CN107937428A - 一种整合microRNA和CAR功能的载体构建方法 - Google Patents
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Abstract
本发明涉及一种整合microRNA和CAR功能的载体构建方法，能在表达microRNA(以hsa‑miR‑301a为例)模拟物或阻遏物的同时表达CD19‑CAR的基于pFIV载体的pFIV‑microRNA(miR‑301a)‑CD19CAR系统；通过引入microRNA模拟物或阻遏物到携带有CAR序列的载体中，实现同时表达microRNA基因调控功能和肿瘤特异性抗原CD19的CAR，从而实现双重对T细胞的基因编辑和修饰功能，从而制备出新一代的具有更高安全性和有效性的CAR，更加适用于临床研究。
Description
技术领域
本发明涉及pFIV-miR-301a-CD19CAR系统构建技术，特别是涉及一种共同表达hsa-miR-301a模拟物或阻遏物，和表达CD19-CAR的基于pFIV载体的pFIV-miR-301a-CD19-CAR系统构建方法。
背景技术
嵌合抗原受体技术(Chimeric Antigen Receptor，CAR)是利用基因编辑技术，使T细胞表达肿瘤特异性嵌合抗原受体，以非主要组织相容性复合体(MHC)限制的方式结合肿瘤抗原，对肿瘤起到杀伤作用。CAR的结构主要包括四部分：细胞外的抗体单链可变区(可特异性结合肿瘤相关抗原)，胞外铰链区，跨膜结合区和活化T细胞的胞内信号区。第I代CAR介导的T细胞激活是通过CD3ζ链或FcRγ的酪氨酸激活基序完成，但是第I代CAR无法完全激活T细胞，也难以维持T细胞的持久增殖及细胞杀伤作用。第II代CAR增加了共刺激结构分子如CD28或CD137(4-1BB)，引入CD28信号肽可以引起T细胞的增殖，提高IL-2和IFN-γ的水平，而引入CD137信号肽使得T细胞的扩增及杀伤肿瘤活性更持久。第III代CAR除了CD3ζ和CD28两种信号分子，还增加了多种不同的信号分子结构来调节T细胞增殖和杀伤毒性等，如经典的CD28和4-1BB联合使用，以及包含共刺激结构分子OX40(TNFRSF4)和ICOS的联合使用，都可以增加体内肿瘤细胞消退。迄今为止，引入共刺激分子信号的第二代和第三代CAR，提高了T细胞的细胞毒性、体内增殖活性、持续存活时间和增加细胞因子释放等功能。与普通的免疫细胞治疗相比，CAR-T技术具有许多独特的优势，如CAR能非MHC依赖性识别肿瘤抗原，不需要经过APC。肿瘤表面蛋白类和脂类抗原都可以作为靶点；有共刺激分子，能有效增强T细胞增殖。因此该技术被认为是有望彻底攻克人类癌症的独特方法。
目前，CAR-T免疫治疗已在许多临床实验上取得巨大进展。其中，针对B相关抗原CD19阳性的B细胞恶性肿瘤，如急性B-淋巴细胞白血病(B-ALL)，慢性B-淋巴细胞白血病(B-CLL)，B细胞霍奇金氏淋巴瘤(B-HL)和非霍奇金氏淋巴瘤(B-NHL)，CAR-T-CD19T细胞免疫治疗疗效显著，已有报告显示CAR-T-CD19治疗在成人和儿童复发和难治性B-ALL中可以获得高达90％的完全缓解率(Completeremission，CR)。研究报告也显示，几种用不同结构设计的CAR-CD19的临床试验治疗都显示了相似的良好疗效。有些患者经CAR-T治疗后还获得了持久性缓解，不需要额外的其他治疗。虽然CAR-T细胞免疫疗法在多种恶性肿瘤的治疗中取得了很好的疗效，但在治愈肿瘤患者的同时也带来诸多不良反应，如CAR-T细胞在清除肿瘤的同时也会攻击正常组织，治疗同时会产生大量细胞因子导致患者多器官功能衰竭等，临床已有致死病例的报道。目前，CAR-T细胞免疫治疗还处于发展阶段，在血液系统恶性肿瘤和实体瘤的治疗中还有很大的发展空间，对CAR结构进行优化改进，在提升CAR-T细胞抗肿瘤能力的同时减少其不良反应，将为CAR-T细胞免疫治疗的大规模临床应用提供新的思路和方向。
