CN110117585B - 一种细菌RNase E截短体及其应用 - Google Patents
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Abstract
本发明公开了一种细菌RNase E截短体及其应用，细菌RNase E截短体的蛋白序列如SEQ ID NO.1所示，本发明利用RNase E的N端(氨基酸残基1～529)可降解RNA的功能域，体外合成该截短体(REt)的核苷酸编码序列，在其5'端添加SV40核定位序列后使其表达于癌细胞核内，在细胞核内直接降解包括mRNA在内的多种RNAs，干扰癌蛋白的生物合成，从而抑制癌细胞的生长增殖和迁移侵袭，预期达到抑制肿瘤的效果。
Description
技术领域
本发明涉及一种细菌RNase E截短体及其应用，属于RNase E截短体及其应用技术领域。
背景技术
《2018年全球癌症统计数据》报告指出，2018年全球约新增癌症1810万例和癌症死亡960万例，在新增1810万例病例中，中国癌症发病率和死亡率位居全球第一。据统计分析，2018年，我国新增病例约380.4万，死亡病例229.6万,这就意味着全球每新增100个癌症患者，中国就占了21人。在我国，发病率最高的前5位癌症分别是肺癌、乳腺癌、胃癌、结直肠癌、肝癌。目前，虽然癌症治疗手段和方式多样化，包括手术切除、化疗、放疗和生物免疫治疗等，但疗效仍有待于进一步提高。
RNase E(RE)是由David等在大肠杆菌中发现并鉴定的一种核糖核酸内切酶，最初认为是9S rRNA产生5S rRNA必不可少的活性酶。后来，研究发现RE是一种广泛存在于细菌中的、可水解单链非特异性RNA的核糖核酸内切酶，它对RNA中富含A/U序列具有偏爱性，可大量降解mRNA、tRNA、rRNA和sRNA等多种RNAs，由rne基因编码1061个氨基酸组成的分子量约180KDa的酸性RNA水解酶。RE是大肠杆菌中分子量最大的蛋白质之一，是细菌内RNA降解体的主要成分，在多种RNAs的加工、成熟和降解中起着关键作用，是细菌存活所需的必需酶之一。RE可分为两个大致相等的部分，即包含有RNA水解酶活性在内的118KDa的保守N-末端结构域(NTD；氨基酸残基1～529)和非结构蛋白的C-末端结构域(CTD；氨基酸残基530～1061)。现有文献报道，其N端催化结构域是细菌存活所必需的，C端对于细胞活力来说是可有可无的。X射线晶体学分析表明，RE的N端催化域由3个子结构域组成：由S1 RNA结合区(S1)、5'传感器、RNase H(H)和DNase I相关的折叠组成的大结构域、锌链间隔区和小结构域，其结构与RNase G高度相似。RE的C端非催化区在进化上并不是高度保守的，大部分本质上属于非结构蛋白，氨基酸残基601～700形成一个富含精氨酸的片段，在体外结合RNA(RNA结合域，RBD)，并被认为能增强RE在体内降解RNA的活性；氨基酸残基701～1061构成RE与其他水解酶相互作用的脚手架结构，使核苷酸磷酸化酶(PNPase)、RNase螺旋酶B(RhlB)和烯醇化酶(enolase)在RE上组装并共同参与RNA的水解。
目前，肿瘤治疗仍然是一个世界级难题，肿瘤治疗常用化疗、放疗和手术治疗。虽然生物治疗如肿瘤免疫治疗也用于肿瘤临床治疗，但效果仍然有待于进一步提高。因此，寻找新的有效的肿瘤治疗方法并结合现有的治疗方法成为现阶段的重要任务。
发明内容
本发明要解决的技术问题是：提供一种细菌RNase E截短体及其应用，干扰癌蛋白生物合成，从而抑制癌细胞的生长增殖和迁移侵袭，预期达到抑制肿瘤的效果。
本发明采取的技术方案为：一种细菌RNase E截短体，细菌RNase E截短体的蛋白序列如SEQ ID NO.1所示。
编码一种细菌RNase E截短体的基因，细菌RNase E截短体的编码基因序列如SEQID NO.2所示。
一种细菌RNase E截短体(REt)的编码基因，含有细菌RNase E截短体的编码基因的载体：用Xho I和EcoR I双酶切线性化骨架载体pLVX-AcGFP-N1和目的基因，经连接、转化、筛选、扩大培养和鉴定，获得慢病毒基因表达载体pLVX-REt-AcGFP。
一种细菌RNase E截短体的应用，细菌RNase E截短体在抑制肿瘤方面的应用。
本发明的有益效果：与现有技术相比，本发明利用RE蛋白截短体REt表达于细胞核内，水解肝癌细胞细胞核内RNAs，从而抑制肝癌细胞的生长增殖、迁移侵袭。
说明书附图
图1为pMD18-RE两端测序序列；
图2为pMD18-REt两端测序序列；
图3为慢病毒表达载体pLVX-RE-AcGFP；；
图4为慢病毒表达载体pLVX-REt-AcGFP；
图5为克隆的RE基因蛋白编码区；
图6为克隆的RE基因编码的蛋白序列；
图7为克隆的REt基因蛋白编码区；
图8为克隆的REt基因编码的蛋白序列；
图9为核内表达蛋白RE和REt的肝癌细胞；
图10为肝癌细胞表达RE及REt蛋白；
图11为肝癌细胞核内表达REt蛋白抑制细胞周期进程；
图12为肝癌细胞核内表达REt蛋白抑制细胞生长增殖速率；
图13为肝癌细胞核内表达REt蛋白抑制细胞迁移速率；
图14为肝癌细胞核内表达REt蛋白抑制细胞侵袭速率。
图15为肝癌细胞内CCNA2和HuR mRNAs及其蛋白表达水平
具体实施方式
下面结合具体的实施例对本发明进行进一步介绍。
实施例1：一种细菌RNase E截短体，细菌RNase E截短体的蛋白序列如SEQ IDNO.1所示。
编码一种细菌RNase E截短体的基因，细菌RNase E截短体的编码基因序列如SEQID NO.2所示。
一种细菌RNase E截短体的编码基因，含有细菌RNase E截短体的编码基因的载体：用Xho I和EcoR I双酶切线性化骨架载体pLVX-AcGFP-N1和目的基因，经连接、转化、筛选、扩大培养和鉴定，获得慢病毒基因表达载体pLVX-REt-AcGFP。
一种细菌RNase E截短体的应用，细菌RNase E截短体在抑制肿瘤方面的应用。
一种细菌RNase E截短体的制备过程步骤如下：
1、RNase E基因克隆与改造
首先，抽提大肠杆菌基因组，作为体外扩增RNase E基因(RE)的PCR模板。根据NCBI上大肠杆菌DH5αRE基因序列号EU902419设计PCR上下游引物，并在上游引物5'端引入SV40核定位信号的核苷酸序列。用高保真DNA聚合酶体外扩增RE基因，***克隆载体中并进行DNA测序。然后，以包含RE基因的克隆载体DNA为模板，通过设计下游引物，用PCR扩增出5'端具有SV40核定位信号的、可编码RE N端1-585个氨基酸的核苷酸序列(REt)，***克隆载体中，DNA测序鉴定。
2、慢病毒表达载体构建
