CN114480399A - 降低CPB1基因表达的siRNA、重组载体及其应用 - Google Patents
降低CPB1基因表达的siRNA、重组载体及其应用 Download PDFInfo
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Abstract
本发明属于分子生物学和基因工程技术领域,涉及一种降低CPB1基因表达的siRNA、重组载体及其应用。具体地,涉及一种siRNA,所述siRNA特异性地降低CPB1基因表达。由于本发明提供的siRNA能够特异性地降低CPB1基因表达,使得该siRNA可应用于制备治疗和/或预防胰腺癌的药物。
Description
技术领域
本发明属于分子生物学和基因工程技术领域,更具体地,涉及一种降低CPB1基因表达的siRNA、所述siRNA对应的shRNA,编码所述shRNA的DNA,包括该DNA的重组载体,包括该重组载体的重组慢病毒,包括所述siRNA、shRNA、编码shRNA的DNA、重组载体和重组慢病毒的宿主细胞,以及它们的应用。
背景技术
胰腺癌是消化道常见恶性肿瘤之一,在肿瘤领域素有“癌症之王”的称号。据柳叶刀杂志记载,胰腺癌确诊后的五年生存率约10%,是预后最差的恶性肿瘤之一。
胰腺癌临床症状隐匿且不典型,是诊断和治疗都很困难的消化道恶性肿瘤,约90%为起源于腺管上皮的导管腺癌。其发病率和死亡率近年来明显上升。胰腺癌早期的确诊率不高,手术死亡率较高,而治愈率很低。胰腺癌发病率男性高于女性,男女之比为1.5~2:1,男性患者远较绝经前的妇女多见,绝经后妇女的发病率与男性相仿。
胰腺癌由于恶性程度高,手术切除率低,预后不良。尽管手术仍然是首要的治疗方法,但由于胰腺癌常常发现较晚,而丧失根治的机会,因此需要对胰腺癌进行综合治疗。迄今同大多数肿瘤一样,还没有一种高效和可完全应用的综合治疗方案。现在的综合治疗仍然是以外科治疗为主,放疗、化疗为辅,并在探讨结合免疫和分子等生物治疗的新方法。
CPB1(carboxpeptidase B1)为胰腺羧肽酶基因,位于3q24。Yamamoto 等认为胰腺羧肽酶是胰腺功能紊乱的重要血清标志物。CPB1基因产物的功能主要有:胰腺羧肽酶A、B活性、选择性地与某些金属离子结合等。结合现有治疗手段仍不能满足临床胰腺癌个体化治疗需求的现状,基于CPB1的开发和应用势在必行。
发明内容
本发明的目的是提供一种降低CPB1基因表达的siRNA、重组载体及其应用,以特异性地降低CPB1基因表达,从而能够治疗和/或预防胰腺癌。
本发明第一方面是的提供一种siRNA,所述siRNA特异性地降低CPB1基因表达。
具体地,本发明的重点在于提供新的胰腺癌治疗靶标,而不限于具体的siRNA序列,可以根据本领域常规的各种方法设计针对该靶标的siRNA。所述siRNA通常具有19~27bp的核苷酸序列。
具体地,所述siRNA的核苷酸序列至少包括以下一组核苷酸序列:
(1)第一组核苷酸序列。
所述第一组核苷酸序列如SEQ ID NO:1和SEQ ID NO:2所示,所述SEQ ID NO:1为5'-GUGGCCUUCAAAUUCCGAACA-3',所述SEQ ID NO:2为5'-GUAUAAGCUGAUGCGUCCUGA-3'。
(2)第二组核苷酸序列。
所述第二组核苷酸序列如SEQ ID NO:3和SEQ ID NO:4所示,所述SEQ ID NO:3为5'-CAUUCCAGAAGCCUGUGACAU-3',所述SEQ ID NO:4为5'-GGUAGGUGAUAUGACUCUCAC-3'。
(3)第三组核苷酸序列。
所述第三组核苷酸序列如SEQ ID NO:5和SEQ ID NO:6所示,所述SEQ ID NO:5为5'-ACCCACCACAUUUAACCCUUG-3',所述SEQ ID NO:6为5'-CAAGUAGUGUGGUAGGGAUGU-3'。
本发明第二方面提供提供一种shRNA。
具体地,所述shRNA是具有茎环结构的单链RNA,所述shRNA的核苷酸序列至少包括以下一组核苷酸序列。
(1)第五组核苷酸序列。
所述第五组核苷酸序列如SEQ ID NO:9和SEQ ID NO:10所示,
