CN107184991A - Prdm15基因序列在制备nf‑kb抑制剂中的应用 - Google Patents
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Abstract
本发明公开了一种PRDM15基因序列在制备NF‑κB抑制剂中的应用。NF‑κB信号通路的功能失调与很多疾病都密切相关，PRDM15基因为发现为治疗这些疾病提供了新的靶点信息。
Description
技术领域
本发明涉及生物技术领域，具体指一种PRDM15基因序列在制备NF-κB抑制剂中的应用。
背景技术
转录因子NF-κB广泛存在于不同种类的细胞中，它可以调控细胞的免疫应答，参与炎症反应，抑制细胞凋亡，调控细胞分化等功能。在哺乳动物细胞内NF-κB家族有5个同源蛋白：RelA(p65)，RelB， cRel，还有p52和p50的前提蛋白NF-κB1(p105)，NF-κB2(p100)(Oeckinghaus A,Hayden M S,Ghosh S.Crosstalk in NF-[kappa]B signaling pathways[J].Nature immunology,2011,12(8):695-708.)。所有 NF-κB家族蛋白都具有Rel(relhomology region)同源结构域，可以和DNA结合，形成二聚体以及与抑制蛋白IκBα结合。NF-κB家族蛋白互相之间可以形成同源或者异源二聚体，通常所说的NF-κB蛋白是p65：p50组成的异源二聚体(陈丹英,翟中和,舒红兵.NF-κB激活的调节机理[J].科学通报,2003,48(18):1893-1911.)。NF-κB能够被多种刺激因子激活，包括肿瘤坏死因子(TNFα，tumornecrosis factorα)，细菌及细菌脂多糖LPS(lipopolysaccharide)，白细胞介素-1(IL-1，interlukin-1)，病毒颗粒和病毒基因，双链RNA(dsRNA)，生理生化胁迫等。在正常生理状态下，NF-κB二聚体与抑制蛋白IκBα结合，掩盖了NF-κB的核定位序列NLS，使得NF-κB滞留在细胞质中 (Oeckinghaus A,Hayden M S,Ghosh S.Crosstalk in NF-[kappa]B signalingpathways[J].Nature immunology,2011,12(8):695-708.)。当细胞受到外界刺激，如TNFα、IL-1、LPS，IκBα蛋白被Iκκs(IκB kinase) 复合物磷酸化修饰发生泛素化降解，NF-κB蛋白被释放并迁移到细胞核内，激活下游靶基因的表达。
PRDM蛋白家族成员的N端都具有保守的PR结构域，该结构域与组蛋白甲基转移酶的SET结构域同源。(Fog C K,Galli G G,Lund A H.PRDM proteins:important playersin differentiation and disease[J]. Bioessays,2012,34(1):50-60.)因此，PRDM家族的蛋白也被归属为SET 家族蛋白的亚家族。在灵长类动物中PRDM家族蛋白有17个成员。但是，和很多具有SET结构域的蛋白不一样，目前发现只有少数几个PRDM家族蛋白具有组蛋白甲基化酶活性(Hohenauer T,Moore A W.The Prdm family:expanding roles in stemcells and development[J]. Development,2012,139(13):2267-2282.)。关于为什么大部分PRDM家族的蛋白并没有组蛋白甲基转移酶活性这一点，目前的研究还不是特别清楚。
PRDM2和PRDM8可以介导组蛋白H3K9的二甲基化修饰，与基因表达抑制相关；PRDM9可以介导组蛋白H3K4的三甲基化修饰，与基因表达激活相关。PRDM1，PRDM5，PRDM6这些自身没有组蛋白甲基化酶活性的PRDM家族蛋白可以和G9a，LSD1，HDAC1， HDAC2，HDAC3等相互作用，调控组蛋白修饰(I,Wu J,Fejér G,et al.PRDI-BF1recruits the histoneH3methyltransferase G9a in transcriptional silencing[J].Nature immunology,2004,5(3): 299-308.)(Involvement of Histone Demethylase LSD1in Blimp-1-Mediated Gene Repression during Plasma Cell Differentiation)。很多PRDM家族蛋白也可能对非组蛋白底物进行翻译后修饰。