将编码RE和REt的核苷酸序列分别***到慢病毒表达载体pLVX-AcGFP-N1中，分别构建慢病毒表达载体pLVX-RE-AcGFP和pLVX-REt-AcGFP，并进行DNA测序。
3、病毒包装与稳转细胞株构建
质粒抽提pLVX-RE-AcGFP和pLVX-REt-AcGFP并去内毒素，将其分别与慢病毒包装辅助载体(pLP1、pLP2、pLP/VSVG)分别共转染入HEK-293FT细胞中，包装出的慢病毒感染癌细胞，通过嘌呤霉素药物筛选、单克隆细胞集落形成、细胞荧光观察、挑取单克隆、扩大培养等步骤，获得细胞核内表达蛋白RE和REt的稳转细胞株，并用蛋白免疫印迹验证。
4、细胞特性分析
测定核内表达RE和REt的癌细胞的细胞生长增殖速率、细胞周期分析、细胞形态观察、细胞迁移、细胞侵袭能力分析。
REt蛋白序列SEQ ID NO.1
MRPKKKRKVEASMKRMLINATQQEELRVALVDGQRLYDLDIESPGHEQKKANIYKGKITRIEPSLEAAFVDYGAERHGFLPLKEIAREYFPANYSAHGRPNIKDVLREGQEVIVQIDKEERGNKGAALTTFISLAGSYLVLMPNNPRAGGISRRIEGDDRTELKEALASLELPEGMGLIVRTAGVGKSAEALQWDLSFRLKHWEAIKKAAESRPAPFLIHQESNVIVRAFRDYLRQDIGEILIDNPKVLELARQHIAALGRPDFSSKIKLYTGEIPLFSHYQIESQIESAFQREVRLPSGGSIVIDSTEALTAIDINSARATRGGDIEETAFNTNLEAADEIARQLRLRDLGGLIVIDFIDMTPVRHQRAVENRLREAVRQDRARIQISHISRFGLLEMSRQRLSPSLGESSHHVCPRCSGTGTVRDNESLSLSILRLIEEEALKENTQEVHAIVPVPIASYLLNEKRSAVNAIETRQDGVRCVIVPNDQMETPHYHVLRVRKGEETPTLSYMLPKLHEEAMALPSEEEFAERKRPEQPALATFAMPDVPPAPTPAEPAAPVVAPSPKAAPATPAAPAQPGLLSRFFGALKALFSGG
REt基因编码序列SEQ ID NO.2
ATGAGGCCCAAGAAGAAGCGGAAGGTGGAGGCTTCCATGAAAAGAATGTTAATCAACGCAACTCAGCAGGAAGAGTTGCGCGTTGCCCTTGTAGATGGGCAGCGTCTGTATGACCTGGATATCGAAAGTCCAGGGCACGAGCAGAAAAAGGCAAACATCTACAAAGGTAAAATCACCCGCATTGAACCGAGTCTGGAAGCTGCTTTTGTTGATTACGGCGCTGAACGTCACGGTTTCCTCCCACTAAAAGAAATTGCCCGCGAATATTTCCCTGCTAACTACAGTGCTCATGGTCGTCCCAACATTAAAGATGTGTTGCGTGAAGGTCAGGAAGTCATTGTTCAGATCGATAAAGAAGAGCGCGGAAACAAAGGCGCGGCATTAACCACCTTTATCAGTCTGGCGGGTAGCTATCTGGTTCTGATGCCGAACAACCCGCGCGCGGGTGGCATTTCTCGCCGTATCGAAGGCGACGACCGTACCGAATTAAAAGAAGCACTGGCAAGCCTTGAACTGCCGGAAGGCATGGGGCTTATCGTGCGCACCGCTGGCGTCGGCAAATCTGCTGAGGCGCTGCAATGGGATTTAAGCTTCCGTCTGAAACACTGGGAAGCCATCAAAAAAGCCGCTGAAAGCCGCCCGGCTCCGTTCCTGATTCATCAGGAGAGCAACGTAATCGTTCGCGCATTCCGCGATTACTTACGTCAGGACATCGGCGAAATCCTTATCGATAACCCGAAAGTGCTCGAACTGGCACGTCAGCATATCGCTGCATTAGGTCGCCCGGATTTCAGCAGCAAAATCAAACTGTACACCGGCGAGATCCCGCTGTTCAGCCACTACCAGATCGAGTCGCAGATCGAATCCGCCTTCCAGCGTGAAGTCCGTCTGCCATCCGGTGGTTCCATTGTTATCGACAGCACCGAAGCGTTAACTGCCATCGACATCAACTCCGCACGCGCGACCCGCGGCGGCGATATCGAAGAAACCGCGTTTAACACCAACCTCGAAGCTGCCGATGAGATTGCTCGTCAGCTGCGCCTGCGTGACCTCGGCGGCCTGATTGTTATCGACTTCATCGACATGACGCCAGTACGCCACCAGCGTGCGGTAGAAAACCGTCTGCGTGAAGCGGTGCGTCAGGACCGTGCGCGTATTCAAATCAGCCATATTTCTCGCTTTGGCCTGCTGGAAATGTCCCGTCAGCGCCTGAGCCCATCACTGGGTGAATCCAGTCATCACGTTTGTCCGCGTTGTTCTGGTACTGGCACCGTGCGTGACAACGAATCGCTGTCGCTCTCTATTCTGCGTCTGATCGAAGAAGAAGCGCTGAAAGAGAACACCCAGGAAGTTCACGCCATTGTTCCTGTGCCAATCGCTTCTTACCTGCTGAATGAAAAACGTTCTGCGGTGAATGCCATTGAAACGCGTCAGGACGGTGTTCGCTGCGTGATTGTGCCAAACGATCAGATGGAAACCCCGCACTACCACGTGCTGCGCGTGCGTAAAGGGGAAGAAACCCCAACCTTAAGCTACATGCTGCCGAAGCTGCATGAAGAAGCGATGGCGCTGCCGTCTGAAGAAGAGTTCGCTGAACGTAAGCGTCCGGAACAACCTGCGCTGGCAACCTTTGCCATGCCGGATGTGCCGCCAGCGCCAACGCCAGCTGAACCTGCCGCGCCTGTCGTAGCTCCATCACCAAAAGCTGCACCGGCAACACCAGCAGCTCCTGCACAACCTGGGCTGTTGAGCCGCTTCTTCGGCGCACTGAAAGCGCTGTTCAGCGGTGGT
RE蛋白序列SEQ ID NO.3