所述SEQ ID NO:9为5'-GUCGUCUGUUUCUACACGGGCUCCAUUAUCGAUGUCAUAGAUGAGCCGCGAACGGAUU-3'。
所述SEQ IDNO:10为
5'-CUAUGGGUCAGUACUCGCAAGGAGACUGACGUCUCAACAUAAUUCAAAGUAAUGACCU-3'。
(2)第六组核苷酸序列。
所述第六组核苷酸序列如SEQ ID NO:11和SEQ ID NO:12所示,
所述SEQ ID NO:11为
5'-AUGGCCAUAAGCACCACGGCAUGCUUGAGUUUCUGAGUCGCUGGUUGCAGACUUCAUU-3'。
所述SEQ IDNO:12为
5'-AAAUUGCCUGUAAUUCCAUAUUCCAAACGGUCAGACUUCCGAGAGAGAUGUGGAACCA-3'。
(3)第七组核苷酸序列。
所述第七组核苷酸序列如SEQ ID NO:13和SEQ ID NO:14所示,
所述SEQ ID NO:13为
5'-GCUGGAGAGAGGAUGUUCAUCAGUCACAGACAUCUCCUUAAUUGGGGUCCUUUGCCCU-3'。
所述SEQ IDNO:14为
5'-AGAGCAAAAACACUGCUUGUCGGAACCCAGGAAACAAUUGUGUGUCUAAGCAUCCUUU-3'。
本发明第三方面提供一种编码本发明的第二方面提供的shRNA的DNA。
具体地,所述DNA的核苷酸序列至少包括以下一组核苷酸序列:
(1)第九组核苷酸序列。
所述第九组核苷酸序列如SEQ ID NO:17和SEQ ID NO:18所示,
所述SEQ ID NO:17为
5'-GTCGTCTGTTTCTACACGGGCTCCATTATCGATGTCATAGATGAGCCGCGAACGGATT-3 '。
所述SEQ IDNO:18为
5'-CTATGGGTCAGTACTCGCAAGGAGACTGACGTCTCAACATAATTCAAAGTAATGACCT-3 '。
(2)第十组核苷酸序列。
所述第十组核苷酸序列如SEQ ID NO:19和SEQ ID NO:20所示,
所述SEQ ID NO:19为
5'-ATGGCCATAAGCACCACGGCATGCTTGAGTTTCTGAGTCGCTGGTTGCAGACTTCATT-3 '。
所述SEQ IDNO:20为
5'-AAATTGCCTGTAATTCCATATTCCAAACGGTCAGACTTCCGAGAGAGATGTGGAACCA-3 '。
(3)第十一组核苷酸序列。
所述第十一组核苷酸序列如SEQ ID NO:21和SEQ ID NO:22所示,
所述SEQ ID NO:21为
5'-GCTGGAGAGAGGATGTTCATCAGTCACAGACATCTCCTTAATTGGGGTCCTTTGCCCT-3 '。
所述SEQID NO:22为
5'-AGAGCAAAAACACTGCTTGTCGGAACCCAGGAAACAATTGTGTGTCTAAGCATCCTTT-3 '。
本发明第四方面提供一种重组载体。
具体地,所述重组载体为在pX330质粒的多克隆位点Not I和EcoR I插入如编码本发明第三方面提供的编码shRNA的DNA得到的重组载体。
本发明的第五方面提供一种重组慢病毒。
具体地,所述重组慢病毒由本发明的第四方面提供的重组载体与病毒包装辅助质粒pHelper 1.0载体和病毒包装辅助质粒pHelper 2.0载体共转染哺乳动物细胞得到。
本发明的第六方面提供一种宿主细胞。
具体地,包括本发明的第一方面提供的siRNA、本发明的第二方面提供的shRNA、本发明的第三方面提供的编码shRNA的DNA、本发明的第四方面提供的重组载体和本发明的第五方面提供的重组慢病毒中的至少一种。本发明对所述宿主细胞的具体种类没有特别限定。
本发明的第七方面提供本发明的第一方面提供的siRNA、本发明的第二方面提供的shRNA、本发明的第三方面提供的编码shRNA的DNA、本发明的第四方面提供的重组载体和本发明的第五方面提供的重组慢病毒中的至少一种在制备治疗和/或预防胰腺癌的药物中的应用。