目前发现PRDM家族蛋白在维持胚胎干细胞的干性和干细胞分化中都有很重要的作用。全基因组ChIP-Seq测序发现，PRDM14与 OCT4，NANOG这些超级转录因子在控制干细胞更新和分化的基因上具有共定位。还有研究表明PRDM1和PRDM14可以影响生殖细胞的特化过程，PRDM9与减数分裂过程中的染色体联会有关。PRDM 家族蛋白还和血管生成，神经系统发育，癌症发生过程密切相关。
发明内容
本发明的目的在于提供一种PRDM15基因序列在制备NF-κB抑制剂中的应用，通过抑制NF-κB基因的的活性来影响细胞的抗病毒天然免疫反应。
本申请的发明人长期致力于表观遗传修饰酶在抗病毒天然免疫方面的研究。我们之前已经发现组蛋白甲基转移酶MLL1可以调控 NF-κB信号通路(Wang X,Zhu K,Li S,etal.MLL1,a H3K4 methyltransferase,regulates the TNFα-stimulated activation ofgenes downstream of NF-κB[J].J Cell Sci,2012,125(17):4058-4066.)。通过大规模的筛选，我们发现PRDM15基因的过表达可以显著抑制TNFα和仙台病毒引起的NF-κB信号通路激活。敲除PRDM15之后，在TNFα和仙台病毒刺激下，NF-κB通路激活程度显著增加。我们关于PRDM15基因的研究为抗病毒治疗提供了新的靶点。
本发明提供了一种PRDM15基因序列在制备NF-κB抑制剂中的应用，其基因序列如SEQ ID NO.1所示。PRDM15是PRDM家族蛋白，属于SET结构域蛋白家族。PRDM15具有潜在的组蛋白甲级转移酶活性。
在293FRT细胞中过表达PRDM家族蛋白，通过双荧光报告基因系统筛选能够调控NF-κB的基因。结果显示，PRDM15的高表达可以显著抑制NF-κB的活性。此后，我们构建了PRDM15过表达的质粒，然后通过磷酸钙转染的方法将质粒转入293FRT细胞内，在 293FRT细胞中过表达PRDM15基因。我们发现，与表达空载体相比较，过表达PRDM15基因后，在不同时间点，NF-κB下游调控基因 A20和cIAP2的mRNA表达量都有所降低，PRDM15可以抑制TNFα诱导的NF-κB活化。PRDM15基因突变之后，NF-κB激活的抗病毒免疫反应增强。这提示，以PRDM15作为靶点，治疗由NF-κB通路功能失调引起的疾病的可能性。
本发明还提供了包含PRDM15基因序列的质粒在制备NF-κB抑制剂中的应用。
PRDM15质粒的载体有多种选择，本发明中优选的载体信息为 pRK质粒，插入时选取的酶切位点为F：SalI；R：XhoI。
本发明还提供了PRDM15基因的短片段在制备NF-κB抑制剂中的应用，所述PRDM15基因的短片段的序列如SEQ ID NO.3所示，以及包含PRDM15基因的短片段的质粒在制备NF-κB抑制剂中的应用。
本发明的有益效果：通过发现一种新的调控NF-κB信号通路的基因PRDM15，提供了PRDM15基因序列在制备NF-κB抑制剂中的应用。NF-κB信号通路的功能失调与很多疾病都密切相关，PRDM15 基因为发现为治疗这些疾病提供了新的靶点信息。
附图说明
图1为PRDM15抑制TNFα和IL-1β诱导NF-κB激活的效果图。
图2A为PRDM15抑制NF-κB下游靶基因A20表达的效果图。
图2B为PRDM15抑制NF-κB下游靶基因cIAP2表达的效果图。
图3A为外源表达FLAG标签PRDM15抑制Sev诱导的IFNβ基因表达的效果图。
图3B为外源表达FLAG标签PRDM15基因的蛋白表达量 Western Blot检测结果照片。
图4A为外源表达HA标签PRDM15抑制Sev诱导的IFNβ基因表达的效果图。
图4B为外源表达HA标签PRDM15基因的蛋白表达量Western Blot检测结果照片。
图5A为PRDM15敲除细胞系的Western Blot检测结果照片。
图5B为PRDM15野生型和PRDM15敲除细胞系对TNFα介导的NF-κB激活的影响的效果图。
图6A为PRDM15蛋白truncation实验模式图。
图6B为双荧光报告系统检测PRDM15不同片段对NF-κB信号调控效果的比较图。
图6C为PRDM15不同片段肽段的表达的Western Blot检测结果照片。
具体实施方式
下面结合附图和具体实施例对本发明作进一步的详细说明，以下实施例是对本发明的解释而本发明并不局限于以下实施例。
实施例1
(1)构建质粒
构建PRDM15的质粒，载体信息：pRK-Flag，PRDM15基因序列如SEQ ID NO.1所示，得到带FLAG标签的PRDM15质粒。
步骤如下：
①在NCBI数据库查找目的基因PRDM15的cDNA序列
②通过primer3软件设计PRDM15的扩增引物
③在cDNA文库中克隆PRDM15的编码序列，PCR扩增体系如下：
a)cDNA 1ul
