CN111154725A - 一种用于筛选维生素k循环小分子抑制剂的双报告基因细胞体系、制备方法及其应用 - Google Patents
一种用于筛选维生素k循环小分子抑制剂的双报告基因细胞体系、制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系、制备方法及其应用,属于分子细胞生物学技术领域。本发明中用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系可以快速对药物小分子库进行高通量筛选,得到特异性靶向维生素K循环的小分子抑制剂。本发明通过对Maybridge Hit Finder小分子化合物库进行筛选,筛选到21种抑制维生素K循环的小分子化合物,在这21种化合物中,有已知是VKOR蛋白抑制剂的小分子,更进一步证明了该体系筛选的有效性。本发明的双报告基因细胞体系解决了现有方法无法高通量筛选靶向维生素K循环的小分子抑制剂的问题,为开发特异性靶向维生素K循环的新型抗凝血药物提供了技术指导。
Description
技术领域
本发明涉及一种用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系、制备方法及其应用,属于分子细胞生物学技术领域。
背景技术
维生素K在血液正常凝固中发挥重要作用,主要用于防止新生婴儿出血疾病、预防内出血以及维生素K拮抗剂的解毒剂。维生素K通过调节一些凝血因子(如凝血因子Ⅱ、凝血因子Ⅶ、凝血因子Ⅸ)的翻译后修饰参与凝血。维生素K依赖性凝血因子的Gla结构域的谷氨酸残基γ羧基化修饰是其发挥凝血功能的必要条件。维生素K在这一翻译后修饰过程中发挥核心作用,并以三种形式在体内循环,分别是维生素K环氧化物(KO)、维生素K(K)和二氢维生素K(KH2)。
目前已知两个酶在维生素K循环中发挥作用。其中γ-谷氨酰羧化酶(GGCX)利用KH2为辅因子,对维生素K依赖性凝血因子的Gla结构域的谷氨酸残基进行γ羧基化修饰,同时把KH2转化成KO。为了使GGCX的催化反应可以持续进行,KO必须被还原成KH2,而这一反应主要是由具有KO还原酶活性和K还原酶活性的维生素K环氧化物还原酶(VKOR)催化的。VKOR催化KO到K,以及K到KH2,使维生素K可以循环利用。研究表明,VKOR是临床抗凝血药物华法林(warfarin)的特异性作用靶点,华法林通过抑制VKOR的活性,使KO不能转化为K和KH2,从而阻断了维生素K循环,进而抑制凝血因子Gla结构域的谷氨酸残基的γ羧基化修饰,达到抗凝血的目的。此外,杀鼠药溴敌隆也是特异性靶向VKOR的抑制剂。
维生素K循环的关键酶VKOR和GGCX都是开发抗凝血药物(或杀鼠药)的重要靶点。然而目前还没有针对这两个靶点筛选小分子抑制剂的体系。
发明内容
本发明的目的是提供一种用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系,该体系可以快速对药物小分子库进行高通量筛选从而得到特异性靶向维生素K循环的小分子抑制剂。
同时,本发明还提供一种用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系的制备方法。
最后,本发明还提供一种用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系在筛选特异性靶向维生素K循环的小分子抑制剂方面的应用。
为了实现以上目的,本发明所采用的技术方案是:
一种用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系,所述双报告基因细胞体系中含有同时表达FIX-Gla-PC融合基因和报告基因的质粒,所述FIX-Gla-PC融合基因的核苷酸序列如SEQ ID NO.1所示。
本发明中用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系可以快速对药物小分子库进行高通量筛选,得到特异性靶向维生素K循环的小分子抑制剂。本发明的双报告基因细胞体系解决了现有方法无法高通量筛选靶向维生素K循环的小分子抑制剂的技术问题,为开发特异性靶向维生素K循环的新型抗凝血药物提供了技术指导。
所述双报告基因细胞体系的宿主细胞为人胚肾细胞293Trex(invitrogen)。
所述FIX-Gla-PC融合基因编码的蛋白质的氨基酸序列如SEQ ID NO.2所示。
所述报告基因为荧光素酶报告基因,如甲虫(萤火虫)荧光素酶报告基因。甲虫荧光素酶报告基因的核苷酸序列如SEQ ID NO.3所示,该基因编码的蛋白质的氨基酸序列如SEQ ID NO.4所示。
进一步优选的,所述同时表达FIX-Gla-PC融合基因和报告基因(甲虫荧光素酶报告基因)的质粒的核苷酸序列如SEQ ID NO.15所示。
一种用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系的制备方法,包括以下步骤:将同时表达FIX-Gla-PC融合基因和报告基因的质粒转入宿主细胞中,即得。
所述宿主细胞为人胚肾细胞293Trex(invitrogen)。
上述用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系在筛选特异性靶向维生素K循环的小分子抑制剂方面的应用。
所述应用为:将待选小分子化合物加入到含有维生素K的双报告基因细胞体系培养液中进行培养,筛选出能够有效抑制维生素K循环的小分子化合物,作为特异性靶向维生素K循环的小分子抑制剂。
所述双报告基因细胞体系培养液的培养基为含双抗的完全培养基,双抗分别为青霉素和链霉素。含双抗的完全培养基的组成为:10%FBS(胎牛血清,v/v),青霉素100IU/mL,链霉素100μg/mL,DMEM(货号SH30243.01,Hyclone)。
进一步优选的,所述双报告基因细胞体系培养液中维生素K的浓度为1~20μmol/L;更优选为5μmol/L。
本发明的有益效果:
本发明首次构建一个可用于筛选靶向维生素K循环小分子抑制剂的双报告基因细胞体系,目前还没有文献报道可用于筛选维生素K循环小分子抑制剂的体系。
本发明的双报告基因细胞体系可用于高通量筛选小分子化合物库,解决了现有方法无法高通量筛选特异性靶向维生素K循环(维生素K循环示意图如图1所示)的小分子抑制剂的技术问题。本发明通过对Maybridge Hit Finder小分子化合物库进行筛选,筛选到21种抑制维生素K循环的小分子化合物。在这21种化合物中,有已知是VKOR蛋白的抑制剂,如HF13020-E7、HF13052-C5、HF13089-C5、HF13148-C7、HF13169-G5,更进一步证明了该体系筛选的有效性。
本发明将含FIX-Gla-PC融合基因的质粒转入293Trex中,可以获得稳定表达报告基因FIX-Gla-PC的细胞系,通过检测报告基因FIX-Gla-PC分泌到培养基上清中的量,即可反映293Trex细胞中的维生素K循环是否被抑制。293Trex细胞本身不方便检测维生素K循环的活性,转入报告基因FIX-Gla-PC后,更方便在细胞水平检测活性,操作简单,检测灵敏度高。
本发明基于293TRex构建的双报告基因细胞体系具有以下优点:1)报告基因定点插入到细胞系的FRT重组位点,而不是随机插入到细胞的基因组;2)由于是定点插入基因组,筛选到的Hygromycin抗性的克隆细胞也都是稳定表达两个报告基因的阳性细胞;3)由于是定点插入基因组,两个报告基因在细胞中共表达量比较均一,而不是一个表达量高,一个表达量低。
本发明的双报告基因细胞体系用分泌表达的甲虫荧光素酶报告基因作为对照基因,可以有效排除只影响细胞存活、蛋白合成和分泌途径,而不影响维生素K循环的小分子化合物。甲虫荧光素酶报告基因活性检测非常便于荧光值定量和高通量筛选。
附图说明
图1为维生素K循环示意图。
具体实施方式
下述实施例仅对本发明作进一步详细说明,但不构成对本发明的任何限制。
实施例1
本实施例中用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系及其制备方法,具体步骤如下:
1.在pBud CE4.1(invitrogen)载体中构建双表达FIX-gla-PC和甲虫荧光素酶(Metridia Luciferase)报告基因的质粒
1.1将Metridia Luciferase基因克隆到pBud CE4.1的CMV启动子后的多克隆位点
以pBud-VKORC1为模板(注:含有甲虫荧光素酶基因序列,参见文献1),以Mluc-F和Mluc-R为引物,用高保真酶phusion(Thermofisher)扩增甲虫荧光素酶基因;扩增片断用PCR纯化后,用Hind III和Xba I在37℃进行双酶切;回收酶切片断,-20℃备用。质粒pBudCE4.1用Hind III和Xba I在37℃进行双酶切;回收酶切片断,-20℃备用。
SEQ ID NO.5:Mluc-F:5’-ATCAAGCTTATGGACATCAAGGTGGTGTTCAC-3’;
SEQ ID NO.6:Mluc-R:5’-TGCTCTAGATCACCTGTCGCCGGCCATGC-3’。
连接酶切的基因片断和质粒片断,然后转化感受态大肠杆菌DH5α,用zeocin抗性平板筛选,挑单菌落于含zeocin抗生素的LB培养基中;摇菌,提取质粒,测序鉴定阳性克隆;阳性克隆命名为pBud-Mluc。