MRPKKKRKVEASMKRMLINATQQEELRVALVDGQRLYDLDIESPGHEQKKANIYKGKITRIEPSLEAAFVDYGAERHGFLPLKEIAREYFPANYSAHGRPNIKDVLREGQEVIVQIDKEERGNKGAALTTFISLAGSYLVLMPNNPRAGGISRRIEGDDRTELKEALASLELPEGMGLIVRTAGVGKSAEALQWDLSFRLKHWEAIKKAAESRPAPFLIHQESNVIVRAFRDYLRQDIGEILIDNPKVLELARQHIAALGRPDFSSKIKLYTGEIPLFSHYQIESQIESAFQREVRLPSGGSIVIDSTEALTAIDINSARATRGGDIEETAFNTNLEAADEIARQLRLRDLGGLIVIDFIDMTPVRHQRAVENRLREAVRQDRARIQISHISRFGLLEMSRQRLSPSLGESSHHVCPRCSGTGTVRDNESLSLSILRLIEEEALKENTQEVHAIVPVPIASYLLNEKRSAVNAIETRQDGVRCVIVPNDQMETPHYHVLRVRKGEETPTLSYMLPKLHEEAMALPSEEEFAERKRPEQPALATFAMPDVPPAPTPAEPAAPVVAPSPKAAPATPAAPAQPGLLSRFFGALKALFSGGEETKPTEQPAPKAEAKPERQQDRRKPRQNSRRDRNERRDTRSERTEGSDNREENRRNRRQAQQQTAETRESRQQTEVTEKARTTDEQQAPRRERSRRRNDDKRQAQQEAKALNVEEQSVQETEQEERVRPVQPRRKQRQLNQKVRYEQSVAEEAVVAPVVEETVAAEPIVQEAPAPRTELVKVPLPVVAQTAPEQQEENNADNRDNGGMPRRSRRSPRHLRVSGQRRRRYRDERYPTQSPMPLTVACASPELASGKVWIRYPIVRPQDVQVEEQREQEEVQVQPMVTEVPVAAAIEPVVSAPVIEEVAEVVEAPVQVAEPQPEVVETTHPEVIAAAVTEQPQVITESDVAVAQEVAEHAEPVVEPQEETADIEEVAETAEVVVAEPEVVTQPAAPVVAEVAAEVETVAAVEPEITVEHNHATAPMTRAPAPEYVPEAPRHSDWQRPTFAFEGKGAAGGHTATHHASAAPARPQPVE
RE基因编码序列SEQ ID NO.4
ATGAGGCCCAAGAAGAAGCGGAAGGTGGAGGCTTCCATGAAAAGAATGTTAATCAACGCAACTCAGCAGGAAGAGTTGCGCGTTGCCCTTGTAGATGGGCAGCGTCTGTATGACCTGGATATCGAAAGTCCAGGGCACGAGCAGAAAAAGGCAAACATCTACAAAGGTAAAATCACCCGCATTGAACCGAGTCTGGAAGCTGCTTTTGTTGATTACGGCGCTGAACGTCACGGTTTCCTCCCACTAAAAGAAATTGCCCGCGAATATTTCCCTGCTAACTACAGTGCTCATGGTCGTCCCAACATTAAAGATGTGTTGCGTGAAGGTCAGGAAGTCATTGTTCAGATCGATAAAGAAGAGCGCGGAAACAAAGGCGCGGCATTAACCACCTTTATCAGTCTGGCGGGTAGCTATCTGGTTCTGATGCCGAACAACCCGCGCGCGGGTGGCATTTCTCGCCGTATCGAAGGCGACGACCGTACCGAATTAAAAGAAGCACTGGCAAGCCTTGAACTGCCGGAAGGCATGGGGCTTATCGTGCGCACCGCTGGCGTCGGCAAATCTGCTGAGGCGCTGCAATGGGATTTAAGCTTCCGTCTGAAACACTGGGAAGCCATCAAAAAAGCCGCTGAAAGCCGCCCGGCTCCGTTCCTGATTCATCAGGAGAGCAACGTAATCGTTCGCGCATTCCGCGATTACTTACGTCAGGACATCGGCGAAATCCTTATCGATAACCCGAAAGTGCTCGAACTGGCACGTCAGCATATCGCTGCATTAGGTCGCCCGGATTTCAGCAGCAAAATCAAACTGTACACCGGCGAGATCCCGCTGTTCAGCCACTACCAGATCGAGTCGCAGATCGAATCCGCCTTCCAGCGTGAAGTCCGTCTGCCATCCGGTGGTTCCATTGTTATCGACAGCACCGAAGCGTTAACTGCCATCGACATCAACTCCGCACGCGCGACCCGCGGCGGCGATATCGAAGAAACCGCGTTTAACACCAACCTCGAAGCTGCCGATGAGATTGCTCGTCAGCTGCGCCTGCGTGACCTCGGCGGCCTGATTGTTATCGACTTCATCGACATGACGCCAGTACGCCACCAGCGTGCGGTAGAAAACCGTCTGCGTGAAGCGGTGCGTCAGGACCGTGCGCGTATTCAAATCAGCCATATTTCTCGCTTTGGCCTGCTGGAAATGTCCCGTCAGCGCCTGAGCCCATCACTGGGTGAATCCAGTCATCACGTTTGTCCGCGTTGTTCTGGTACTGGCACCGTGCGTGACAACGAATCGCTGTCGCTCTCTATTCTGCGTCTGATCGAAGAAGAAGCGCTGAAAGAGAACACCCAGGAAGTTCACGCCATTGTTCCTGTGCCAATCGCTTCTTACCTGCTGAATGAAAAACGTTCTGCGGTGAATGCCATTGAAACGCGTCAGGACGGTGTTCGCTGCGTGATTGTGCCAAACGATCAGATGGAAACCCCGCACTACCACGTGCTGCGCGTGCGTAAAGGGGAAGAAACCCCAACCTTAAGCTACATGCTGCCGAAGCTGCATGAAGAAGCGATGGCGCTGCCGTCTGAAGAAGAGTTCGCTGAACGTAAGCGTCCGGAACAACCTGCGCTGGCAACCTTTGCCATGCCGGATGTGCCGCCAGCGCCAACGCCAGCTGAACCTGCCGCGCCTGTCGTAGCTCCATCACCGAAAGCTGCACCGGCAACACCAGCAGCTCCTGCACAACCTGGGCTGTTGAGCCGCTTCTTCGGCGCACTGAAAGCGCTGTTCAGCGGTGGTGAAGAAACCAAACCGACCGAGCAACCAGCACCGAAAGCAGAAGCGAAACCGGAACGTCAACAGGATCGTCGCAAGCCTCGTCAGAACAGCCGCCGTGACCGTAATGAGCGCCGCGACACCCGTAGCGAACGTACTGAAGGCAGCGATAATCGCGAAGAAAACCGTCGTAATCGTCGCCAGGCACAGCAGCAGACTGCCGAGACGCGTGAGAGCCGTCAGCAGACTGAGGTAACGGAAAAAGCGCGTACCACCGACGAGCAGCAAGCGCCGCGTCGTGAACGTAGCCGCCGCCGTAACGATGATAAACGTCAGGCGCAACAAGAAGCGAAGGCGCTGAATGTTGAAGAGCAATCTGTTCAGGAAACCGAACAGGAAGAACGTGTACGTCCGGTTCAGCCGCGTCGTAAACAGCGTCAGCTCAATCAGAAAGTGCGTTACGAGCAAAGCGTAGCCGAAGAAGCGGTAGTCGCACCGGTGGTTGAAGAAACTGTCGCTGCCGAACCAATTGTTCAGGAAGCGCCAGCTCCACGCACAGAACTGGTGAAAGTCCCGCTGCCAGTCGTAGCGCAAACTGCACCAGAACAGCAAGAAGAGAACAATGCCGATAACCGTGACAACGGTGGTATGCCGCGTCGTTCTCGCCGCTCGCCTCGTCACCTGCGCGTGAGTGGTCAGCGTCGTCGTCGCTATCGTGACGAGCGTTATCCAACCCAGTCGCCAATGCCGTTGACCGTAGCGTGCGCGTCTCCGGAACTGGCCTCTGGCAAAGTCTGGATCCGCTATCCAATTGTACGTCCGCAAGATGTACAGGTTGAAGAGCAGCGCGAACAGGAAGAAGTACAAGTGCAGCCGATGGTGACTGAGGTCCCTGTCGCCGCCGCTATCGAACCGGTTGTTAGCGCGCCAGTTATTGAAGAAGTGGCTGAAGTCGTAGAAGCCCCCGTTCAGGTTGCCGAACCGCAACCGGAAGTGGTTGAAACGACGCATCCTGAAGTAATTGCCGCCGCGGTAACTGAACAGCCGCAGGTGATTACCGAGTCTGATGTTGCCGTAGCCCAGGAAGTTGCAGAACACGCAGAACCGGTGGTTGAACCGCAGGAAGAGACGGCAGATATTGAAGAAGTTGCCGAAACTGCTGAGGTTGTGGTTGCTGAACCTGAAGTTGTTACTCAACCTGCCGCGCCAGTCGTCGCTGAAGTCGCAGCAGAAGTTGAAACGGTAGCCGCGGTTGAACCTGAGATCACCGTTGAGCATAACCACGCTACCGCGCCAATGACACGCGCTCCGGCACCGGAATATGTTCCGGAGGCACCACGTCACAGTGACTGGCAGCGCCCTACTTTTGCCTTCGAAGGTAAAGGTGCCGCAGGTGGTCATACGGCAACGCATCATGCCTCTGCCGCTCCTGCGCGTCCGCAACCTGTTGAG
为了说明本发明的有益效果，进行如下实验研究：
一、实验试剂、材料和仪器
1、实验试剂和材料
无内毒素质粒小题中量试剂盒、大肠杆菌DH5α感受态细菌购自北京天根生化科技有限公司。Matrigel、胰蛋白酶、嘌呤霉素购自北京索莱宝科技有限公司。
GXLDNA聚合酶、TaKaRa Z-TaqTM DNA聚合酶、TaKaRa MiniBEST Agarose Gel DNA ExtractionKit Ver.4.0)、pMD18-T载体、Xho I和EcoR I购自宝日医生物技术(北京)有限公司。pLVX-AcGFP-N1购自美国Clontech。DAPI、病毒包装辅助质粒***(pLP1、pLP2、pLP/VSVG)、脂质体2000、DMEM细胞培养基、RPMI-1640细胞培养基购自美国赛默飞世尔。HEK-293FT细胞、HepG2细胞购自中科院上海细胞库。新生牛血清购自杭州四季青生物工程材料有限公司。PVDF膜购自美国Millipore。E-plate16、CIM-Plate 16购自杭州艾森生物有限公司。ECL发光液购自上海天能科技有限公司。抗体购自北京博迈德生物有限公司。Trizol购自美国Invitrogen。荧光定量PCR试剂盒购自美国Bio-Rad。HEK-293FT细胞、Hep G2肝癌细胞购自中科院上海细胞库。
其他常规试剂购自北京索莱宝科技有限公司、天津化学试剂有限公司、国药集团化学试剂有限公司。
引物合成和DNA测序委托Invitrogen广州分公司完成。
2、实验仪器
PCR仪(ABI)、高速冷冻离心机(Centrifuge)、荧光倒置显微镜(Nikon)、细胞CO2培养箱(Thermo)、流式细胞分析仪(Sysmex)、xCELLigence RTCADP分析仪(ACEA)、多功能成像***(Bio-rad)、荧光定量PCR仪(Bio-rad)。
二、实验方法与流程
(一)实验方法
1、细菌基因组抽提
LB细菌培养基培养大肠杆菌DH5α，当OD450大于1.0时，取1.5ml菌液，12000rpm离心5min，弃去上清液，细菌沉淀用天根细胞DNA抽提试剂盒按说明书抽提细菌DNA。
2、引物合成
根据NCBI序列号EU902419，合成RNase E基因(RE)PCR扩增引物，序列如下：
上游引物：
5'-CTCGAGCCACCATGAGGCCCAAGAAGAAGCGGAAGGTGGAGGCTTCCATGAAAAGAATGTTAATCAACGCAACTC-3'
下游引物：
5'-GAATTCCCTCAACAGGTTGCGGACGCGCAGGAG-3'
合成的RNase E截短体基因(REt)PCR扩增引物，序列如下：
上游引物：
5'-CTCGAGCCACCATGAGGCCCAAGAAGAAGCGGAAGGTGGAGGCTTCCATGAAAAGAATGTTAATCAACGCAACTC-3'
下游引物：
5'-CACTGAAAGCGCTGTTCAGCGGTGGTGGAATTC-3'
3、PCR扩增目的基因RE及其截断体REt
利用Takara高保真
GXL DNA聚合酶，按试剂盒操作说明书配制50μl反应液体系添加细菌基因组模板、上下游引物、dNTPs、DNA聚合酶，按预变性(95℃，2min)、扩增(95℃，15s；55℃，30s；72℃，3min；28个循环)进行反应。反应结束后加入0.5μl TaKaRaZ-TaqTM DNA聚合酶，按扩增(95℃，15s；55℃，30s；72℃，3min；4个循环)、稳定(72℃，5min)进行反应。
4、慢病毒表达载体构建
