光素酶报告基因的转录。
图7为在革兰氏阴性菌大肠杆菌重要表面抗原LPS(30μg/mL)刺激条件下日本鳗鲡c-Rel基因启动子的活性变化图。
其中,横坐标pGL3-Basic表示空载体pGL3-Basic转染EPC细胞的荧光素酶相对活性(作为对照组);
pGL3-c-Rel-pro为荧光素酶重组载体pGL3-c-Rel-pro转染EPC细胞的荧光素酶相对活性(作为实验组)。
如图7所示,经LPS刺激24h重组载体pGL3-c-Rel-pro转染EPC细胞中的荧光素酶相对活性为空载体pGL3-Basic转染EPC细胞的15.8倍,说明了日本鳗鲡c-Rel基因启动子可被LPS诱导激活。
图8为在水产动物重要致病菌嗜水气单胞菌(106cfu/mL)刺激条件下日本鳗鲡c-Rel基因启动子的活性变化图。
其中,横坐标pGL3-Basic表示空载体pGL3-Basic转染EPC细胞的荧光素酶相对活性(作为对照组);
pGL3-c-Rel-pro为荧光素酶重组载体pGL3-c-Rel-pro转染EPC细胞的荧光素酶相对活性(作为实验组)。
如图8所示,经嗜水气单胞菌刺激6h重组载体pGL3-c-Rel-pro转染EPC细胞中的荧光素酶相对活性为空载体pGL3-Basic转染EPC细胞的4.5倍,说明了日本鳗鲡c-Rel基因启动子可被嗜水气单胞菌诱导激活。
图9为信号通路重要炎症因子Caspase-1过表达条件下日本鳗鲡c-Rel基因启动子的活性变化图。
其中,横坐标pcDNA3.1表示空载体pcDNA3.1、荧光素酶重组载体pGL3-c-Rel-pro以及海肾荧光素酶报告基因载体pRL-TK共同转染EPC细胞24h后的荧光素酶相对活性(作为对照组);
横坐标表示pcDNA-Caspase1表示真核表达质粒pcDNA-Caspase1、荧光素酶重组载体pGL3-c-Rel-pro以及海肾荧光素酶报告基因载体pRL-TK共同转染EPC细胞24h后的荧光素酶相对活性(作为实验组)。
如图9所示,与pcDNA3.1空载体相比较,信号通路重要炎症因子真核表达质粒pcDNA-Caspase1转染EPC细胞后,能够显著上调pGL3-c-Rel-pro启动子荧光素酶活性高达173.6倍,说明了日本鳗鲡c-Rel基因启动子可被炎症因子Caspase-1正向调控并激活。
具体实施方式
为了更好地理解本发明,下面结合实施例和附图对本发明做进一步的详细说明,但本领域技术人员了解,下述实施例不是对本发明保护范围的限制,任何在本发明基础上做出的改变和变化,都在本发明的保护范围之内。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1日本鳗鲡转录因子c-Rel基因启动子的克隆
一、采用TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0试剂盒对日本鳗鲡肌肉组织基因组DNA进行提取和纯化。具体操作如下:
1、取10mg的日本鳗鲡肌肉组织用刀片切碎后置于2ml离心管中,加入180μL的Buffer GL、20μL的Proteinase K和10μL的RNase A(10mg/mL),于56℃水浴温浴过夜裂解。
2、向裂解液中加入200μL Buffer GB和200μL100%乙醇,充分吸打混匀。将SpinColumn安置于Collection Tube上,溶液移至Spin Column中,12,000rpm离心2分钟,弃滤液。
3、将500μL的BufferWA加入至Spin Column中,12,000rpm离心1分钟,弃滤液。
4、将700μL的BufferWB(用之前加入指定体积的100%乙醇)沿管壁四周加入至Spin Column中,12,000rpm离心1分钟,弃滤液。再次将700μL的Buffer WB沿管壁四周加入至Spin Column中,12,000rpm离心1分钟,弃滤液。
5、将Spin Column安置于Collection Tube上,12,000rpm离心2分钟。将SpinColumn安置于新的1.5mL的离心管上,在Spin Column膜的中央处加入加热至65℃的150μL的灭菌水室温静置5分钟。12,000rpm离心2分钟洗脱DNA。
6、提取得到的基因组DNA进行吸光度测定其浓度。
二、采用两轮降落PCR的方法扩增出日本鳗鲡c-Rel基因启动子序列。具体步骤如下:
1、通过日本鳗鲡转录因子c-Rel基因开放阅读框第一外显子序列(SEQ ID NO:2)比对分析日本鳗鲡基因组,对分析预测具有c-Rel基因5’侧翼区序列进行扩增,上游引物“5'-TAAACTGATCCGGGTCCCTTTGT-3'”(SEQ ID NO:3)为c-Rel基因5’侧翼区序列,下游引物“5'-TGGGGTAGGTCCTGTTGTTGTC-3'”(SEQ ID NO:4)为c-Rel基因开放阅读框第一外显子序列,由上海生物工程公司合成。
日本鳗鲡转录因子c-Rel基因开放阅读框第一外显子序列(如SEQ ID NO:2所示):
ATGCTTGTAATTGCCACCCTTTCTGCCTCCTCCTTTGCCCGTCCAGTGGGTGAGCCCAGCGTGCAGATATTTGAGGAACCCAAGCAAAGGGGCATGCGCTTCAGATACAAGTGTGAGGGGCGATCGGCTGGCAGCATCCCCGGTGAGAGGAGCACAGACAACAACAGGACCTACCCCA
2、第一轮PCR采用Takara公司高保真酶GC Buffer(Mg2+plus)进行扩增,反应体系:2×PrimeSTAR HS DNA Polymerase 12.5μL、上游引物0.5μL、下游引物0.5μL、gDNA 0.5μL、灭菌水11μL;降落PCR反应程序为95℃5min;95℃30s,56℃30s,72℃4min,4个循环;95℃30s,54℃30s,72℃4min,4个循环;95℃30s,52℃30s,72℃4min,30个循环;72℃10min;4℃5min。
3、第二轮PCR采用Takara公司10×Ex TaqBuffer(Mg2+plus)进行扩增,反应体系:Ex Taq 0.13μL、10×Ex Taq Buffer(Mg2+plus)2.5μL、dNTP Mixture(2.5mM each)2μL、上游引物0.5μL、下游引物0.5μL、第一轮PCR产物0.5μL、灭菌水18.87μL;降落PCR反应程序为95℃5min;95℃30s,56℃30s,72℃4min,4个循环;95℃30s,54℃30s,72℃4min,4个循环;95℃30s,52℃30s,72℃4min,30个循环;72℃10min;4℃5min。
4、第二轮PCR扩增得到的PCR产物连接到TaKaRa公司pMD19T-Simple vector载体进行序列测定与分析,从而获得含有日本鳗鲡转录因子c-Rel基因启动子序列的pMD19T-c-Rel-pro重组质粒。
日本鳗鲡转录因子c-Rel基因启动子的核苷酸序列如SEQ ID NO:1所示:
TAAACTGATCCGGGTCCCTTTGTTGACTATTTTTTATGAACCCCCCCCCCTCTTTCTCTCTCTTTCTCGTCAAAGCAATATTAAATAACATCATCGTAATATTTTTTACGTAAAACTAAAACAGTTGGTATGACTTTTATTACATATTGGACAAAATATTGGATAAAACAATGACTTGAGATAAAATGTAGCTTAAAAAGTTTTCAAAAAACACGAACAAGGTGTTTTGCCTGTCTGTGTCACTCCTTGTCTATCAGACTGTAGTAGGCCAAACCATCCTCTACAAAAATGATTGCCATTTGCTTGTTGTCCTGTGGGACAACAGATTTATTGCTGTTATTTTGGGGAAGTGGGTACCTCTTACTTGCATGTATTTTCACGCATTTTTGACAAAGTTCTTTTCTTTGCTGCTTGTTGACTGCAGTGATGCACATGTGTTCTGCATGGCTATCTAGTCCTTGGTTTGATCCATGTCAGGGTGTCTCTATTGTCAGTTCTTGCTCTCTGACATGTCAAAACATTGTTTATTAATTTTTACTGTTAATAGGTGAGATGTTACAGTACAGTCGACTCCAGAATTATAGGCACCCAGCATGAAAATGAGCAAAAATGACTGCATAAAACAATACACATAATGATTGAAATTTTCTGCTCTAACATTAGGTGGAACTTTCTTTAACTCTAATAAGAATATTCATATAAAAATTCATGATGAAATTCAATTCAATATTTAAAAAAATGTTTGTTTTTTTCTATCTCAGTCTCCAGAAACTTTTGCCATTTACTGAACACAAAGTTTCAAAAGTTTGAGACAGTTGCAGATTTGCCAAAAAGAAGATGCATGTGCATATTTCCCCCACACACTGTGAGGAAGATGATGAGGGAGGCAAAGAAGAACCCAAGGATCATAGTTTCATAGCTGCACATTTCAGTGCAGTCAGGACTACACCACTGTCTCTTCACAGCAAAAAGGTCTTGGGTTCGAATCTTGGCCTGGGCCTATCTGTTTGTTCTCCCCGTGCTTTCCTCTGGGTCCACAGTCCAAAGAAATGCAGGTATGCTAATTGGCCGTGAATATAAGCGTGTGAGTGAATGGTGAGTGTGCCCTGCCATAGACTGGCGATACTGTAGGTGTATTCCTGCCACTCGCCCAATGCATGCTGGGATGTAATTTGTTCATGATCGTGTTTTTGGATTGAAAATCATTTGTGCTCTTTTCACAAATCATTAAATTCTTTGTCAAGACAAATTAGTCTATCTCTACACACACCCACGCACACACACTTTTGTTATTTTCAGAAAAGGAGTTTAAAACGGCGAGGAAATGTATCAAAATACTAGATCGTGAAGCATAGTCCACAGTGATCTCTTACTTGTATTTAATCTGACGTGCCCATAAGAGTACTTTCGCGGGCGCCGAAGTAGTTGCTGCTGTATGTTTGTAGGTGAGTTTTTTTTTTTTTTTTTAGAATTTACTGTCGTCACTTAATACCGCCCAGATGAGGATTGTACAGCTCCTTCAGGCGGAGATAATGTGGGGAATTCCCCTTCATCGTCTCTGTTTAAAAATGAATACATCACTCTTCTTCTTTTTCAGTCCAGTCGTCTCACTCAAATACTATACCGTGATAAGGGTGTTCACAACATGGATGGTGAGTACTGTCAAAAACCTCAACTTTTGCAATATGTTTCAAAGTAGAAATATTGTGCTGGTCCTACATTTACAAAAGGTGCCAGTGTTAGTTTTCTCTCTTTTTTTCGCATATAATGAAATGGCTCCTTGCTAAAGCAAAGCAAGTTAAGTTCTATGTAGTCGCGTTTCATGTAATGTTTTGAAGTACAGAATCAAGGCTTCATTGAGCAGCAGTGTTCTAACATCTTCGAGTTTGTCAGAATCTTTAGATATTAGATTGTTCGTGTATTTATGATAACTTTTTAAAATTAGTCGTAGAAAATGCATTACCAAACAAATGACAACTTTGTAATGATTGCTAATAACGCTCTATGTTCATGATATTCTGCGATTAAATAATTACTTAATTTAAAAGTTGATTTTAAAAAATGAATGTATCTTTTCGTAAATTAGGCTATCATAGCATAAGCGAAAACGGGATGGTGTAAATGCGGAAGGAAAATTACGTAGGCTGTCCGTGGGTGGCAATGAAAAACGAAAAATCTTTATGATTTCAATATTATTAACGACGAAACCTCTTAAATAACAAAACGTGATTATTTCATACTATTGTCATAACATATAATTAACATCGCACGAGATCGGGTAACAGATTACAATGAAATCCCAGTCACGGGAAACTTGTGAAGAGACCTCATAACATCCACCTTACAATTTGTCAATGAAATGTGGGTCATAGTATTTTGATTTAGCCAGATTAAATAGATTTTTAGATTTCCATATAATGAATGTCATTTAGAATTACGAAATTACTTCGCCGTGCTCAGTGTGTGTTGTTGCTTGGTTGTTTGTGCGTACTTTTCCGCAGGCGCGTGGCGCGGGTGAGTGTTTGCCTAGGTGTATTTCTGTGGCATGCGAGTTCGGATGTGTGGAATGTGTGAACTTGCGCTCGTGCATTTTTTGGAATGAATGGTTTGTTTCTCCAATAGAAGTGCAGATTGTATATGCTGTTGACAGGTGCAGATCTTCTGAAATGAAATCTAGTTTTGGGGGACTTTAAAAACTGAATTTAGTGTTCTCATTAGATTTAAGATATTTATGATAATCGTCATTAAAATTTTCTGTGATTTCAGTTGCTTTATGCAACACTCACAGCCTTTCTTTTTCCATCCCTGGCTCATACCCAATGAATATTTTTGTGGTGAAAAGTCGGCGGAACACCATTTAGGCGTTAAGGTGAAACCCCGACTGCGTTTGGTTTGAAAGTGAAAGTTTTCTAGCCGACTAGGACGTAGCCAGGCTATATCCAGGTAAAACCTGGGCTGTATTATGGTTATCCTTGTCTGTTTATATCAAAACGTCTCTTAACCTAGACCTCCACCCCTCTGGACGTCTAGATTCCAGCTTTTGCTCACTGAGTATATTCATTTGTTCATTTTCTACAGCTCATATGCTTCCACTGCAGAGGGGTTCTCATCTCTTAGGTAAGTAAGACTTCCTGCTCCACGATTTGCTAAAAAAAAAAAAAAAAATTAAAAACACACAGTTAAGTTTTTTAATTATTGGAAGCACCAGTGGAATTAGTTCCGTTATTTTTGTGATCTCCATCTCAAAGTTAAGCTTTCAGTTTGGTGTGTCTC
实施例2日本鳗鲡转录因子c-Rel基因启动子序列的转录因子结合位点分析
上网登录基因5′侧翼区转录因子结合位点在线预测软件Alibaba2(http://gene-regulation.com/pub/programs/alibaba2/index.html)将已经克隆测试验证获得的c-Rel基因启动子序列复制后以fasta格式粘贴在对话框内,点击START进行转录因子结合位点的预测分析。部分结果如图1~图5所示。
日本鳗鲡转录因子c-Rel基因启动子主要转录因子结合位点如下:
实施例3日本鳗鲡转录因子c-Rel基因启动子的活性分析
一、含有日本鳗鲡转录因子c-Rel基因启动子片段的重组荧光素酶报告基因载体pGL3-c-Rel-pro的构建。
1、将日本鳗鲡转录因子c-Rel基因启动子片段插入Promega公司荧光素酶报告基因载体pGL3-Basic中,使萤火虫荧光素酶(Luciferase)报告基因的表达由日本鳗鲡c-Rel基因启动子启动子控制,构建得到的荧光素酶重组载体命名为pGL3-c-Rel-pro。具体步骤如下:
合成带有SacI酶切位点的上游引物:
5'-CGAGCTCTAAACTGATCCGGGTCCCTTTGT-3'(SEQ ID NO:5),
带有XhoI酶切位点的下游引物:
5'-CCGCTCGAGGAGACACACCAAACTGAAAGCTTAACTT-3'(SEQ ID NO:6)。
2、采用Takara公司高保真酶GC Buffer(Mg2+plus)进行扩增,反应体系:2×PrimeSTAR HS DNA Polymerase 12.5μL、上游引物0.5μL、下游引物0.5μL、pMD19T-c-Rel-pro重组质粒0.5μL、灭菌水11μL;降落PCR反应程序为95℃5min;95℃30s,56℃30s,72℃4min,4个循环;95℃30s,54℃30s,72℃4min,4个循环;95℃30s,52℃30s,72℃4min,30个循环;72℃10min;4℃5min。用Omega公司胶回收试剂盒进行PCR产物回收。
3、回收后的PCR产物和载体pGL3-Basic分别进行SacI/XhoI双酶切(ThermoScientific Fermentas Fast Digest)。双酶切反应体系总共40μL,包括4μL 10×FastDigest GreenBuffer,酶SacI及酶XhoI各2μL,载体/PCR产物各1.5μg,灭菌水补至40μL,将以上体系于PCR管中混匀后进行酶切反应,反应程序为:37℃,60min;80℃,20min;4℃,5min。用Omega公司胶回收试剂盒分别回收上述经SacI/XhoI双酶切的PCR产物和载体pGL3-Basic,并用Takara公司T4连接酶连接经双酶切的PCR产物和载体pGL3-Basic,连接反应体系为20μL,包括2μL 10×T4 Buffer,1μL T4 DNA连接酶,40ng双酶切的载体pGL3-Basic,300ng双酶切的PCR产物,灭菌水补至20μL,将以上体系于PCR管中混匀后进行16℃连接过夜。