酶切体系和条件:4μg DNA片段(质粒或PCR产物),4μL 10×buffer R,Hind III和Xba I各1μL,加水至40μL;酶切4-6小时。
连接体系和条件:T4连接酶1μL,10×连接缓冲液1μL,酶切的PCR片断和质粒各4μL;4℃过夜连接。
1.2将FIX-gla-PC克隆到pBud CE4.1的EF1α启动子后的多克隆位点
采用基于PCR的非酶切依赖的快速克隆方法进行,步骤如下:
以pBud-Mluc为模板,以pBUD-F和pBUD-R为引物,用高保真酶phusion(Thermofisher)进行PCR扩增反应,获得质粒扩增片断1。
SEQ ID NO.7:pBUD-F:5’-TAAGCCTATCCCTAACCCTCTCCTCGGT-3’;
SEQ ID NO.8:pBUD-R:5’-CATGGTGGCGGCGGATCCGAGCTCGGTACCTT-3’。
以pcDNA3.1-FIX-gla-PC(从Tie实验室获得)为模板(参见文献3),以FIX-PC-F和FIX-PC-R为引物,用高保真酶phusion进行PCR扩增反应,获得融合基因扩增片断2。
SEQ ID NO.9:FIX-PC-F:5’-ATCCGCCGCCACCATGGCATGGCAGCTCACAAGCCT-3’;
SEQ ID NO.10:FIX-PC-R:5’-GTTAGGGATAGGCTTAAGGTGCCCAGCTCTTCTGG-3’。
按快速克隆方法(参见文献2)混合两个PCR扩增片断1-2,用DH5α感受态细胞进行转化,然后用zeocin抗性平板筛选,挑单菌落于含zeocin抗生素的LB培养基中;摇菌,提取质粒,测序鉴定阳性克隆;阳性克隆命名为Pbud-F9-gla-PC(核苷酸序列如SEQ ID NO.16所示)备用。
2.构建Pbud-F9-gla-PC和pcDNA5/FRT/TO-TOPO的融合质粒
采用基于PCR的非酶切依赖的快速克隆方法进行,步骤如下:
以Pbud-F9-gla-PC为模板,以CMV-F和BGH-R为引物,用高保真酶phusion(Thermofisher)获得扩增片断3;
SEQ ID NO.11:CMV-F:5’-CGCAAAT GGGCGGTAGG CGTG-3’;
SEQ ID NO.12:BGH-R:5’-CAACTAGAAG GCACAGTCGA GG-3’。
以pcDNA5/FRT/TO-TOPO为模板,以D5-CMV-R(CMV-F反向)和D5-BGH-F(BGH-R反向)为引物,用高保真酶phusion(Thermofisher)获得扩增片断4。
SEQ ID NO.13:D5-CMV-R:5’-CACGCCTACC GCCCATTTGC GTC-3’;
SEQ ID NO.14:D5-BGH-F:5’-CCTCGACTGTGC CTTCTAGTTG-3’。
按快速克隆方法(参见文献2)混合两个PCR扩增片断3-4,用DH5α感受态细胞进行转化,然后用Amp+抗性平板筛选阳性克隆;挑选阳性克隆,用Amp+和zeocin的双抗性的液体LB培养基培养。
PCR体系和条件:10mmol/L正向引物和反向引物各1μL,5×buffer H 10μL,10ng/μL质粒模板0.5μL,10mmol/L dNTP 2μL,加水至25μL;预变性98℃1分钟,循环条件;98℃变性20s,55℃退火30s,72℃延伸30s到2分钟(延伸按15s/kb的条件设置),18个循环;最后延伸5分钟。
酶切体系和条件:PCR产物中直接加0.5μL Dpn I;37℃酶切2小时,后80℃变性30分钟。
转化:两个PCR片断各取3μL,充分混匀;之后取1μL混合物,加入30μL DH5α感受态细胞进行转化。
采用质粒提取试剂盒提取质粒,质粒通过测序进行鉴定。挑选测序完全正确的阳性质粒,命名为pcDNA5-F9-Met(核苷酸序列如SEQ ID NO.15所示),用于双报告基因表达稳定细胞体系的构建。
3.构建双报告基因表达稳定细胞体系
用上述构建的表达质粒转染293TRex细胞系(invitrogen)(参见文献4),用hygromycin筛选稳定表达FIX-gla-PC和Metridia Luciferase双报告基因的细胞体系,命名为293TRex-F9-Met。具体步骤如下:
3.1细胞培养
在10cm培养皿中铺293TRex,第二天细胞密度长到覆盖10cm皿底面积的60-70%。
3.2细胞转染
按lipo3000试剂转染方法进行,取9μg质粒pOGG44和1μg质粒pcDNA5-F9-Met,加入0.5mL Opti-MEM,之后加入20μL P3000TM,备用;取7.5μL转染试剂lipofectamine 3000,加入0.5mL Opti-MEM,轻轻混匀;将混匀的转染试剂加入准备好的质粒混合物中,轻轻混匀,室温孵育5分钟。将质粒转染试剂混合物加入铺好293TRex的10cm皿中,轻轻混匀后,将细胞放回二氧化碳培养箱中,继续培养24小时。
3.3传代培养
转染24小时后,用含双抗的完全培养基(组成为:10%FBS(胎牛血清,v/v),青霉素100IU/mL,链霉素100μg/mL,DMEM(货号SH30243.01,Hyclone)),按1皿传9皿的比例对细胞进行传代,留2皿细胞。
3.4筛选稳定表达双报告基因的细胞体系
传代的细胞继续培养24小时后,在培养基中直接加筛选抗生素hygromycin和blasticidin,终浓度分别为300μg/mL和15μg/mL;继续培养24小时,换新鲜的筛选培养基(DMEM(货号SH30243.01,Hyclone),10%FBS(胎牛血清),hygromycin 150μg/mL,blasticidin 15μg/mL的完全培养基),hygromycin和blasticidin的浓度分别为150μg/mL和15μg/mL;培养48小时后,再换一次新鲜的筛选培养基,此时细胞已非常稀少;之后每隔3-4天换一次新鲜的筛选培养基,2周后出现阳性克隆,4-5周获得大量阳性克隆的细胞,即为双报告基因细胞体系。
本实施例构建了pBud CE4.1和pcDNA5/FRT/TO-TOPO的融合质粒,可以借助pcDNA5/FRT/TO-TOPO质粒的FRT重组位点,在293TRex细胞中构建稳定表达双报告基因的细胞株。
实施例2
本实施例中用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系在筛选特异性靶向维生素K循环的小分子抑制剂方面的应用,包括以下步骤:
1)将Maybridge Hit Finder小分子化合物库(14400种小分子化合物)中的小分子化合物分别铺在96孔细胞培养板中,每孔加150nL的小分子药物(浓度为10mmol/L),置于-20℃冰箱备用;
2)在铺好小分子化合物的96孔板中铺293TRex-F9-Met细胞,每孔的细胞量为30000-40000个,体积为150μL;相当于2-2.5×105个/mL浓度的细胞悬液,培养基为含双抗的完全培养基(组成为:10%FBS(胎牛血清,v/v),青霉素100IU/mL,链霉素100μg/mL,DMEM(货号SH30243.01,Hyclone)),维生素K浓度为5μmol/L;
3)培养24小时后,收集96孔细胞培养板中培养基上清,分别用ELSIA检测报告基因FIX-gla-PC的分泌量(参见文献3),荧光素酶报告基因实验检测甲虫荧光素酶(MetridiaLuciferase)的活性(参见文献1和5);
4)计算每孔FIX-gla-PC的分泌量与甲虫荧光素酶的荧光值的比值(FIX-gla-PC的分泌量/甲虫荧光素酶的荧光值,命名为R),用加药组的比值(R drug,缩写为Rd)除以不加药组的比值(R no treatment,缩写为Rn),得到比值(Rd/Rn);将比值Rd/Rn≤0.3的小分子化合物判定为阳性结果,共筛选到21种小分子化合物,如下表1所示,其中华法林为阳性对照。
表1 Maybridge Hit Finder小分子化合物库的筛选结果
21种小分子化合物的结构式如下:
21种小分子化合物中,除已知的VKOR蛋白抑制剂外,其他均可作为特异性靶向维生素K循环的小分子抑制剂。
上述未详细记载的手段均为本领域的常规技术,可参考申请日以前的文献实现。
参考文献如下:
文献1:Shen G,Cui W,Zhang H,Zhou F,Huang W,Liu Q,Yang Y,Li S,BowmanGR,Sadler JE,Gross ML,Li W,Guomin S,Weidong C,Hao Z,Fengbo Z,Wei H,Qian L,Yihu Y,Bowman GR,et al.Warfarin prevents blood coagulation by trapping humanvitamin K epoxide reductase in an intermediate state during electrontransfer.Nat Struct Mol Biol 2016;24:69-76.;
文献2:Li,et al.FastCloning:a highly simplified,purification-free,sequence-and ligation-independent PCR cloning method.BMC Biotechnology,2011,11:92.;