对PCR产物进行1％琼脂糖凝胶电泳，目的条带用试剂盒(TaKaRa MiniBESTAgarose Gel DNA Extraction Kit Ver.4.0)回收，并对纯化产物用pMD18-T载体进行TA克隆。重组质粒导入DH5α感受态细菌中，经氨苄青霉素筛选培养、扩大培养、质粒抽提、Xho I和EcoR I双酶切鉴定后，进行DNA测序。测序正确的重组质粒命名为pMD18-RE、pMD18-REt。
选取慢病毒表达载体pLVX-AcGFP-N1作为骨架载体，用Xho I和EcoR I分别对骨架载体和目的基因进行双酶切，1％琼脂糖凝胶电泳分离和获取目的条带，凝胶纯化后获得线性化骨架载体和目的基因片段，并用T4 DNA ligase 4℃连接过夜，重组DNA质粒导入DH5α感受态细菌中，经氨苄青霉素筛选培养、扩大培养、质粒抽提、Xho I和EcoR I双酶切鉴定，DNA测序鉴定，重组质粒命名为pLVX-RE-AcGFP和pLVX-REt-AcGFP。
5、慢病毒颗粒包装及收集
用无内毒素质粒小提中量试剂盒抽提纯化重组慢病毒表达载体pLVX-RE-AcGFP和pLVX-REt-AcGFP，并分别与病毒包装辅助质粒***(pLP1、pLP2、pLP/VSVG)用脂质体2000共转染HEK-293FT细胞，其转染用量和步骤如表1所示。
表1慢病毒包装各组分用量及具体过程
6、细胞培养、病毒感染、细胞筛选
肝癌细胞Hep G2用含10％新生牛血清的RPMI-1640培养基培养(RPMI-1640完全培养基)，HEK-293FT用DMEM完全培养基培养，细胞融合至85％-90％用0.25％胰酶消化传代。
病毒感染前16h，用0.25％胰酶消化肝癌细胞Hep G2，按5×105细胞铺入6孔板中。病毒感染前更换为1ml RPMI-1640完全培养基，并加入1ml病毒液，轻摇板子混匀，感染后24h更换为2ml RPMI-1640完全培养基。感染后72h，在荧光显微镜下观察细胞荧光。
细胞感染后72h，在细胞培养基中加入终浓度2μg/ml的嘌呤霉素，筛选培养2天后更换为0.5μg/ml的嘌呤霉素维持培养，直至单克隆细胞集落形成。挑取单克隆细胞集落扩大培养，并进行细胞种子冻存。
7、荧光显微镜检
细胞在荧光显微镜下观察绿色荧光，并拍照记录。
8、Western blot
取适量的细胞，用万分之一天平对其定量，然后按40μl上样缓冲液/mg细胞的比例在100℃裂解细胞5min，震荡混匀后瞬时离心。样品制备完成后用5％浓缩胶-10％分离胶进行SDS-PAGE，完成后将蛋白通过湿转法转至PVDF膜上，对印迹膜经5％BSA封闭、一抗孵育、二抗孵育后，用ECL发光液在伯乐凝胶成像***中显色。
9、细胞周期分析
用0.25％胰酶消化细胞，RPMI-1640完全培养基终止细胞消化并收集入离心管，1000rpm/min离心弃去上清，细胞用PBS清洗2次，用预冷的250μl PBS制成细胞悬液，加入预冷的750μl无水乙醇，轻轻混匀，-20℃固定24h，1000rpm/min离心2min，弃去上清，PBS清洗细胞一次，加入2ml细胞周期分析缓冲液，并加入终浓度4μg/ml的DAPI，冰浴30min，用sysmex公司CyFlow Cube8进行测定，并用Denovo FCS Express 6软件分析数据。
10、细胞增殖
用0.25％胰酶消化和收集细胞后，经血球计数板细胞计数后，细胞最终用RPMI-1640完全培养基稀释成20000/ml备用。
先在E-plate16的每个细胞培养孔中加入100μl RPMI-1640完全培养基，并将E-plate16放入xCELLigence RTCA DP多功能实时无标记细胞分析仪(RTCA分析仪)上自检，证实每一个孔电路连接和电阻正常后，取下E-plate16，在对应的孔中加入100μl需要测定的细胞悬液，使每个孔有2000个细胞。将E-plate16放入RTCA分析仪中，按仪器使用说明书设定程序，实时监测细胞生长增殖速度。
11、细胞迁移
用0.25％胰酶消化和收集细胞后，经血球计数板细胞计数后，细胞浓度最终稀释为5×105/ml，取1ml细胞悬液铺入12孔板中，混匀后培养至细胞完全贴壁，用直尺和10μl白枪头在培养孔中划“|”痕迹，PBS洗去漂浮的细胞后，按0h、24h、48h用倒置显微镜记录划痕宽度。
12、细胞侵袭
在冰浴中溶化matrigel，用预冷的枪头和无血清RPMI-1640细胞培养基将其按1:11稀释。
组装CIM-Plate 16(艾森生物)，在上层孔中加入50μl稀释后的基质胶，37℃细胞培养箱中放置4h，弃去未凝固的基质胶液体。
拆分CIM-Plate 16，在下层孔中每孔加入170μl RPMI-1640完全培养基，上层孔中每孔加入60μl无血清RPMI-1640细胞培养基，组装后放入RTCA分析仪中，监测每个孔电路连接和电阻是否正常。
细胞用0.25％胰蛋白酶消化和收集，经细胞计数后定容为5×105/ml，从RTCA分析仪中取出CIM-Plate 16，每孔加入100μl混合均匀的细胞悬液，再次放入RTCA分析仪中，按说明书设计程序，实时监测细胞浸润情况。
13、荧光定量PCR
用Trizol试剂从细胞中抽提总RNA，经反转录PCR合成cDNA。以cDNA为模板，用特异性引物(表2)按三步法[94℃30s,60℃30s,72℃30s；40cycles]进行荧光定量PCR分析。
表2荧光定量PCR引物
三、实验结果
1、成功克隆RE基因及其截短体REt，并构建慢病毒表达载体
以细菌DH5α的基因组为模板，成功克隆细菌RE基因蛋白编码区，并获得截短体编码基因REt。用Xho I和EcoR I双酶切线性化骨架载体pLVX-AcGFP-N1和目的基因，经连接、转化、筛选、扩大培养和鉴定，最后获得慢病毒基因表达载体pLVX-RE-AcGFP(图1)和pLVX-REt-AcGFP(图2)，需要说明的是，在基因RE或者REt编码序列前导入了SV40核定位信号，使RE或者REt表达的蛋白质定位于细胞核内，其测序结果的5'端和3'端如图所示(图3和图4)，图中下划线代表限制性核酸内切酶酶切位点和起始密码子ATG。另外，多次DNA测序结果显示，细菌DH5α来源的RE基因蛋白编码区与NCBI公布的DNA序列具有高度保守性，但并非完全一致，其所编码的蛋白序列也有个别差异，在蛋白质一级结构上，执行RNA水解功能的N端高度保守，而负责与其他蛋白结合的C端保守性相对较弱(图5、图6、图7和图8)，在比对序列中，上一行序列是NCBI基因序列号EU902419公布的序列及其编码蛋白序列，下一行是以实验室DH5α为模板高保真PCR扩增序列及其编码蛋白序列。
2、构建核内表达RE或REt的稳转肝癌细胞株