本发明的有益效果在于:提供了一种降低CPB1基因表达的siRNA、重组载体及其应用,以特异性地降低CPB1基因表达,从而能够治疗和/或预防胰腺癌。
本发明的其它特征和优点将在随后具体实施方式部分予以详细说明。
附图说明
通过结合附图对本发明示例性实施方式进行更详细的描述,本发明的上述以及其它目的、特征和优势将变得更加明显。
图1为CPB1在正常细胞和胰腺癌细胞中的表达差异。
HPDE6-C7为正常细胞,CAPAN-2和PATU 8988t为胰腺癌细胞。
图2为敲低CPB1对胰腺癌细胞增殖的影响。
shCtrl为对照组,shCPB1为实验组,上方的曲线为对照组,下方的曲线为实验组。
图3为敲低CPB1对荷瘤小鼠胰腺癌生长的影响。
NC为对照组,shCPB1为实验组。
具体实施方式
下面将更详细地描述本发明的优选实施方式。虽然以下描述了本发明的优选实施方式,然而应该理解,可以以各种形式实现本发明而不应被这里阐述的实施方式所限制。
实施例中未注明具体条件者,皆按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
实施例1:本实施例用于说明在胰腺癌细胞系中CPB1高表达。
1、RNA的提取和逆转录定量PCR(RT-qPCR)
1.1 总RNA提取。
本实施例在低温下进行。细胞培养在6孔板中进行,去掉培养基,PBS漂洗3次,每孔加入Trizol 1000μL,摇床10min;收集到1.5ml离心管中,每管加入200μL氯仿,剧烈混匀30sec,静置15min,4℃12000rpm离心15min;轻轻吸取上层液体400μL至另一新离心管中,加入等体积异丙醇,轻轻颠倒混匀,4℃,12000rpm离心10min;弃上清,加入1mL75%酒精洗涤沉淀物,4℃,12000rpm离心10min;尽可能弃掉上清,室温下晾干10min,每管加入10μL无RNase水,溶解,分光光度计定量。
1.2 反转录
每25μL反转录体系包括100pmol随机引物,2μg总RNA,M-MLV反转录酶1μL,RNase抑制剂0.625μL,dNTPs(10mM)1.25μL,5×M-MLV缓冲液5μL,其余用ddH2O补齐至25μL。反应条件为:37℃,1h,95℃,5min。
1.3 定量PCR
每20μL反应体系包含2×PCR Mix(ABI)10μL,上、下游引物各0.4μL,cDNA1μL,ddH2O 8.2μL。反应条件为:94℃,2min,94℃,15s,60℃,40s,40个循环。实验中用到的引物序列见表1。
表1:定量PCR引物序列
图1出示了CPB1在正常细胞和胰腺癌细胞中的表达差异。
HPDE6-C7为正常细胞,CAPAN-2和PATU 8988t为胰腺癌细胞。
如图1所示,RT-qPCR结果显示:在CAPAN-2和SPATU 8988t胰腺癌细胞中CPB1基因表达较对照组HPDE6-C7细胞显著升高。
实施例2:本实施例用于说明敲低CPB1基因表达可以抑制胰腺癌增殖和/或生长。
1、RNAi慢病毒克隆的制备
1.1 靶点设计
针对CPB1基因序列,根据RNAi序列设计原则,设计3条RNAi靶点序列,CPB1基因的3种siRNA的核苷酸序列,以及阴性对照(NC)的核苷酸序列。3种siRNA以及阴性对照对应的名称分别为CPB1-si-1a、CPB1-si-1b、CPB1-si-2a、CPB1-si-2b、CPB1-si-3a、CPB1-si-3b,NC-si-a和NC-si-b为针对阴性对照组设计的序列。详见表2。
表2:3个RNA干扰靶点(siRNA)和阴性对照的核苷酸序列
表3示出了实施例所用3种shRNA的核苷酸序列,实施例所用shRNA为CPB1-sh-1a、CPB1-sh-1b、CPB1-sh-2a、CPB1-sh-2b、CPB1-sh-3a、CPB1-sh-3b的核苷酸序列,NC-sh-a和NC-sh-b为对照组的核苷酸序列。详见表3。
表3:3种shRNA和阴性对照的核苷酸序列