文献3:Tie J-K,Jin D-Y,Straight DL,Stafford DW.Functional study of thevitamin K cycle in mammalian cells.Blood 2011;117:2967-74.;
文献4:Wanrooij S,Goffart S,JLO,Yasukawa T,SpelbrinkJN.Expression of catalytic mutants of the mtDNA helicase Twinkle andpolymerase POLG causes distinct replication stalling phenotypes.Nucleic AcidsRes 2007;35:3238-51.;
文献5:Tie JK,Jin DY,Tie K,Stafford DW.Evaluation of warfarinresistance using transcription activator-like effector nucleases-mediatedvitamin K epoxide reductase knockout HEK293 cells.J Thromb Haemost 2013;11:1556-64.。
序列表
SEQUENCE LISTING
<110> 河南科技大学
<120> 一种用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系、制备方法及其应用
<170> SIPOSequenceListing 1.0
<160> 16
<211> 1392
<212> DNA
<213> 人源F9和PROC基因重组
<221> FIX-Gla-PC融合基因
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atggcatggc agctcacaag cctcctcctg ttcgtggcca cctggggaat ttccggcaca 60
ccagctcctc ttgactcagt gttctccagc agcgagcgtg cccaccaggt gctgcggatt 120
cgaaaacgtt ataattcagg taaattggaa gagtttgttc aagggaacct tgagagagaa 180
tgtatggaag aaaagtgtag ttttgaagaa gcacgagaag tttttgaaaa cacggaaaga 240
acaactgaat tttggaagca gtatgttgac ggtgaccagt gcttggtctt gcccttggag 300
cacccgtgcg ccagcctgtg ctgcgggcac ggcacgtgca tcgacggcat cggcagcttc 360
agctgcgact gccgcagcgg ctgggagggc cgcttctgcc agcgcgaggt gagcttcctc 420
aattgctctc tggacaacgg cggctgcacg cattactgcc tagaggaggt gggctggcgg 480
cgctgtagct gtgcgcctgg ctacaagctg ggggacgacc tcctgcagtg tcaccccgca 540
gtgaagttcc cttgtgggag gccctggaag cggatggaga agaagcgcag tcacctgaaa 600
cgagacacag aagaccaaga agaccaagta gatccgcggc tcattgatgg gaagatgacc 660
aggcggggag acagcccctg gcaggtggtc ctgctggact caaagaagaa gctggcctgc 720
ggggcagtgc tcatccaccc ctcctgggtg ctgacagcgg cccactgcat ggatgagtcc 780
aagaagctcc ttgtcaggct tggagagtat gacctgcggc gctgggagaa gtgggagctg 840
gacctggaca tcaaggaggt cttcgtccac cccaactaca gcaagagcac caccgacaat 900
gacatcgcac tgctgcacct ggcccagccc gccaccctct cgcagaccat agtgcccatc 960
tgcctcccgg acagcggcct tgcagagcgc gagctcaatc aggccggcca ggagaccctc 1020
gtgacgggct ggggctacca cagcagccga gagaaggagg ccaagagaaa ccgcaccttc 1080
gtcctcaact tcatcaagat tcccgtggtc ccgcacaatg agtgcagcga ggtcatgagc 1140
aacatggtgt ctgagaacat gctgtgtgcg ggcatcctcg gggaccggca ggatgcctgc 1200
gagggcgaca gtggggggcc catggtcgcc tccttccacg gcacctggtt cctggtgggc 1260
ctggtgagct ggggtgaggg ctgtgggctc cttcacaact acggcgttta caccaaagtc 1320
agccgctacc tcgactggat ccatgggcac atcagagaca aggaagcccc ccagaagagc 1380
tgggcacctt aa 1392
<211> 463
<212> PRT
<213> 人源F9和PROC基因重组
<221> FIX-Gla-PC融合基因编码的蛋白质
<400> 2
MAWQLTSLLL FVATWGISGT PAPLDSVFSS SERAHQVLRI RKRYNSGKLE EFVQGNLERE 60
CMEEKCSFEE AREVFENTER TTEFWKQYVD GDQCLVLPLE HPCASLCCGH GTCIDGIGSF 120
SCDCRSGWEG RFCQREVSFL NCSLDNGGCT HYCLEEVGWR RCSCAPGYKL GDDLLQCHPA 180
VKFPCGRPWK RMEKKRSHLK RDTEDQEDQV DPRLIDGKMT RRGDSPWQVV LLDSKKKLAC 240
GAVLIHPSWV LTAAHCMDES KKLLVRLGEY DLRRWEKWEL DLDIKEVFVH PNYSKSTTDN 300
DIALLHLAQP ATLSQTIVPI CLPDSGLAER ELNQAGQETL VTGWGYHSSR EKEAKRNRTF 360
VLNFIKIPVV PHNECSEVMS NMVSENMLCA GILGDRQDAC EGDSGGPMVA SFHGTWFLVG 420
LVSWGEGCGL LHNYGVYTKV SRYLDWIHGH IRDKEAPQKS WAP 463
<211> 660
<212> DNA
<213> 甲虫
<221> 甲虫荧光素酶报告基因
<400> 3
atggacatca aggtggtgtt caccctggtg ttcagcgccc tggtgcaggc caagagcacc 60
gagttcgacc ccaacatcga catcgtgggc ctggaaggca agttcggcat caccaacctg 120
gaaaccgacc tgttcaccat ctgggagacc atggaagtga tgatcaaggc cgacatcgcc 180
gacaccgacc gggccagcaa cttcgtggcc accgagaccg acgccaaccg gggcaagatg 240
cccggcaaga agctgcccct ggccgtcatc atggaaatgg aagccaacgc cttcaaggcc 300
ggctgcaccc ggggctgcct gatctgcctg agcaagatca agtgcaccgc caagatgaag 360
gtgtacatcc ccggcaggtg ccacgactac ggcggcgaca agaaaaccgg ccaggccggc 420
atcgtgggcg ccatcgtgga catccccgag atcagcggct tcaaagaaat ggcccccatg 480
gaacagttca tcgcccaggt ggacagatgc gccagctgca ccaccggctg cctgaagggc 540
ctggccaacg tgaagtgcag cgagctgctg aagaagtggc tgcccgaccg ctgcgccagc 600
ttcgccgaca agatccagaa agaggtgcac aacatcaagg gcatggccgg cgacaggtga 660
<211> 219
<212> PRT
<213> 甲虫