将重组构建的慢病毒表达载体和病毒包装辅助载体(pLP1、pLP2、pLP/VSVG)通过脂质体共转染法导入HEK-293FT细胞中，包装产生的慢病毒颗粒感染人肝癌细胞Hep G2，感染后72h，通过嘌呤霉素筛选后获得单克隆细胞集落，扩大培养后用荧光显微镜记录。细胞核内表达绿色荧光，证明外源基因可表达并定位于肝癌细胞核内(图9)。由于RE和REt是GFP融合蛋白，用对应的GFP抗体对细胞内蛋白进行免疫印迹分析，结果证明RE和REt的确在细胞内表达(图10)。
3、核内表达REt使肝癌细胞形态发生变化
如图9所示，核内表达RE的肝癌细胞，少数细胞的形态发生改变；但是，当肝癌细胞核内表达REt后，肝癌细胞由原来的梭形和多边形转变成圆形，证明REt能够改变肝癌细胞形态。
4、REt抑制肝癌细胞周期进程
如图11所示，当细胞核内表达RE蛋白后，其细胞周期与对照组细胞之间未发生显著变化，而当细胞核内表达REt后，细胞周期明显发生改变，G2/M期细胞数明显增多，证明REt可以使肝癌细胞周期阻滞在G2/M期。图11中：A，对照细胞；B，核内表达RE蛋白的肝癌细胞；C，核内表达REt蛋白的肝癌细胞。
5、REt抑制肝癌细胞增殖速率
肝细胞核内表达REt蛋白后，肝癌细胞的生长增殖速率显著下降(图12)，而细胞核内表达RE蛋白后，细胞的生长增殖速率与对照细胞无显著差异。
6、REt抑制肝癌细胞迁移速率
肝癌细胞核内表达REt蛋白后，细胞划痕愈合实验证明肝癌细胞的迁移能力显著下降，而表达RE蛋白的肝癌细胞的迁移能力与对照细胞之间无显著差异(图13)。
7、REt抑制肝癌细胞侵袭
肝癌细胞核内表达REt蛋白后，细胞浸润实验证明肝癌细胞的侵袭能力显著下降，而表达RE蛋白的肝癌细胞的侵袭能力与对照细胞之间无显著差异(图14)。
8、REt可降解肝癌细胞内mRNAs及其蛋白表达
肝癌细胞核内表达REt蛋白后，细胞内与细胞周期相关的基因Cyclin A2(CCNA2)及癌基因HuR的mRNAs表达水平及蛋白表达水平显著下降(图15)。图11中：A，细胞内mRNAs表达水平；B，细胞内蛋白质表达水平。
四、实验结论
肝癌细胞Hep G2核内表达REt使其形态改变、细胞周期阻滞在G2/M期、细胞生长增殖能力下降、细胞迁移和侵袭能力下降，证明REt具有抑制癌细胞生长增殖和迁移侵袭的能力。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。
序列表
<110> 贵州医科大学
<120> 一种细菌RNase E截短体及其应用
<160> 4
<210> 1
<211> 597
<212> DNA
<213> REt蛋白序列
<400> 1
MRPKKKRKVEASMKRMLINATQQEELRVALVDGQRLYDLDIESPGHEQKKANIYKGKITR 60
IEPSLEAAFVDYGAERHGFLPLKEIAREYFPANYSAHGRPNIKDVLREGQEVIVQIDKEE 120
RGNKGAALTTFISLAGSYLVLMPNNPRAGGISRRIEGDDRTELKEALASLELPEGMGLIV 180
RTAGVGKSAEALQWDLSFRLKHWEAIKKAAESRPAPFLIHQESNVIVRAFRDYLRQDIGE 240
ILIDNPKVLELARQHIAALGRPDFSSKIKLYTGEIPLFSHYQIESQIESAFQREVRLPSG 300
GSIVIDSTEALTAIDINSARATRGGDIEETAFNTNLEAADEIARQLRLRDLGGLIVIDFI 360
DMTPVRHQRAVENRLREAVRQDRARIQISHISRFGLLEMSRQRLSPSLGESSHHVCPRCS 420
GTGTVRDNESLSLSILRLIEEEALKENTQEVHAIVPVPIASYLLNEKRSAVNAIETRQDG 480
VRCVIVPNDQMETPHYHVLRVRKGEETPTLSYMLPKLHEEAMALPSEEEFAERKRPEQPA 540
LATFAMPDVPPAPTPAEPAAPVVAPSPKAAPATPAAPAQPGLLSRFFGALKALFSGG 597
<210> 2
<211> 1791
<212> DNA
<213> REt基因编码序列
<400> 2
ATGAGGCCCAAGAAGAAGCGGAAGGTGGAGGCTTCCATGAAAAGAATGTTAATCAACGCA 60
ACTCAGCAGGAAGAGTTGCGCGTTGCCCTTGTAGATGGGCAGCGTCTGTATGACCTGGAT 120
ATCGAAAGTCCAGGGCACGAGCAGAAAAAGGCAAACATCTACAAAGGTAAAATCACCCGC 180
ATTGAACCGAGTCTGGAAGCTGCTTTTGTTGATTACGGCGCTGAACGTCACGGTTTCCTC 240
CCACTAAAAGAAATTGCCCGCGAATATTTCCCTGCTAACTACAGTGCTCATGGTCGTCCC 300
AACATTAAAGATGTGTTGCGTGAAGGTCAGGAAGTCATTGTTCAGATCGATAAAGAAGAG 360
CGCGGAAACAAAGGCGCGGCATTAACCACCTTTATCAGTCTGGCGGGTAGCTATCTGGTT 420
CTGATGCCGAACAACCCGCGCGCGGGTGGCATTTCTCGCCGTATCGAAGGCGACGACCGT 480
ACCGAATTAAAAGAAGCACTGGCAAGCCTTGAACTGCCGGAAGGCATGGGGCTTATCGTG 540
CGCACCGCTGGCGTCGGCAAATCTGCTGAGGCGCTGCAATGGGATTTAAGCTTCCGTCTG 600
AAACACTGGGAAGCCATCAAAAAAGCCGCTGAAAGCCGCCCGGCTCCGTTCCTGATTCAT 660
CAGGAGAGCAACGTAATCGTTCGCGCATTCCGCGATTACTTACGTCAGGACATCGGCGAA 720
ATCCTTATCGATAACCCGAAAGTGCTCGAACTGGCACGTCAGCATATCGCTGCATTAGGT 780
CGCCCGGATTTCAGCAGCAAAATCAAACTGTACACCGGCGAGATCCCGCTGTTCAGCCAC 840
TACCAGATCGAGTCGCAGATCGAATCCGCCTTCCAGCGTGAAGTCCGTCTGCCATCCGGT 900
GGTTCCATTGTTATCGACAGCACCGAAGCGTTAACTGCCATCGACATCAACTCCGCACGC 960
GCGACCCGCGGCGGCGATATCGAAGAAACCGCGTTTAACACCAACCTCGAAGCTGCCGAT 1020
GAGATTGCTCGTCAGCTGCGCCTGCGTGACCTCGGCGGCCTGATTGTTATCGACTTCATC 1080