表4示出了所用编码3种shRNA的DNA的核苷酸序列。实施例所用编码shRNA的DNA为CPB1-d-1a、CPB1-d-1b、CPB1-d-2a、CPB1-d-2b、CPB1-d-3a、CPB1-d-3b的核苷酸序列,NC-d-a和NC-d-b为对照组的核苷酸序列。详见表4。
表4:3种shRNA的DNA和阴性对照的核苷酸序列
1.2 载体酶切
根据表5配制50μL酶切体系。按列表顺序依次加入各种试剂,用移液器轻轻吹打混匀,瞬时离心,置于37℃反应3h。对载体酶切产物进行琼脂糖凝胶电泳,回收目的条带。
表5:载体酶切体系
1.3 shRNA的DNA退火形成双链DNA。
合成后成对的shRNA的DNA干粉溶解于退火缓冲液中,90℃水浴15min,自然冷却至室温。
1.4 载体连接。
通过T4 DNA ligase (T4 DNA连接酶)将双酶切线性化的载体和退火双链DNA连接,16℃连接1~3h。
表6:载体连接体系
1.5 转化。
将10μL连接反应产物加入到100μL感受态细胞中,轻弹管壁数下混匀,在冰上放置30min;42℃热激90s,冰浴孵育2min;加入500μL LB培养基,置于37℃摇床振荡培养1h;取适量菌液均匀涂布在含有相应抗生素的平板上,在恒温培养箱中倒置培养12~16h。
1.6 测序鉴定。
将鉴定出的阳性克隆转化子接种于适量含相应抗生素的LB液体培养基中,37℃培养12-16h,取适量菌液进行测序、鉴定。
1.7 质粒转染与慢病毒收获 。
病毒包装共涉及三个质粒:携带靶点序列的工具载体质粒pX330载体、病毒包装辅助质粒Helper 1.0载体和病毒包装辅助质粒Helper 2.0载体。采用上述三个质粒共转染293T细胞。
转染前24h,用胰蛋白酶消化对数生长期的293T细胞,以含10%血清的培养基调整细胞密度约5×106/15ml,重新接种于10cm细胞培养皿,37℃、5%CO2培养箱内培养;24h待细胞密度达70%~80%时即可用于转染;转染前2h更换为无血清培养基;取灭菌离心管,加入各DNA溶液(pX330质粒20μg、pHelper 1.0载体质粒15μg、pHelper 2.0载体质粒10μg),与相应体积的转染试剂混合均匀,调整总体积为1ml,在室温下温育15min;混合液缓慢滴加至293T细胞培养液中,混匀,于37℃、5%CO2细胞培养箱中培养;培养6h后弃去含有转染混和物的培养基,加入10ml的PBS清洗一次,轻柔晃动培养皿以洗涤残余的转染混和物后倒弃。
缓慢加入含10%血清的细胞培养基20ml,于37℃、5%CO2培养箱内继续培养48~72h。
1.8 慢病毒浓缩和纯化与质检。
根据细胞状态,收集转染后48h的293T细胞上清液;于4℃、4000g离心10min,除去细胞碎片;以0.45μm滤器过滤上清液于40ml超速离心管中;分别配平样品,将带有病毒上清液的超速离心管逐一放入至超速离心机内,4℃,25000rpm,离心2h;离心结束后,弃去上清,尽量去除残留在管壁上的液体,加入病毒保存液,轻轻反复吹打重悬;经充分溶解后,高速离心10000rpm、5min后,取上清按要求分装。
慢病毒的质量控制要点包括物理状态检测、无菌检测及病毒滴度检测。
2、慢病毒转染。
为保证基因干扰效率,本实施例针对CPB1基因设计3种RNA干扰靶点(siRNA),并将携带不同靶点的3个质粒等比例混合进行慢病毒包装,从而确保病毒感染细胞后目的基因的敲减效率。
细胞传代培养到6孔板中12~16小时后:将病毒液0.15ml与新鲜细胞培养液混匀,比例为0.15ml病毒上清液中加入0.5ml新鲜培养液和0.65μL polybrene(终浓度4ng/mL);预混好的病毒感染液加入到目的细胞培养皿中,此时细胞密度不宜超过50%。过夜培养后更换成新鲜培养液。
感染3天后,取对数生长期细胞,进行细胞增殖实验。
3、细胞增殖实验。
将上述对照组和敲低CPB1表达的处于对数生长期的细胞进行胰酶消化,制成细胞悬液;将细胞悬液(细胞数约为3000)接种于96孔板中,分别于第1天、2天、3天、4天、5天计算细胞数量,绘制生长曲线。
图2示出了敲低CPB1对胰腺癌细胞增殖的影响。其中,shCtrl为对照组,shCPB1为实验组,上方的曲线为对照组,下方的曲线为实验组。
如图2所示,细胞增殖实验结果显示,敲低CPB1基因的表达,在第4天和第5天均显著抑制胰腺癌细胞CAPAN-2的增殖。