4、上述连接产物转化大肠杆菌E.coli DH5α感受态细胞,经菌落PCR筛选阳性克隆,用Omega公司无内毒素小量质粒试剂盒提取质粒,并经测序确认启动子片段插入的正确性,从而获得含有日本鳗鲡转录因子c-Rel基因启动子片段的重组荧光素酶报告基因载体pGL3-c-Rel-pro。
二、采用双荧光素酶报告基因检测系统分析日本鳗鲡转录因子c-Rel基因启动子的基础活性。
1、把状态较好的EPC细胞接种至48孔细胞板中(1×105/孔),加入L15培养基(L15基础培养基含有10%Gibco澳洲胎牛血清),转入恒温培养箱28℃中过夜培养,使其贴壁并恢复至对数生长期的状态,贴壁量达到80%左右时,进行转染实验。在转染前2h更换细胞培养基。
2、转染时,按每孔0.5μL Lipofectamine 3000Reagent转染试剂和20μL Opti-MEM低血清培养基配制转染试剂稀释液,混匀后室温孵育5min。再按每孔20μL Opti-MEM低血清培养基与每孔所需质粒充分混匀,其中对照组含有20ng海肾荧光素酶内参报告基因载体pRL-TK、300ng荧光素酶报告基因载体pGL3-Basic载体,实验组含有20ng海肾荧光素酶内参报告基因载体pRL-TK、300ng重组荧光素酶报告基因载体pGL3-c-Rel-pro,随后加入0.5μLP3000TM试剂并混匀。将所配的质粒稀释液逐滴滴加到转染试剂稀释液中,混匀为转染复合物溶液,室温孵育15min后,缓慢加入EPC细胞培养孔,于恒温培养箱(28℃)中培养。
3、24h后收集转染细胞,用双荧光素酶报告基因检测系统分别读取萤火虫荧光素酶和海肾荧光素酶的酶活性值,通过计算二者酶活性值的比值得出转染细胞中荧光素酶的相对活性。荧光素酶酶活测定的方法参考Promega公司双萤光素酶报告基因检测系统说明书进行。每个实验设三次重复,每次重复设三个平行;误差靶代表平均值的标准误差。利用双尾成组T检验统计分析实验组与对照组的显著性差异,“*”p<0.05,“**”p<0.01。
具体步骤为:
(1)配制实验所需试剂:1×PLB裂解液:1体积5×Passive Lysis Buffer加4体积双蒸水混匀配制而成;Start试剂(LARⅠ):将Luciferase Assay Substrate粉末完全溶解于10mL Luciferase AssayBufferⅡ溶液,用1.5mL离心管分装后,于-80℃冰箱保存;Stop试剂:视实验量将1体积的50×Stop&Substrate用49体积的Stop&/>Buffer稀释。
(2)缓慢吸去48孔细胞培养板中的细胞培养液,于每孔加入65μL的1×PLB裂解液。
(3)将48孔细胞培养板置于细胞振荡器上,振荡裂解15min。
(3)将裂解后的细胞液转移至1.5mL离心管,离心(13000rpm,4℃,10min)。
(4)取3μL离心后的上清液于透光性良好的1.5mL离心管中。
(5)加入10μL Start试剂,用GloMax 20/20发光检测仪检测样品中的萤火虫荧光素酶活性数值。随后加入10μL Stop试剂检测样品中的海参荧光素酶活性数值。两者活性的比值即为各样品的荧光素酶相对活性。
以空载体pGL3-Basic和pRL-TK共同转染的EPC细胞作为对照组,计算出pGL3-c-Rel-pro启动子的相对活性(图6)。
如图6所示,重组载体pGL3-c-Rel-pro转染EPC细胞中的荧光素酶相对活性为空载体pGL3-Basic转染EPC细胞的11.5倍,说明了日本鳗鲡c-Rel基因启动子可以较好地启动荧光素酶报告基因的转录。
三、免疫刺激实验
将pGL3-Basic和日本鳗鲡c-Rel基因启动子荧光素酶重组载体pGL3-c-Rel-pro分别与海肾荧光素酶内参报告基因载体pRL-TK共同转染EPC细胞,转染12h后,在细胞培养液中分别加入LPS(30μg/mL)以及嗜水气单胞菌(106cfu/mL)进行免疫刺激,分别在刺激12h和6h后分别收集转染细胞进行荧光素酶相对活性测定。
在LPS(30μg/mL)刺激条件下日本鳗鲡c-Rel基因启动子的活性变化见图7。重组载体pGL3-c-Rel-pro转染EPC细胞中的荧光素酶相对活性为空载体pGL3-Basic转染EPC细胞的15.8倍,说明了日本鳗鲡c-Rel基因启动子可被LPS诱导激活,“*”p<0.05,“**”p<0.01。