b)ddH2O 32.5ul
c)5x buffer 10ul
d)dNTP 4ul
e)F-primer 1ul
f)R-primer 1ul
g)rTaq enzyme 0.5ul
④PCR反应程序：
⑤PCR结束之后，产物进行琼脂糖凝胶电泳，切割目的条带并回收产物。
⑥通过SalI和Xhol切割载体，酶切体系如下：
a)目的载体，1ug
b)SalI，1ul
c)Xhol，1ul
d)10x buffer，2ul
e)ddH2O，15ul
4℃酶切12小时以上。
⑦酶切反应结束之后，通过琼脂糖凝胶电泳回收酶切之后的载体。
⑧目的DNA和酶切之后的载体连接：
a)目的DNA片段，1ul
b)载体DNA，1ul
c)10x T4DNAligase buffer，1ul
d)T4DNAligase，1ul
e)ddH2O，6ul
16C°连接12小时以上。
⑨将连接产物转化至感受态大肠杆菌中，进行扩增。
a)取出新制备的感受态菌株DH5α，于冰上快速解冻，加入一定量的质粒混匀，4℃静至10min；
b)然后42℃热激90s，再迅速放回冰盒中，静至1～2min；
c)最后将菌液涂至相应抗性的平板，37℃培养12-16h；
d)挑取单克隆至LB或TB液体培养基中，37℃，220rpm培养过夜；
e)离心收集菌液，并用试剂盒提取质粒，Nanodrop测定浓度。
(2)转染
在293FRT细胞中转染实施例1中得到的带FLAG标签的 PRDM15质粒、连有κB原件的质粒PGL3-NF-κB及内参质粒 PGL3-TK，转染步骤如下。
磷酸钙转染的操作流程：
1.转染前的一天，以合适的密度，将细胞接种到6孔板。
2.12～14小时之后，在显微镜下观察细胞。当细胞的密度达到合适的状态时(30％～40％)，准备转染。
3.将转染试剂2×HBS和CaCl2分装至100μl每孔。CaCl2稀释至 0.25M浓度，加入相应的质粒吹打均匀。
4.将包含质粒的CaCl2溶液一边旋转一边逐滴加入2×HBS中，用移液器向悬液内吹打8～10次。对于逆转录病毒，目的质粒：ZV77 比例为2μg：1μg。对于慢病毒感染，目的质粒：psPAX：pMD.G比例为2μg：1μg：1μg。
5.将配置好的悬液，逐滴加入细胞培养皿中，轻轻摇晃均匀，在37℃培养。
6.转染之后6～8小时，更换新鲜的培养基。
(3)IL-1β和TNFα处理
转染20h后分别加入10ng/mL的IL-1β和TNFα处理12h，收集细胞检测双荧光素酶活性，结果如图1所示，PBS对照、IL-1β和TNFα的三组中左侧柱形代表空载体vector，右侧为PRDM15质粒，PRDM15 可以抑制TNFα和IL-1β诱导的NF-κB激活。*P值<0.05；**P值<0.01。
实施例2
在HCT116细胞中分别转染空载体和实施例1中得到的带FLAG 标签的PRDM15质粒(转染过程同实施例1)，20h后加入TNFα处理0小时，1小时，2小时，4小时，提取RNA，逆转录，实时荧光定量PCR检测NF-κB下游基因A20和cIAP2的表达，结果如图2A、 2B所示，PRDM15可以抑制NF-κB下游靶基因A20及cIAP2。*P 值<0.05；**P值<0.01。
实施例中的RNA提取、逆转录、实时荧光定量PCR步骤如下：
(1)细胞总RNA的提取：
1.将细胞用加入1％β-巯基乙醇的裂解液裂解。
2.细胞裂解液用1mL的注射器抽打10-20次，打断DNA，提高 RNA的得率。
3.加入等体积的70％乙醇，吹打混匀。
4.将裂解液加入到吸附柱，12000rpm离心30秒。
5.加入500μl去蛋白液，12000rpm离心30秒。
6.加入500μl漂洗液，12000rpm，离心30秒。重复此步骤一次。
7.12000rpm离心2分钟，室温下干燥5分钟，除去残留的乙醇，防止影响洗脱效果以及后续的实验。
8.加入50～100μl，预热的DEPC水，12000rpm离心1分钟洗脱 RNA。
9.重复洗脱一次。
10.提取的RNA立即放置于冰上，进行后续的实验。RNA可以在-80℃保存。
逆转录合成cDNA：
1.取1μg总的RNA，加入0.5μl Random引物，0.5μl Oligo(dT)，用DEPC水补充至13.5μL。
2.72℃孵育2分钟，迅速放置于冰上。
3.加入0.5μLRNase抑制剂，1μL dNTPs，1μL RevTra逆转录酶， 4μL 5X buffer，吹打混匀。
4.42℃反应1小时，95℃处理5分钟灭活逆转录酶。
得到的cDNA产物可放置于-80℃。
(2)实时定量PCR检测：
1.准备工作：准备好无菌枪头、96孔板、压膜刮，预热机器；
2.配制引物混合液：将正反引物等量混匀，稀释至5μmol；
3.点样：将引物、模板、mix依次加入96孔板中，封膜，离心混匀；