miRNA是真核生物中具有调节功能的非编码RNA，其长度约为21-25个碱基。成熟的miRNA通过碱基互补配对的方式识别靶分子mRNA 3’端非编码区(3’-UTR)上的特异性序列，导致mRNA降解或者翻译阻遏，从而在翻译水平实现对基因表达的调控，并参与调控多种疾病的发生、发展过程。miRNA在固有免疫和适应性免疫细胞中可通过特定的表达谱调节细胞的分化和功能，从而参与机体促肿瘤和抗肿瘤免疫的调节，如参与T细胞调节的miR-17-92家族，miR-155和miR-181a等，参与巨噬细胞调节的miR-125b，miR-146和miR-155等，以及与损伤分子模式相关的miR-34c和miR-214。因此，将miRNA表达调控和CAR的功能联系在一起，有如下优势：(1)与传统的T细胞共刺激分子活性结构域相比，miRNA分子小(只有21-25个碱基)，miRNA和CAR的整合不会导致因慢病毒质粒过大而产生的CAR表达减低及其脱靶效应；(2)利用多个miRNA可以靶向多个T细胞激活或抑制性转录因子或受体，设计不同miRNA的组合可以最大限度激活T细胞杀伤肿瘤功能；(3)CAR结构中引入microRNA功能可以反向调控细胞因子风暴，对设计合成具有更安全的、有效的新一代整合miRNA的CAR，将有助于我们设计更有效、安全的新一代CAR技术，制备标准化的CAR-T细胞，为临床T细胞免疫治疗提供新的思路和策略。
发明内容
本发明的目的是提供一种整合microRNA和CAR功能的载体构建方法，能在表达microRNA(以hsa-miR-301a为例)模拟物或阻遏物的同时表达CD19-CAR的基于pFIV载体的pFIV-microRNA(miR-301a)-CD19CAR系统；通过引入microRNA模拟物或阻遏物到携带有CAR序列的载体中，实现同时表达microRNA基因调控功能和肿瘤特异性抗原CD19的CAR，从而实现双重对T细胞的基因编辑和修饰功能，从而制备出新一代的具有更高安全性和有效性的CAR，更加适用于临床研究。
本发明的上述技术目的是通过以下技术方案得以实现的：
(1)查找miRBase数据中hsa-miR-301a(Accession ID：MIMAT0000688)的序列，通过优化设计并合成hsa-miR-301a的模拟物和阻遏物；构建含有microRNA(以hsa-miR-301a为例)模拟物(pre-miR-301a)和阻遏物(sponge-miR-301a)的载体，并由H1启动子驱动。其中pre-miR-301a促进hsa-miR-301a表达升高，sponge-miR-301a抑制has-miR-301a表达，其基因序列如SEQ ID NO.1和SEQ ID NO.2
(2)CAR(以CD19-CAR为例)由EF1α驱动并包括：CD19的单链抗体可变区(singlechain variable fragment,scFv)基因片段序列，CD8来源的连接和穿膜片段序列，传递信号的效应分子CD3ζ序列和胞内信号分子CD28和4-1BB片段序列组成。
(3)根据(1)和(2)的序列，采用合成、酶切、连接和转化的方法，分别将hsa-miR-301a(模拟物或阻遏物)和CD19-CAR结构序列连接到含有H1和EF1α的第三代FIV载体上(命名为：pFIV-miR-301a-CD19CAR)，
(4)将连接产物转入大肠杆菌感受态DH5α中进行大量扩增并提取质粒。采用质粒载体引物进行PCR和测序验证连接序列的正确性。其中miR-301a模拟物或阻遏物测序引物序列为：5’-TGTCTTTGGATTTGGGAATCTTAT-3’；CD19-CAR测序引物为：5’-ATTTATTGTATCTGTGGGAGCCTC-3’
(5)共转染质粒pFIV-miR-301a-CD19CAR和FIV慢病毒包装质粒(pFIV-34N和pVSV-G质粒)于293T细胞，收集293T细胞上清，并制备慢病毒颗粒，感染Jurkat细胞。
(6)采用荧光定量PCR检测miR-301a表达，流式细胞技术检测myc-tag标签来显示CD19-CAR在细胞膜的表达水平。
本发明的有益效果是：
整合microRNA调控功能于CAR结构上，通过表达microRNA模拟物或阻遏物来促进CAR编辑T细胞的功能，制备新一代的具有更高安全性和有效性的CAR，更加适用于临床研究。
附图说明
图1为本发明构建后的基因时序图，见SEQ ID NO.3的信息；
图2为本发明构建hsa-miR-301a模拟物(pre-miR-301a)和阻遏物(sponge-miR-301a)后PCR鉴定克隆电泳图；其中，pre-miR-301a片段为274bp，sponge-miR-301a为230，control为176bp；