<221> 甲虫荧光素酶报告基因编码的蛋白质
<400> 4
MDIKVVFTLV FSALVQAKST EFDPNIDIVG LEGKFGITNL ETDLFTIWET MEVMIKADIA 60
DTDRASNFVA TETDANRGKM PGKKLPLAVI MEMEANAFKA GCTRGCLICL SKIKCTAKMK 120
VYIPGRCHDY GGDKKTGQAG IVGAIVDIPE ISGFKEMAPM EQFIAQVDRC ASCTTGCLKG 180
LANVKCSELL KKWLPDRCAS FADKIQKEVH NIKGMAGDR 219
<211> 32
<212> DNA
<213> 人工序列
<221> 引物Mluc-F
<400> 5
atcaagctta tggacatcaa ggtggtgttc ac 32
<211> 29
<212> DNA
<213> 人工序列
<221> 引物Mluc-R
<400> 6
tgctctagat cacctgtcgc cggccatgc 29
<211> 28
<212> DNA
<213> 人工序列
<221> 引物pBUD-F
<400> 7
taagcctatc cctaaccctc tcctcggt 28
<211> 32
<212> DNA
<213> 人工序列
<221> 引物pBUD-R
<400> 8
catggtggcg gcggatccga gctcggtacc tt 32
<211> 36
<212> DNA
<213> 人工序列
<221> 引物FIX-PC-F
<400> 9
atccgccgcc accatggcat ggcagctcac aagcct 36
<211> 35
<212> DNA
<213> 人工序列
<221> 引物FIX-PC-R
<400> 10
gttagggata ggcttaaggt gcccagctct tctgg 35
<211> 21
<212> DNA
<213> 人工序列
<221> 引物CMV-F
<400> 11
cgcaaatggg cggtaggcgt g 21
<211> 22
<212> DNA
<213> 人工序列
<221> 引物BGH-R
<400> 12
caactagaag gcacagtcga gg 22
<211> 23
<212> DNA
<213> 人工序列
<221> 引物D5-CMV-R
<400> 13
cacgcctacc gcccatttgc gtc 23
<211> 22
<212> DNA
<213> 人工序列
<221> 引物D5-BGH-F
<400> 14
cctcgactgt gccttctagt tg 22
<211> 9452
<212> DNA
<213> 人工序列
<221> 同时表达FIX-Gla-PC融合基因和报告基因的质粒pcDNA5-F9-Met
<400> 15
ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca 60
aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg 120
cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag 180
taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt 240
acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 300
acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 360
ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 420
attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 480
gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg 540
ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc 600
caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa 660
tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 720
tatataagca gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt 780
aatacgactc actataggga gacccaagct tgcattcctg caggtcgacg ccgccaccat 840
ggacatcaag gtggtgttca ccctggtgtt cagcgccctg gtgcaggcca agagcaccga 900
gttcgacccc aacatcgaca tcgtgggcct ggaaggcaag ttcggcatca ccaacctgga 960
aaccgacctg ttcaccatct gggagaccat ggaagtgatg atcaaggccg acatcgccga 1020
caccgaccgg gccagcaact tcgtggccac cgagaccgac gccaaccggg gcaagatgcc 1080
cggcaagaag ctgcccctgg ccgtcatcat ggaaatggaa gccaacgcct tcaaggccgg 1140
ctgcacccgg ggctgcctga tctgcctgag caagatcaag tgcaccgcca agatgaaggt 1200
gtacatcccc ggcaggtgcc acgactacgg cggcgacaag aaaaccggcc aggccggcat 1260
cgtgggcgcc atcgtggaca tccccgagat cagcggcttc aaagaaatgg cccccatgga 1320
acagttcatc gcccaggtgg acagatgcgc cagctgcacc accggctgcc tgaagggcct 1380
ggccaacgtg aagtgcagcg agctgctgaa gaagtggctg cccgaccgct gcgccagctt 1440
cgccgacaag atccagaaag aggtgcacaa catcaagggc atggccggcg acaggtgata 1500
atctagagga tccgaacaaa aactcatctc agaagaggat ctgaatatgc ataccggtca 1560
tcatcaccat caccattgag tttgatcccc gggaattcag acatgataag atacattgat 1620
gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt 1680
gatgctattg ctttatttgt aaccattata agctgcaata aacaagttgg ggtgggcgaa 1740
gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat 1800
tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgta gcacgtgtca 1860
gtcctgctcc tcggccacga agtgcacgca gttgccggcc gggtcgcgca gggcgaactc 1920
ccgcccccac ggctgctcgc cgatctcggt catggccggc ccggaggcgt cccggaagtt 1980
cgtggacacg acctccgacc actcggcgta cagctcgtcc aggccgcgca cccacaccca 2040
ggccagggtg ttgtccggca ccacctggtc ctggaccgcg ctgatgaaca gggtcacgtc 2100
gtcccggacc acaccggcga agtcgtcctc cacgaagtcc cgggagaacc cgagccggtc 2160
ggtccagaac tcgaccgctc cggcgacgtc gcgcgcggtg agcaccggaa cggcactggt 2220
caacttggcc atggtttagt tcctcacctt gtcgtattat actatgccga tatactatgc 2280
cgatgattaa ttgtcaacac gtgctgatca gatccgaaaa tggatataca agctcccggg 2340
agctttttgc aaaagcctag gcctccaaaa aagcctcctc actacttctg gaatagctca 2400
gaggcagagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga 2460
gaatgggcgg aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg 2520
gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac 2580
tttccacacc tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg 2640
gagcctgggg actttccaca ccctcgtcga gctagcttcg tgaggctccg gtgcccgtca 2700
gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg 2760
aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct 2820
ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt 2880
tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg 2940
gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca cctggctcca 3000
gtacgtgatt cttgatcccg agctggagcc aggggcgggc cttgcgcttt aggagcccct 3060
tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg 3120
tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga 3180
tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaggatctg 3240
cacactggta tttcggtttt tgggcccgcg gccggcgacg gggcccgtgc gtcccagcgc 3300
acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct 3360
caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg 3420
gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc 3480
cctgctccag ggggctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca 3540
cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag 3600
taccgggcgc cgtccaggca cctcgattag ttctggagct tttggagtac gtcgtcttta 3660
ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa 3720
gttaggccag cttggcactt gatgtaattc tcgttggaat ttgccctttt tgagtttgga 3780
tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg 3840
tcgtgaacac gtggtcgcgg ccgcttcgaa ggtaccgagc tcggatccgc cgccaccatg 3900
gcatggcagc tcacaagcct cctcctgttc gtggccacct ggggaatttc cggcacacca 3960
gctcctcttg actcagtgtt ctccagcagc gagcgtgccc accaggtgct gcggattcga 4020
aaacgttata attcaggtaa attggaagag tttgttcaag ggaaccttga gagagaatgt 4080
atggaagaaa agtgtagttt tgaagaagca cgagaagttt ttgaaaacac ggaaagaaca 4140
actgaatttt ggaagcagta tgttgacggt gaccagtgct tggtcttgcc cttggagcac 4200
ccgtgcgcca gcctgtgctg cgggcacggc acgtgcatcg acggcatcgg cagcttcagc 4260
tgcgactgcc gcagcggctg ggagggccgc ttctgccagc gcgaggtgag cttcctcaat 4320
tgctctctgg acaacggcgg ctgcacgcat tactgcctag aggaggtggg ctggcggcgc 4380
tgtagctgtg cgcctggcta caagctgggg gacgacctcc tgcagtgtca ccccgcagtg 4440
aagttccctt gtgggaggcc ctggaagcgg atggagaaga agcgcagtca cctgaaacga 4500
gacacagaag accaagaaga ccaagtagat ccgcggctca ttgatgggaa gatgaccagg 4560
cggggagaca gcccctggca ggtggtcctg ctggactcaa agaagaagct ggcctgcggg 4620
gcagtgctca tccacccctc ctgggtgctg acagcggccc actgcatgga tgagtccaag 4680
aagctccttg tcaggcttgg agagtatgac ctgcggcgct gggagaagtg ggagctggac 4740
ctggacatca aggaggtctt cgtccacccc aactacagca agagcaccac cgacaatgac 4800
atcgcactgc tgcacctggc ccagcccgcc accctctcgc agaccatagt gcccatctgc 4860
ctcccggaca gcggccttgc agagcgcgag ctcaatcagg ccggccagga gaccctcgtg 4920
acgggctggg gctaccacag cagccgagag aaggaggcca agagaaaccg caccttcgtc 4980
ctcaacttca tcaagattcc cgtggtcccg cacaatgagt gcagcgaggt catgagcaac 5040
atggtgtctg agaacatgct gtgtgcgggc atcctcgggg accggcagga tgcctgcgag 5100
ggcgacagtg gggggcccat ggtcgcctcc ttccacggca cctggttcct ggtgggcctg 5160
gtgagctggg gtgagggctg tgggctcctt cacaactacg gcgtttacac caaagtcagc 5220
cgctacctcg actggatcca tgggcacatc agagacaagg aagcccccca gaagagctgg 5280
gcaccttaat ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt 5340
tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 5400
cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 5460
tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc 5520
aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc 5580
tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 5640
acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 5700
ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 5760
ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 5820
ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact 5880
tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag 5940
ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc 6000
ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac 6060
aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt 6120
gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc 6180
acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc 6240
atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg 6300
caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat 6360
gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc 6420
gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat 6480
ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc 6540
gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg 6600
ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga 6660
tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg 6720
gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga 6780
tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc 6840
gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg 6900
gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg 6960
ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg 7020
ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc 7080
aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 7140
tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg 7200
gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 7260
aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 7320
acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 7380
cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 7440
tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 7500
tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 7560
gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 7620
aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 7680
ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 7740
gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 7800
ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 7860
ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 7920
cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 7980
attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 8040
ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 8100
aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 8160
gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 8220
tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 8280
ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 8340
taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 8400
atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 8460
actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 8520
cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 8580
agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 8640
gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 8700
gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 8760
gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 8820
gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 8880
cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 8940
ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 9000
accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 9060
aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 9120
aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 9180
caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 9240
ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 9300
gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 9360
cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct 9420
gctctgatgc cgcatagtta agccagtatc tg 9452
<211> 6556
<212> DNA
<213> 人工序列
<221> 质粒Pbud-F9-gla-PC
<400> 16
gcgcgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag 60
ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 120
gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 180
caatagggac tttccattga cgtcaatggg tggactattt acggtaaact gcccacttgg 240
cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 300
ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 360
tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 420
gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 480
gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 540
tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc 600
taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga 660
cccaagcttg cattcctgca ggtcgacgcc gccaccatgg acatcaaggt ggtgttcacc 720
ctggtgttca gcgccctggt gcaggccaag agcaccgagt tcgaccccaa catcgacatc 780
gtgggcctgg aaggcaagtt cggcatcacc aacctggaaa ccgacctgtt caccatctgg 840
gagaccatgg aagtgatgat caaggccgac atcgccgaca ccgaccgggc cagcaacttc 900
gtggccaccg agaccgacgc caaccggggc aagatgcccg gcaagaagct gcccctggcc 960
gtcatcatgg aaatggaagc caacgccttc aaggccggct gcacccgggg ctgcctgatc 1020
tgcctgagca agatcaagtg caccgccaag atgaaggtgt acatccccgg caggtgccac 1080
gactacggcg gcgacaagaa aaccggccag gccggcatcg tgggcgccat cgtggacatc 1140
cccgagatca gcggcttcaa agaaatggcc cccatggaac agttcatcgc ccaggtggac 1200
agatgcgcca gctgcaccac cggctgcctg aagggcctgg ccaacgtgaa gtgcagcgag 1260
ctgctgaaga agtggctgcc cgaccgctgc gccagcttcg ccgacaagat ccagaaagag 1320
gtgcacaaca tcaagggcat ggccggcgac aggtgataat ctagaggatc cgaacaaaaa 1380
ctcatctcag aagaggatct gaatatgcat accggtcatc atcaccatca ccattgagtt 1440
tgatccccgg gaattcagac atgataagat acattgatga gtttggacaa accacaacta 1500
gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa 1560
ccattataag ctgcaataaa caagttgggg tgggcgaaga actccagcat gagatccccg 1620
cgctggagga tcatccagcc ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag 1680
aaggcggcgg tggaatcgaa atctcgtagc acgtgtcagt cctgctcctc ggccacgaag 1740
tgcacgcagt tgccggccgg gtcgcgcagg gcgaactccc gcccccacgg ctgctcgccg 1800
atctcggtca tggccggccc ggaggcgtcc cggaagttcg tggacacgac ctccgaccac 1860
tcggcgtaca gctcgtccag gccgcgcacc cacacccagg ccagggtgtt gtccggcacc 1920
acctggtcct ggaccgcgct gatgaacagg gtcacgtcgt cccggaccac accggcgaag 1980
tcgtcctcca cgaagtcccg ggagaacccg agccggtcgg tccagaactc gaccgctccg 2040
gcgacgtcgc gcgcggtgag caccggaacg gcactggtca acttggccat ggtttagttc 2100
ctcaccttgt cgtattatac tatgccgata tactatgccg atgattaatt gtcaacacgt 2160
gctgatcaga tccgaaaatg gatatacaag ctcccgggag ctttttgcaa aagcctaggc 2220
ctccaaaaaa gcctcctcac tacttctgga atagctcaga ggcagaggcg gcctcggcct 2280
ctgcataaat aaaaaaaatt agtcagccat ggggcggaga atgggcggaa ctgggcggag 2340
ttaggggcgg gatgggcgga gttaggggcg ggactatggt tgctgactaa ttgagatgca 2400
tgctttgcat acttctgcct gctggggagc ctggggactt tccacacctg gttgctgact 2460
aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc 2520
ctcgtcgagc tagcttcgtg aggctccggt gcccgtcagt gggcagagcg cacatcgccc 2580
acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg 2640
cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg 2700
ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc 2760
gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat 2820
ggcccttgcg tgccttgaat tacttccacc tggctccagt acgtgattct tgatcccgag 2880
ctggagccag gggcgggcct tgcgctttag gagccccttc gcctcgtgct tgagttgagg 2940
cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg 3000
ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt 3060
tctggcaaga tagtcttgta aatgcgggcc aggatctgca cactggtatt tcggtttttg 3120
ggcccgcggc cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc 3180
tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg 3240
tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg 3300
caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctccaggg ggctcaaaat 3360
ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct 3420
ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc 3480
tcgattagtt ctggagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg 3540
cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga 3600
tgtaattctc gttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc 3660
agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgaacacgt ggtcgcggcc 3720
gcttcgaagg taccgagctc ggatccgccg ccaccatggc atggcagctc acaagcctcc 3780
tcctgttcgt ggccacctgg ggaatttccg gcacaccagc tcctcttgac tcagtgttct 3840
ccagcagcga gcgtgcccac caggtgctgc ggattcgaaa acgttataat tcaggtaaat 3900
tggaagagtt tgttcaaggg aaccttgaga gagaatgtat ggaagaaaag tgtagttttg 3960