4.上机：将装有样品的96孔板置于样品槽中，设置程序后开始 Real-Time，待反应完毕后分析数据。
实施例3
1)在293T细胞中转染带FLAG标签的PRDM15质粒(转染过程同实施例1)、PGL3-NF-κB和PGL3-TK，转染20h后加入仙台病毒(SeV)刺激12h，用promega公司的试剂盒检测双荧光素酶活性，结果如图3A所示，外源表达FLAG标签PRDM15可以抑制Sev诱导的IFNβ基因表达。
2)对检测完双荧光素酶活性后的细胞裂解液进行收集，用 Western Blot检测PRDM15和内参β-actin的蛋白表达。
(Western Blot)免疫印迹实验过程如下：
1.蛋白样品的制备：弃DMEM，用PBS洗细胞一次，弃PBS，然后用细胞刮子收集细胞，3000rpm离心1～5min，弃上清，加入50 μl PBS和50μl 2×SDS loading buffer，95℃煮样10min。
2.SDS-PAGE胶的制作：配制10％分离胶混合液，混匀后灌入胶版中，用H2O进行液封，待其凝固后配制5％的浓缩胶，混匀后灌入胶版中，插入梳子，待浓缩胶凝固后拔出梳子备用；
3.电泳：固定好胶板，检查其是否漏水，确定其不漏水后将其置于电泳槽中，加入电泳缓冲液，在点样孔中点上蛋白样品，接通电源，先以低压跑胶，带样品过浓缩胶后换高压跑胶。
4.转膜：电泳完毕后取出蛋白胶，浸与转膜液中，在转膜以上铺好用转膜液润湿的滤纸、蛋白胶、NC膜、滤纸，然后进行转膜。
5.封闭：转膜完毕后，NC膜用TBST漂洗3次，然后用15ml 5％的脱脂牛奶室温封闭15min；
6.一抗孵育：封闭完毕后用TBST将NC膜洗3次，每次10min，用10mlTBST配制抗体，然后加入到置有NC膜的抗体孵育盒中，室温孵育一个小时，然后用TBST漂洗，洗3次，每次10min；
7.二抗孵育：用10ml TBST配制抗体，然后加入到置有NC膜的抗体孵育盒中，室温孵育一个小时，然后用TBST漂洗，洗3次，每次10min；
显影：用辣根过氧化物化学发光底物其将NC膜润湿，暗室显影。结果如图3B所示，外源FLAG标签PRDM15基因的蛋白表达量检测结果，在转入FLAG-PRDM15质粒的细胞中，都能检测到PRDM15 的蛋白表达，证明我们转染是成功。
实施例4
1)在293T细胞中转染带HA标签的PRDM15质粒(载体信息为pRK-Ha，质粒构建及转染过程同实施例1)、PGL3-NF-κB和 PGL3-TK，转染20h后加入仙台病毒(SeV)刺激12h，用promega 公司的试剂盒检测双荧光素酶活性，结果如图4A所示，外源表达 HA标签PRDM15可以抑制Sev诱导的IFNβ基因表达。
2)对检测完双荧光素酶活性后的细胞裂解液进行收集，用Western Blot检测PRDM15和内参β-actin的蛋白表达。结果如图4B 所示，外源HA标签PRDM15基因的蛋白表达量检测结果，在转入 HA-PRDM15质粒的细胞中，都能检测到PRDM15的蛋白表达，证明我们转染是成功。
实施例5
利用CRISPR-Cas构建PRDM15敲除细胞系单克隆细胞株，在 PRDM15野生型和敲除的细胞系里面加入10ng/mL TNF-α，处理 30min，Western Blot检测内源PRDM15的蛋白表达，结果如图5A所示，
在PRDM15野生型和PRDM15敲除的细胞系里转染带FLAG标签的PRDM15质粒(实施例1中构建的)、PGL3-NF-κB及PGL3-TK， 20h后加入TNFα处理12h，收集细胞检测双荧光素酶活性，结果如图5B所示，PRDM15敲除之后可以增强TNFα介导的NF-κB激活。
实施例6
(1)根据不同的功能结构域构建PRDM15的截短片段表达质粒。实验模式图如图6A。
分别截取86～491aa段(基因序列如SEQ ID NO.2所示)， 492～862aa段(基因序列如SEQ ID NO.3所示)，863～1178aa段(基因序列如SEQ ID NO.4所示)，三段短片段，以pRK-Flag为载体，构建三个质粒(质粒构建过程同实施例1)。
(2)在293T细胞中分别转染带有Flag标签的PRDM15全长(FL) 以及截短的片段，24h后往培养基中分别加入10ng/mL的IL-1β和 TNFα处理细胞12h，收集细胞检测荧光素酶活性。P值是与转空载体的细胞相比较计算出来的，*P值<0.05；**P值<0.01。
结果图5B所示，PRDM15的492～862aa段短片段具有近似PRDM15全长的抑制效果。
(3)检测完双荧光素酶活性后，收集细胞裂解液，Western Blot 检测PRDM15不同片段肽段和内参β-actin的蛋白表达，结果如图 6C所示。
SEQUENCE LISTING
<110> 武汉奥秘克生物科技有限公司
<120> PRDM15基因序列在制备NF-kB抑制剂中的应用
<130> 2017
<160> 4