GACATGACGCCAGTACGCCACCAGCGTGCGGTAGAAAACCGTCTGCGTGAAGCGGTGCGT 1140
CAGGACCGTGCGCGTATTCAAATCAGCCATATTTCTCGCTTTGGCCTGCTGGAAATGTCC 1200
CGTCAGCGCCTGAGCCCATCACTGGGTGAATCCAGTCATCACGTTTGTCCGCGTTGTTCT 1260
GGTACTGGCACCGTGCGTGACAACGAATCGCTGTCGCTCTCTATTCTGCGTCTGATCGAA 1320
GAAGAAGCGCTGAAAGAGAACACCCAGGAAGTTCACGCCATTGTTCCTGTGCCAATCGCT 1380
TCTTACCTGCTGAATGAAAAACGTTCTGCGGTGAATGCCATTGAAACGCGTCAGGACGGT 1440
GTTCGCTGCGTGATTGTGCCAAACGATCAGATGGAAACCCCGCACTACCACGTGCTGCGC 1500
GTGCGTAAAGGGGAAGAAACCCCAACCTTAAGCTACATGCTGCCGAAGCTGCATGAAGAA 1560
GCGATGGCGCTGCCGTCTGAAGAAGAGTTCGCTGAACGTAAGCGTCCGGAACAACCTGCG 1620
CTGGCAACCTTTGCCATGCCGGATGTGCCGCCAGCGCCAACGCCAGCTGAACCTGCCGCG 1680
CCTGTCGTAGCTCCATCACCAAAAGCTGCACCGGCAACACCAGCAGCTCCTGCACAACCT 1740
GGGCTGTTGAGCCGCTTCTTCGGCGCACTGAAAGCGCTGTTCAGCGGTGGT 1791
<210> 3
<211> 1073
<212> DNA
<213> RE蛋白序列
<400> 3
MRPKKKRKVEASMKRMLINATQQEELRVALVDGQRLYDLDIESPGHEQKKANIYKGKITR 60
IEPSLEAAFVDYGAERHGFLPLKEIAREYFPANYSAHGRPNIKDVLREGQEVIVQIDKEE 120
RGNKGAALTTFISLAGSYLVLMPNNPRAGGISRRIEGDDRTELKEALASLELPEGMGLIV 180
RTAGVGKSAEALQWDLSFRLKHWEAIKKAAESRPAPFLIHQESNVIVRAFRDYLRQDIGE 240
ILIDNPKVLELARQHIAALGRPDFSSKIKLYTGEIPLFSHYQIESQIESAFQREVRLPSG 300
GSIVIDSTEALTAIDINSARATRGGDIEETAFNTNLEAADEIARQLRLRDLGGLIVIDFI 360
DMTPVRHQRAVENRLREAVRQDRARIQISHISRFGLLEMSRQRLSPSLGESSHHVCPRCS 420
GTGTVRDNESLSLSILRLIEEEALKENTQEVHAIVPVPIASYLLNEKRSAVNAIETRQDG 480
VRCVIVPNDQMETPHYHVLRVRKGEETPTLSYMLPKLHEEAMALPSEEEFAERKRPEQPA 540
LATFAMPDVPPAPTPAEPAAPVVAPSPKAAPATPAAPAQPGLLSRFFGALKALFSGGEET 600
KPTEQPAPKAEAKPERQQDRRKPRQNSRRDRNERRDTRSERTEGSDNREENRRNRRQAQQ 660
QTAETRESRQQTEVTEKARTTDEQQAPRRERSRRRNDDKRQAQQEAKALNVEEQSVQETE 720
QEERVRPVQPRRKQRQLNQKVRYEQSVAEEAVVAPVVEETVAAEPIVQEAPAPRTELVKV 780
PLPVVAQTAPEQQEENNADNRDNGGMPRRSRRSPRHLRVSGQRRRRYRDERYPTQSPMPL 840
TVACASPELASGKVWIRYPIVRPQDVQVEEQREQEEVQVQPMVTEVPVAAAIEPVVSAPV 900
IEEVAEVVEAPVQVAEPQPEVVETTHPEVIAAAVTEQPQVITESDVAVAQEVAEHAEPVV 960
EPQEETADIEEVAETAEVVVAEPEVVTQPAAPVVAEVAAEVETVAAVEPEITVEHNHATA 1020
PMTRAPAPEYVPEAPRHSDWQRPTFAFEGKGAAGGHTATHHASAAPARPQPVE 1073
<210> 4
<211> 3219
<212> DNA
<213> RE基因编码序列
<400> 4
ATGAGGCCCAAGAAGAAGCGGAAGGTGGAGGCTTCCATGAAAAGAATGTTAATCAACGCA 60
ACTCAGCAGGAAGAGTTGCGCGTTGCCCTTGTAGATGGGCAGCGTCTGTATGACCTGGAT 120
ATCGAAAGTCCAGGGCACGAGCAGAAAAAGGCAAACATCTACAAAGGTAAAATCACCCGC 180
ATTGAACCGAGTCTGGAAGCTGCTTTTGTTGATTACGGCGCTGAACGTCACGGTTTCCTC 240
CCACTAAAAGAAATTGCCCGCGAATATTTCCCTGCTAACTACAGTGCTCATGGTCGTCCC 300
AACATTAAAGATGTGTTGCGTGAAGGTCAGGAAGTCATTGTTCAGATCGATAAAGAAGAG 360
CGCGGAAACAAAGGCGCGGCATTAACCACCTTTATCAGTCTGGCGGGTAGCTATCTGGTT 420
CTGATGCCGAACAACCCGCGCGCGGGTGGCATTTCTCGCCGTATCGAAGGCGACGACCGT 480
ACCGAATTAAAAGAAGCACTGGCAAGCCTTGAACTGCCGGAAGGCATGGGGCTTATCGTG 540
CGCACCGCTGGCGTCGGCAAATCTGCTGAGGCGCTGCAATGGGATTTAAGCTTCCGTCTG 600
AAACACTGGGAAGCCATCAAAAAAGCCGCTGAAAGCCGCCCGGCTCCGTTCCTGATTCAT 660
CAGGAGAGCAACGTAATCGTTCGCGCATTCCGCGATTACTTACGTCAGGACATCGGCGAA 720
ATCCTTATCGATAACCCGAAAGTGCTCGAACTGGCACGTCAGCATATCGCTGCATTAGGT 780