图3a为本发明构建hsa-miR-301a模拟物(pre-miR-301a)的序列验证图，图3b为和构建阻遏物(sponge-miR-301a)的序列验证图，见SEQ ID NO.1和SEQ ID NO.2的信息；测序验证pre-miR-301a请详见图3a中阴影部分，测序验证sponge-miR-301a请详见图3b中阴影部分；
图4为本发明构建CD19-CAR后的部分序列验证图，见SEQ ID NO.4的信息；
图5为本发明转染Jurkat细胞后，miR-301a表达的验证图；
图6为本发明转染Jurkat细胞后，CD19-CAR表达的验证图。
具体实施方式
下面结合附图和具体的实施例对本发明的技术方案做进一步介绍。
本实施例是一种共同表达hsa-miR-301a模拟物或阻遏物，和表达CD19-CAR的基于pFIV载体的pFIV-miR-301a-CD19CAR系统构建方法，其步骤为：
(1)查找miRBase数据中hsa-miR-301a(Accession ID：MIMAT0000688)的序列，通过设计优化并合成hsa-miR-301a的模拟物和阻遏物；其中pre-miR-301a促进hsa-miR-301a表达升高，sponge-miR-301a抑制hsa-miR-301a表达，其设计优化基因序列如SEQ ID NO.1和SEQ ID NO.2。
(2)合成的hsa-miR-301a的模拟物(阻遏物)两条单链的磷酸化和退火延伸，反应体系为：hsa-miR-301a的模拟物(阻遏物)单链1，hsa-miR-301a的模拟物(阻遏物)单链2，T4PolynucleotideKinase,10mM ATP。反应条件为：37℃30分钟，95℃2分钟，放置室温。
(3)BamHI和EcoRI酶切pFIV质粒，使用Qiagen PCRpurification试剂盒纯化线性质粒，T4DNA连接酶将hsa-miR-301a的模拟物(阻遏物)与线性pFIV质粒连接，并转化大肠杆菌感受态DH5α，提取质粒。
(4)设计合成由EF1α驱动的CD19-CAR，主要包括：CD19的单链抗体可变区scFv基因片段序列，CD8来源的连接和穿膜片段序列，传递信号的效应分子CD3ζ序列和胞内信号分子CD28和4-1BB片段序列组成，其设计优化基因序列如SEQ ID NO.3。使用XbaI和SalI从pUC57质粒上切下CD19-CAR，并经T4DNA连接酶连接到含有has-miR-301a的模拟物(阻遏物)的pFIV载体上，转化大肠杆菌感受态DH5α，提取质粒。
(5)构建后的基因时序图如图1所示，采用质粒载体引物进行PCR和测序验证连接序列的正确性；其中hsa-miR-301a模拟物或阻遏物测序引物序列为：5’-TGTCTTTGGATTTGGGAATCTTAT-3’；CD19-CAR测序引物为：5’-ATTTATTGTATCTGTGGGAGCCTC-3’。
(6)使用转染试剂lipofectamine3000，共转染质粒pFIV-miR-301a-CD19CAR和FIV慢病毒包装质粒(pFIV-34N和pVSV-G质粒)于293T细胞，收集293T细胞上清，并制备慢病毒颗粒，感染Jurkat细胞。
(7)慢病毒感染Jurkat细胞48小时后，收集细胞，提取RNA，采用荧光定量PCR检测miR-301a表达；收集细胞，清洗后，myc-tag荧光抗体标记，流式细胞技术检测myc-tag标签来显示CD19-CAR在细胞膜的表达水平。
实验证明基因序列与所设计合成的基因序列(图1)完全一致；图2是插入后挑选阳性克隆进行PCR鉴定图；图3为测序验证的hsa-miR-301a的模拟物(阻遏物)基因序列；图4是测序验证的CD19-CAR的基因序列；图5为本发明转染Jurkat细胞后，miR-301a表达的验证图；图6为本发明转染Jurkat细胞后，CD19-CAR表达的验证图。