<170> PatentIn version 3.5
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agcagggcaa ggtcatccct tcctcccaac ttggagatca gacgactgga agatggagcc 180
gagggggtgt tcgccatcac tcagctcgtc aagcggacac agttcggtcc ctttgagtcc 240
aggagggtcg ccaaatggga aaaggagtct gcatttcccc tgaaggtgtt ccagaaggac 300
gggcaccccg tgtgcttcga cacctccaac gaggatgact gcaactggat gatgctggtg 360
cggccagcgg cggaggccga gcaccagaac ctgacggcct accagcacgg cagcgacgtg 420
tacttcacca cctccagaga catccccccg ggtaccgagc tgcgcgtgtg gtatgcggcc 480
ttctatgcca agaagatgga caagcccatg ctgaagcagg ccggctctgg cgtccacgct 540
gcaggcaccc cagaaaacag cgcccccgtg gagtcggagc ccagccagtg ggcgtgtaaa 600
gtgtgttctg ccaccttcct ggagctgcag ctcctcaatg aacatctgtt gggccactta 660
gaacaagcca aaagccttcc tccaggcagc caaagcgagg cagcagctcc cgagaaggag 720
caggacacac cccgggggga accccctgca gtgcccgaga gcgagaatgt tgccaccaaa 780
gaacagaaga aaaagcctcg aagggggaga aaacccaaag tgtccaaagc tgagcagcct 840
ctagtcatcg tggaagacaa ggaacccaca gagcaagtgg cagagatcat taccgaggtc 900
cctccggatg agcctgtgag tgcaacgcca gatgagcgga tcatggagct ggttctgggg 960
aagctggcca ccaccaccac tgacaccagc tcggttccaa agttcaccca tcatcagaat 1020
aacaccatca cgctcaagag gagcttaatt ctctcaagca gacacggcat ccggcgcaag 1080
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gagcagtgtg ggaagtcctt tgccaggaag gacatgctga aggagcacat gcgtgtgcac 2220
gacaatgtcc gcgagtacct gtgtgccgag tgtgggaaag gcatgaagac caagcacgcg 2280
ctgcgccacc acatgaagct gcacaagggc atcaaggagt acgagtgcaa ggagtgccac 2340
cgcaggttcg cgcagaaggt caacatgctc aagcactgca agcggcacac ggggattaaa 2400
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aagctcatcc acacagtggg caagcagtgg acgtgctccg tgtgcgacaa gaagtacgtg 2520
accgagtaca tgctgcagaa gcacgttcag ctcacacacg acaaggtgga ggcgcagagc 2580
tgccagctgt gcgggaccaa ggtgtccacc agggcctcca tgagccgaca catgcggcgc 2640
aagcaccccg aggtgctcgc ggtgaggatc gatgacctgg accacctccc ggagaccacc 2700
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gccgagcacc agaacctgac ggcctaccag cacggcagcg acgtgtactt caccacctcc 180
agagacatcc ccccgggtac cgagctgcgc gtgtggtatg cggccttcta tgccaagaag 240