aagaagcacg agaagttttt gaaaacacgg aaagaacaac tgaattttgg aagcagtatg 4020
ttgacggtga ccagtgcttg gtcttgccct tggagcaccc gtgcgccagc ctgtgctgcg 4080
ggcacggcac gtgcatcgac ggcatcggca gcttcagctg cgactgccgc agcggctggg 4140
agggccgctt ctgccagcgc gaggtgagct tcctcaattg ctctctggac aacggcggct 4200
gcacgcatta ctgcctagag gaggtgggct ggcggcgctg tagctgtgcg cctggctaca 4260
agctggggga cgacctcctg cagtgtcacc ccgcagtgaa gttcccttgt gggaggccct 4320
ggaagcggat ggagaagaag cgcagtcacc tgaaacgaga cacagaagac caagaagacc 4380
aagtagatcc gcggctcatt gatgggaaga tgaccaggcg gggagacagc ccctggcagg 4440
tggtcctgct ggactcaaag aagaagctgg cctgcggggc agtgctcatc cacccctcct 4500
gggtgctgac agcggcccac tgcatggatg agtccaagaa gctccttgtc aggcttggag 4560
agtatgacct gcggcgctgg gagaagtggg agctggacct ggacatcaag gaggtcttcg 4620
tccaccccaa ctacagcaag agcaccaccg acaatgacat cgcactgctg cacctggccc 4680
agcccgccac cctctcgcag accatagtgc ccatctgcct cccggacagc ggccttgcag 4740
agcgcgagct caatcaggcc ggccaggaga ccctcgtgac gggctggggc taccacagca 4800
gccgagagaa ggaggccaag agaaaccgca ccttcgtcct caacttcatc aagattcccg 4860
tggtcccgca caatgagtgc agcgaggtca tgagcaacat ggtgtctgag aacatgctgt 4920
gtgcgggcat cctcggggac cggcaggatg cctgcgaggg cgacagtggg gggcccatgg 4980
tcgcctcctt ccacggcacc tggttcctgg tgggcctggt gagctggggt gagggctgtg 5040
ggctccttca caactacggc gtttacacca aagtcagccg ctacctcgac tggatccatg 5100
ggcacatcag agacaaggaa gccccccaga agagctgggc accttaatct agagggcccg 5160
tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 5220
cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 5280
atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 5340
ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg 5400
gctctatggc ttctgaggcg gaaagaacca gtggcggtaa tacggttatc cacagaatca 5460
ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 5520
aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 5580
cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 5640
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 5700
gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 5760
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 5820
cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 5880
ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 5940
gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 6000
gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 6060
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 6120
ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 6180
tcacgttaag ggattttggt catgacatta acctataaaa ataggcgtat cacgaggccc 6240
tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag 6300
acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca 6360
gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg 6420
agagtgcacc atatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca 6480
tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct 6540
cttcgctatt acgcca 6556
Claims (10)
1.一种用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系,其特征在于:所述双报告基因细胞体系中含有同时表达FIX-Gla-PC融合基因和报告基因的质粒,所述FIX-Gla-PC融合基因的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系,其特征在于:所述双报告基因细胞体系的宿主细胞为人胚肾细胞293Trex。
3.根据权利要求1所述的用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系,其特征在于:所述FIX-Gla-PC融合基因编码的蛋白质的氨基酸序列如SEQ ID NO.2所示。
4.根据权利要求1所述的用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系,其特征在于:所述报告基因为甲虫荧光素酶报告基因,甲虫荧光素酶报告基因的核苷酸序列如SEQ ID NO.3所示,其编码的蛋白质的氨基酸序列如SEQ ID NO.4所示。
5.根据权利要求2-4中任一项所述的用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系,其特征在于:所述同时表达FIX-Gla-PC融合基因和报告基因的质粒的核苷酸序列如SEQ ID NO.15所示。
6.如权利要求1-5中任一项所述的用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系的制备方法,其特征在于:包括以下步骤:将同时表达FIX-Gla-PC融合基因和报告基因的质粒转入宿主细胞中,即得。
7.如权利要求1-5中任一项所述的用于筛选维生素K循环小分子抑制剂的双报告基因细胞体系在筛选特异性靶向维生素K循环的小分子抑制剂方面的应用。
8.根据权利要求7所述的应用,其特征在于:所述应用为:将待选小分子化合物加入到含有维生素K的双报告基因细胞体系培养液中进行培养,筛选出抑制维生素K循环的小分子化合物,作为特异性靶向维生素K循环的小分子抑制剂。
9.根据权利要求8所述的应用,其特征在于:所述双报告基因细胞体系培养液的培养基为含双抗的完全培养基,双抗分别为青霉素和链霉素。
10.根据权利要求8所述的应用,其特征在于:所述双报告基因细胞体系培养液中维生素K的浓度为1~20μmol/L。
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