在水气单胞菌(106cfu/mL)刺激条件下日本鳗鲡c-Rel基因启动子的活性变化见图8。pGL3-c-Rel-pro转染EPC细胞中的荧光素酶相对活性为空载体pGL3-Basic转染EPC细胞的4.5倍,说明了日本鳗鲡c-Rel基因启动子可被嗜水气单胞菌诱导激活,“*”p<0.05,“**”p<0.01。
四、炎症信号因子过表达对日本鳗鲡c-Rel的调控作用实验
将空载体pcDNA3.1和真核表达质粒pcDNA-Caspase1分别与荧光素酶重组载体pGL3-c-Rel-pro、海肾荧光素酶报告基因载体pRL-TK共同转染EPC细胞24h后收集转染细胞进行荧光素酶相对活性测定。
日本鳗鲡Caspase-1过表达条件下日本鳗鲡c-Rel基因启动子的活性变化见图9。与pcDNA3.1空载体相比较,信号通路重要炎症因子真核表达质粒pcDNA-Caspase1转染EPC细胞后,能够显著上调pGL3-c-Rel-pro启动子荧光素酶活性高达173.6倍,说明了日本鳗鲡c-Rel基因启动子可被炎症因子Caspase-1正向调控并激活,“*”p<0.05,“**”p<0.01。
虽然以上描述了本发明的具体实施方式,但是熟悉本技术领域的技术人员应当理解,我们所描述的具体的实施例只是说明性的,而不是用于对本发明的范围的限定,熟悉本领域的技术人员在依照本发明的精神所作的等效的修饰以及变化,都应当涵盖在本发明的权利要求所保护的范围内。
序列表
<110> 集美大学
<120> 一种日本鳗鲡转录因子c-Rel基因启动子及其应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3273
<212> DNA
<213> 日本鳗鲡(Anguilla japonica)
<400> 1
taaactgatc cgggtccctt tgttgactat tttttatgaa cccccccccc tctttctctc 60
tctttctcgt caaagcaata ttaaataaca tcatcgtaat attttttacg taaaactaaa 120
acagttggta tgacttttat tacatattgg acaaaatatt ggataaaaca atgacttgag 180
ataaaatgta gcttaaaaag ttttcaaaaa acacgaacaa ggtgttttgc ctgtctgtgt 240
cactccttgt ctatcagact gtagtaggcc aaaccatcct ctacaaaaat gattgccatt 300
tgcttgttgt cctgtgggac aacagattta ttgctgttat tttggggaag tgggtacctc 360
ttacttgcat gtattttcac gcatttttga caaagttctt ttctttgctg cttgttgact 420
gcagtgatgc acatgtgttc tgcatggcta tctagtcctt ggtttgatcc atgtcagggt 480
gtctctattg tcagttcttg ctctctgaca tgtcaaaaca ttgtttatta atttttactg 540
ttaataggtg agatgttaca gtacagtcga ctccagaatt ataggcaccc agcatgaaaa 600
tgagcaaaaa tgactgcata aaacaataca cataatgatt gaaattttct gctctaacat 660
taggtggaac tttctttaac tctaataaga atattcatat aaaaattcat gatgaaattc 720
aattcaatat ttaaaaaaat gtttgttttt ttctatctca gtctccagaa acttttgcca 780
tttactgaac acaaagtttc aaaagtttga gacagttgca gatttgccaa aaagaagatg 840
catgtgcata tttcccccac acactgtgag gaagatgatg agggaggcaa agaagaaccc 900
aaggatcata gtttcatagc tgcacatttc agtgcagtca ggactacacc actgtctctt 960
cacagcaaaa aggtcttggg ttcgaatctt ggcctgggcc tatctgtttg ttctccccgt 1020