atggacaagc ccatgctgaa gcaggccggc tctggcgtcc acgctgcagg caccccagaa 300
aacagcgccc ccgtggagtc ggagcccagc cagtgggcgt gtaaagtgtg ttctgccacc 360
ttcctggagc tgcagctcct caatgaacat ctgttgggcc acttagaaca agccaaaagc 420
cttcctccag gcagccaaag cgaggcagca gctcccgaga aggagcagga cacaccccgg 480
ggggaacccc ctgcagtgcc cgagagcgag aatgttgcca ccaaagaaca gaagaaaaag 540
cctcgaaggg ggagaaaacc caaagtgtcc aaagctgagc agcctctagt catcgtggaa 600
gacaaggaac ccacagagca agtggcagag atcattaccg aggtccctcc ggatgagcct 660
gtgagtgcaa cgccagatga gcggatcatg gagctggttc tggggaagct ggccaccacc 720
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ggggagcaca agcgggttta ccagtgcaat atctgcagca agatcttcca gaacagcagc 900
aacctgagca ggcacgtgcg ctcgcatggt gacaagctgt ttaagtgcga agagtgtgca 960
aaattgttca gccgcaaaga gagcctaaag cagcacgttt cctacaagca cagcaggaac 1020
gaggtggacg gcgagtacag gtaccgctgc ggcacttgtg agaagacctt ccgcatcgag 1080
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ttcagattct tctccaccaa cagcaacctc tccaagcaca agaagaagca cggcgacaag 1200
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<213> 人
<400> 3
tgcagcaaga tgttctaccg caaggacgtc atgctggacc accagcgccg gcacctggaa 60
ggagtgcggc gagtgaagcg agaggacctg gaggccggtg gggagaacct ggtccgttac 120
aagaaggagc cttccgggtg cccggtgtgt ggcaaggtgt tctcctgccg gagcaatatg 180
aacaagcacc tgctcaccca cggcgacaag aagtacacct gcgagatctg cgggcgcaag 240
ttcttccgcg tggatgtgct cagggaccac atccatgtcc acttcaagga catcgcgttg 300
atggatgacc accagaggga agagtttatc ggcaagatcg ggatctcctc ggaagaaaac 360
gatgacaatt ctgacgagag cgcagactcg gagcctcaca agtacagctg caagcggtgc 420
cagctcacct tcggccgggg gaaggagtac ctgaagcaca tcatggaggt gcacaaggag 480
aagggctatg gctgcagcat ctgcaaccgg cgctttgcac tgaaggccac ctaccacgcc 540
cacatggtca tccaccgtga aaacctgccg gaccccaacg tgcagaagta catccacccc 600
tgcgagatct gcgggcggat cttcaacagc atcgggaacc tggagcgcca caagctcatc 660
cacacaggtg tgaagagcca cgcctgcgag cagtgtggga agtcctttgc caggaaggac 720
atgctgaagg agcacatgcg tgtgcacgac aatgtccgcg agtacctgtg tgccgagtgt 780
gggaaaggca tgaagaccaa gcacgcgctg cgccaccaca tgaagctgca caagggcatc 840