4、裸鼠胰腺癌荷瘤实验。
将对照组和敲低CPB1的CAPAN-2胰腺癌细胞系分别制成细胞悬液,进行裸鼠皮下种植。每组6只小鼠,每只小鼠接种100μL,5×106个细胞。1.5个月后,检测肿瘤大小和体积,进行统计学分析。
图3示出了敲低CPB1对荷瘤小鼠胰腺癌生长的影响。其中,NC为对照组,shCPB1为实验组。
如图3所示,小鼠荷瘤实验结果显示,CPB1低表达组胰腺癌肿瘤体积显著低于对照组,可见,降低CPB1基因的表达能够显著抑制胰腺癌细胞生长。
以上已经描述了本发明的各实施例,上述说明是示例性的,并非穷尽性的,并且也不限于所披露的各实施例。在不偏离所说明的各实施例的范围和精神的情况下,对于本技术领域的普通技术人员来说许多修改和变更都是显而易见的。
序列表
<110> 江苏医药职业学院
<120> 降低CPB1基因表达的siRNA、重组载体及其应用
<141> 2022-03-17
<160> 28
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Claims (2)
1.一种siRNA在制备抑制胰腺癌细胞CAPAN-2增殖的药物中的应用,其特征在于,所述siRNA特异性地降低CPB1基因表达,所述siRNA为由以下核苷酸序列组成的组合:
(1)第一组核苷酸序列
所述第一组核苷酸序列如SEQ ID NO:1和SEQ ID NO:2所示,
所述SEQ ID NO:1为5'-GUGGCCUUCAAAUUCCGAACA-3';
所述SEQ ID NO:2为5'-GUAUAAGCUGAUGCGUCCUGA-3';
(2)第二组核苷酸序列
所述第二组核苷酸序列如SEQ ID NO:3和SEQ ID NO:4所示,
所述SEQ ID NO:3为5'-CAUUCCAGAAGCCUGUGACAU-3';
所述SEQ ID NO:4为5'-GGUAGGUGAUAUGACUCUCAC-3';
(3)第三组核苷酸序列
所述第三组核苷酸序列如SEQ ID NO:5和SEQ ID NO:6所示,
所述SEQ ID NO:5为5'-ACCCACCACAUUUAACCCUUG-3';
所述SEQ ID NO:6为5'-CAAGUAGUGUGGUAGGGAUGU-3'。
2.一种shRNA在制备抑制胰腺癌细胞CAPAN-2增殖的药物中的应用,其特征在于,所述shRNA为由以下核苷酸序列组成的组合:
(1)第五组核苷酸序列
所述第五组核苷酸序列如SEQ ID NO:9和SEQ ID NO:10所示,
所述SEQ ID NO:9为
5'-GUCGUCUGUUUCUACACGGGCUCCAUUAUCGAUGUCAUAGAUGAGCCGCGAACGGAUU-3';
所述SEQ ID NO:10为
5'-CUAUGGGUCAGUACUCGCAAGGAGACUGACGUCUCAACAUAAUUCAAAGUAAUGACCU-3';
(2)第六组核苷酸序列
所述第六组核苷酸序列如SEQ ID NO:11和SEQ ID NO:12所示,
所述SEQ ID NO:11为
5'-AUGGCCAUAAGCACCACGGCAUGCUUGAGUUUCUGAGUCGCUGGUUGCAGACUUCAUU-3';
所述SEQ IDNO:12为
5'-AAAUUGCCUGUAAUUCCAUAUUCCAAACGGUCAGACUUCCGAGAGAGAUGUGGAACCA-3';
(3)第七组核苷酸序列
所述第七组核苷酸序列如SEQ ID NO:13和SEQ ID NO:14所示,
所述SEQ ID NO:13为
5'-GCUGGAGAGAGGAUGUUCAUCAGUCACAGACAUCUCCUUAAUUGGGGUCCUUUGCCCU-3';
所述SEQ IDNO:14为
5'-AGAGCAAAAACACUGCUUGUCGGAACCCAGGAAACAAUUGUGUGUCUAAGCAUCCUUU-3'。
Priority Applications (1)
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