gctttcctct gggtccacag tccaaagaaa tgcaggtatg ctaattggcc gtgaatataa 1080
gcgtgtgagt gaatggtgag tgtgccctgc catagactgg cgatactgta ggtgtattcc 1140
tgccactcgc ccaatgcatg ctgggatgta atttgttcat gatcgtgttt ttggattgaa 1200
aatcatttgt gctcttttca caaatcatta aattctttgt caagacaaat tagtctatct 1260
ctacacacac ccacgcacac acacttttgt tattttcaga aaaggagttt aaaacggcga 1320
ggaaatgtat caaaatacta gatcgtgaag catagtccac agtgatctct tacttgtatt 1380
taatctgacg tgcccataag agtactttcg cgggcgccga agtagttgct gctgtatgtt 1440
tgtaggtgag tttttttttt tttttttaga atttactgtc gtcacttaat accgcccaga 1500
tgaggattgt acagctcctt caggcggaga taatgtgggg aattcccctt catcgtctct 1560
gtttaaaaat gaatacatca ctcttcttct ttttcagtcc agtcgtctca ctcaaatact 1620
ataccgtgat aagggtgttc acaacatgga tggtgagtac tgtcaaaaac ctcaactttt 1680
gcaatatgtt tcaaagtaga aatattgtgc tggtcctaca tttacaaaag gtgccagtgt 1740
tagttttctc tctttttttc gcatataatg aaatggctcc ttgctaaagc aaagcaagtt 1800
aagttctatg tagtcgcgtt tcatgtaatg ttttgaagta cagaatcaag gcttcattga 1860
gcagcagtgt tctaacatct tcgagtttgt cagaatcttt agatattaga ttgttcgtgt 1920
atttatgata actttttaaa attagtcgta gaaaatgcat taccaaacaa atgacaactt 1980
tgtaatgatt gctaataacg ctctatgttc atgatattct gcgattaaat aattacttaa 2040
tttaaaagtt gattttaaaa aatgaatgta tcttttcgta aattaggcta tcatagcata 2100
agcgaaaacg ggatggtgta aatgcggaag gaaaattacg taggctgtcc gtgggtggca 2160
atgaaaaacg aaaaatcttt atgatttcaa tattattaac gacgaaacct cttaaataac 2220
aaaacgtgat tatttcatac tattgtcata acatataatt aacatcgcac gagatcgggt 2280
aacagattac aatgaaatcc cagtcacggg aaacttgtga agagacctca taacatccac 2340
cttacaattt gtcaatgaaa tgtgggtcat agtattttga tttagccaga ttaaatagat 2400
ttttagattt ccatataatg aatgtcattt agaattacga aattacttcg ccgtgctcag 2460
tgtgtgttgt tgcttggttg tttgtgcgta cttttccgca ggcgcgtggc gcgggtgagt 2520
gtttgcctag gtgtatttct gtggcatgcg agttcggatg tgtggaatgt gtgaacttgc 2580