aaggagtacg agtgcaagga gtgccaccgc aggttcgcgc agaaggtcaa catgctcaag 900
cactgcaagc ggcacacggg gattaaagat ttcatgtgtg aattgtgtgg gaagacattc 960
agcgagagga acaccatgga gacccacaag ctcatccaca cagtgggcaa gcagtggacg 1020
tgctccgtgt gcgacaagaa gtacgtgacc gagtacatgc tgcagaagca cgttcagctc 1080
acacacgaca aggtggaggc gcagagctgc cag 1113
<210> 4
<211> 798
<212> DNA
<213> 人
<400> 4
ctgtgcggga ccaaggtgtc caccagggcc tccatgagcc gacacatgcg gcgcaagcac 60
cccgaggtgc tcgcggtgag gatcgatgac ctggaccacc tcccggagac caccaccatc 120
gacgcctcct ccattggcat cgtccagcct gagctgactc tggagcagga ggatttggcc 180
gaagggaagc acgggaaagc tgccaagcga agtcacaaga gaaagcagaa gccagaagag 240
gaggcgggtg ctccggtgcc cgaggacgcc accttcagcg aatactcaga gaaagagacg 300
gagttcacag gcagtgtagg cgacgagacc aattccgcag tacagagcat tcagcaggta 360
gtggtgaccc tgggtgaccc aaatgtgacc acaccatcga gctcagtcgg cttaaccaac 420
atcaccgtga cccccatcac cactgcggcc gcgactcagt ttaccaatct ccagccggtg 480
gccgtggggc accttaccac ccctgaacgc cagttacagc tggacaactc aatcctgacc 540
gtgacctttg ataccgtcag cggctctgcc atgttgcaca accgccaaaa tgacgtccag 600
atccaccccc agccggaagc ctcgaaccca cagtctgtgg cccatttcat caacctgacg 660
accctggtca actccatcac gcccctgggg agccagctta gtgaccagca cccgctcacg 720
tggcgggcag tgccccagac tgacgtcttg ccaccctcgc agccgcaggc acccccacag 780
caggcggccc agccccaggt gcaggcggag cagcagcagc agcagatgta cagctactga 840
Claims (5)
1.PRDM15基因序列在制备NF-KB抑制剂中的应用，其基因序列如SEQ ID NO.1所示。
2.包含PRDM15基因序列的质粒在制备NF-kB抑制剂中的应用，其基因序列如SEQ IDNO.1所示。
3.根据权利要求2所述包含PRDM15基因序列的质粒在制备NF-kB抑制剂中的应用，其特征在于：所述载体为pRK质粒，插入时选取的酶切位点为F：SalI；R：XhoI。
4.PRDM15基因的短片段在制备NF-KB抑制剂中的应用，所述PRDM15基因的短片段的序列如SEQ ID NO.3所示。
5.包含PRDM15基因的短片段的质粒在制备NF-KB抑制剂中的应用，所述PRDM15基因的短片段的序列如SEQ ID NO.3所示。
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Non-Patent Citations (2)
| Title |
|---|
| ERIKA DI ZAZZO等: "PRDM Proteins: Molecular Mechanisms in Signal Transduction", 《BIOLOGY》 * |
| XIANG WANG等: "MLL1, a H3K4 methyltransferase, regulates the TNFa-stimulated activation of genes downstream of NF-kB", 《JOURNAL OF CELL SCIENCE》 * |
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