CGCCCGGATTTCAGCAGCAAAATCAAACTGTACACCGGCGAGATCCCGCTGTTCAGCCAC 840
TACCAGATCGAGTCGCAGATCGAATCCGCCTTCCAGCGTGAAGTCCGTCTGCCATCCGGT 900
GGTTCCATTGTTATCGACAGCACCGAAGCGTTAACTGCCATCGACATCAACTCCGCACGC 960
GCGACCCGCGGCGGCGATATCGAAGAAACCGCGTTTAACACCAACCTCGAAGCTGCCGAT 1020
GAGATTGCTCGTCAGCTGCGCCTGCGTGACCTCGGCGGCCTGATTGTTATCGACTTCATC 1080
GACATGACGCCAGTACGCCACCAGCGTGCGGTAGAAAACCGTCTGCGTGAAGCGGTGCGT 1140
CAGGACCGTGCGCGTATTCAAATCAGCCATATTTCTCGCTTTGGCCTGCTGGAAATGTCC 1200
CGTCAGCGCCTGAGCCCATCACTGGGTGAATCCAGTCATCACGTTTGTCCGCGTTGTTCT 1260
GGTACTGGCACCGTGCGTGACAACGAATCGCTGTCGCTCTCTATTCTGCGTCTGATCGAA 1320
GAAGAAGCGCTGAAAGAGAACACCCAGGAAGTTCACGCCATTGTTCCTGTGCCAATCGCT 1380
TCTTACCTGCTGAATGAAAAACGTTCTGCGGTGAATGCCATTGAAACGCGTCAGGACGGT 1440
GTTCGCTGCGTGATTGTGCCAAACGATCAGATGGAAACCCCGCACTACCACGTGCTGCGC 1500
GTGCGTAAAGGGGAAGAAACCCCAACCTTAAGCTACATGCTGCCGAAGCTGCATGAAGAA 1560
GCGATGGCGCTGCCGTCTGAAGAAGAGTTCGCTGAACGTAAGCGTCCGGAACAACCTGCG 1620
CTGGCAACCTTTGCCATGCCGGATGTGCCGCCAGCGCCAACGCCAGCTGAACCTGCCGCG 1680
CCTGTCGTAGCTCCATCACCGAAAGCTGCACCGGCAACACCAGCAGCTCCTGCACAACCT 1740
GGGCTGTTGAGCCGCTTCTTCGGCGCACTGAAAGCGCTGTTCAGCGGTGGTGAAGAAACC 1800
AAACCGACCGAGCAACCAGCACCGAAAGCAGAAGCGAAACCGGAACGTCAACAGGATCGT 1860
CGCAAGCCTCGTCAGAACAGCCGCCGTGACCGTAATGAGCGCCGCGACACCCGTAGCGAA 1920
CGTACTGAAGGCAGCGATAATCGCGAAGAAAACCGTCGTAATCGTCGCCAGGCACAGCAG 1980
CAGACTGCCGAGACGCGTGAGAGCCGTCAGCAGACTGAGGTAACGGAAAAAGCGCGTACC 2040
ACCGACGAGCAGCAAGCGCCGCGTCGTGAACGTAGCCGCCGCCGTAACGATGATAAACGT 2100
CAGGCGCAACAAGAAGCGAAGGCGCTGAATGTTGAAGAGCAATCTGTTCAGGAAACCGAA 2160
CAGGAAGAACGTGTACGTCCGGTTCAGCCGCGTCGTAAACAGCGTCAGCTCAATCAGAAA 2220
GTGCGTTACGAGCAAAGCGTAGCCGAAGAAGCGGTAGTCGCACCGGTGGTTGAAGAAACT 2280
GTCGCTGCCGAACCAATTGTTCAGGAAGCGCCAGCTCCACGCACAGAACTGGTGAAAGTC 2340
CCGCTGCCAGTCGTAGCGCAAACTGCACCAGAACAGCAAGAAGAGAACAATGCCGATAAC 2400
CGTGACAACGGTGGTATGCCGCGTCGTTCTCGCCGCTCGCCTCGTCACCTGCGCGTGAGT 2460
GGTCAGCGTCGTCGTCGCTATCGTGACGAGCGTTATCCAACCCAGTCGCCAATGCCGTTG 2520
ACCGTAGCGTGCGCGTCTCCGGAACTGGCCTCTGGCAAAGTCTGGATCCGCTATCCAATT 2580
GTACGTCCGCAAGATGTACAGGTTGAAGAGCAGCGCGAACAGGAAGAAGTACAAGTGCAG 2640
CCGATGGTGACTGAGGTCCCTGTCGCCGCCGCTATCGAACCGGTTGTTAGCGCGCCAGTT 2700
ATTGAAGAAGTGGCTGAAGTCGTAGAAGCCCCCGTTCAGGTTGCCGAACCGCAACCGGAA 2760
GTGGTTGAAACGACGCATCCTGAAGTAATTGCCGCCGCGGTAACTGAACAGCCGCAGGTG 2820
ATTACCGAGTCTGATGTTGCCGTAGCCCAGGAAGTTGCAGAACACGCAGAACCGGTGGTT 2880
GAACCGCAGGAAGAGACGGCAGATATTGAAGAAGTTGCCGAAACTGCTGAGGTTGTGGTT 2940
GCTGAACCTGAAGTTGTTACTCAACCTGCCGCGCCAGTCGTCGCTGAAGTCGCAGCAGAA 3000
GTTGAAACGGTAGCCGCGGTTGAACCTGAGATCACCGTTGAGCATAACCACGCTACCGCG 3060
CCAATGACACGCGCTCCGGCACCGGAATATGTTCCGGAGGCACCACGTCACAGTGACTGG 3120
CAGCGCCCTACTTTTGCCTTCGAAGGTAAAGGTGCCGCAGGTGGTCATACGGCAACGCAT 3180
CATGCCTCTGCCGCTCCTGCGCGTCCGCAACCTGTTGAG 3219
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1.一种细菌RNase E截短体在制备抑制肝癌细胞药物中的应用，其特征在于：细菌RNase E截短体的蛋白序列如SEQ ID NO.1所示。
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