gctcgtgcat tttttggaat gaatggtttg tttctccaat agaagtgcag attgtatatg 2640
ctgttgacag gtgcagatct tctgaaatga aatctagttt tgggggactt taaaaactga 2700
atttagtgtt ctcattagat ttaagatatt tatgataatc gtcattaaaa ttttctgtga 2760
tttcagttgc tttatgcaac actcacagcc tttctttttc catccctggc tcatacccaa 2820
tgaatatttt tgtggtgaaa agtcggcgga acaccattta ggcgttaagg tgaaaccccg 2880
actgcgtttg gtttgaaagt gaaagttttc tagccgacta ggacgtagcc aggctatatc 2940
caggtaaaac ctgggctgta ttatggttat ccttgtctgt ttatatcaaa acgtctctta 3000
acctagacct ccacccctct ggacgtctag attccagctt ttgctcactg agtatattca 3060
tttgttcatt ttctacagct catatgcttc cactgcagag gggttctcat ctcttaggta 3120
agtaagactt cctgctccac gatttgctaa aaaaaaaaaa aaaaattaaa aacacacagt 3180
taagtttttt aattattgga agcaccagtg gaattagttc cgttattttt gtgatctcca 3240
tctcaaagtt aagctttcag tttggtgtgt ctc 3273
<210> 2
<211> 178
<212> DNA
<213> 日本鳗鲡(Anguilla japonica)
<400> 2
atgcttgtaa ttgccaccct ttctgcctcc tcctttgccc gtccagtggg tgagcccagc 60
gtgcagatat ttgaggaacc caagcaaagg ggcatgcgct tcagatacaa gtgtgagggg 120
cgatcggctg gcagcatccc cggtgagagg agcacagaca acaacaggac ctacccca 178
<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
taaactgatc cgggtccctt tgt 23
<210> 4
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tggggtaggt cctgttgttg tc 22
<210> 5
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cgagctctaa actgatccgg gtccctttgt 30
<210> 6
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ccgctcgagg agacacacca aactgaaagc ttaactt 37
Claims (7)
1.一种日本鳗鲡转录因子c-Rel基因启动子,其特征在于,所述启动子的核苷酸序列如SEQ ID NO:1所示。
2.含有权利要求1所述启动子的表达盒、重组载体、转基因细胞系、重组菌或重组病毒。
3.如权利要求2所述的表达盒,其特征在于,所述表达盒由权利要求1所述的启动子、由权利要求1所述启动子启动转录的目的基因和终止子组成。
4.如权利要求2所述的重组载体,其特征在于,所述重组载体为pGL3-basic、pGL2-Basic、pGL4.10、pGLuc。
5.如权利要求2所述的重组菌,其特征在于:所述重组菌为大肠杆菌、枯草杆菌、乳酸菌、酵母菌。
6.如权利要求1所述的日本鳗鲡转录因子c-Rel基因启动子在构建真核表达载体、鱼类细胞或哺乳动物细胞中高效表达外源基因中的应用。
7.如权利要求1所述的日本鳗鲡转录因子c-Rel基因启动子在构建转基因鱼中的应用。
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