实验中，引物、寡核苷酸和全基因序列合成由广州天一辉远基因科技有限公司合成；各种限制性内切酶、T4Polynucleotide Kinase和T4DNA连接酶均购自NEB公司；质粒提取和PCR纯化试剂盒购自Qiagen；转染试剂lipofectamine3000和RPIM1640培养基购自Invitrogen公司；RNA抽提试剂盒和hsa-miR-301a Taqman荧光定量PCR试剂盒购自ThermoFisher；myc-tag荧光抗体购自Cell SignalingTechnology公司。
序列表
<110> 马晓冬
<120> 一种整合microRNA和CAR功能的载体构建方法
<141> 2017-11-28
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 98
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
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gcaatagtat tgtcaaagca tctgaaagca ggtttttg 98
<210> 2
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gatccgcttt gacaatcggt tgcactgaat tgctttgaca atcggttgca ctgg 54
<210> 3
<211> 8892
<212> DNA
<213> 人工序列(Artificial Sequence)
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gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac 180
tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa 240
aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt 300
aggcgtgtac ggtgggaggt ctatataagc agagcttgtg aaacttcgag gagtctcttt 360
gttgaggact tttgagttct cccttgaggc tcccacagat acaataaata tttgagattg 420
aaccctgtcg agtatctgtg taatcttttt tacctgtgag gtctcggaat ccgggccgag 480
aacttcgcag ttggcgcccg aacagggact tgattgagag tgattgagga agtgaagcta 540
gagcaataga aagctgttaa gcagaactcc tgctgaccta aatagggaag cagtagcaga 600
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taaaggccca gataaattac atctggtgac tcttcgcgga ccttcaagcc aggagattcg 720
ccgagggaca gtcaacaagg taggagagat tctacagcaa catggggaat ggacaggggc 780
gagattggaa aatggccatt aagagatgta gtaatgttgc tgtaggagta ggggggaaga 840
gtaaaaaatt tggagaaggg aatttcagat gggccattag aatggctaat gtatctacag 900
gacgagaacc tggtgatata ccagagactt tagatcaact aaggttggtt atttgcgatt 960
tacaagaaag aagagaaaaa tttggatcta gcaaagaaat tgatatggca attcctgcat 1020
tgaggagaaa tggtaggcaa tgtggcatgt ctgaaaaaga ggaggaatga tgaagtatct 1080
cagacttatt ttataaggga gatactgtgc tgagttcttc cctttgagga aggtatgtca 1140
tatcctagac atagtctcaa ttttaaaaga agaggtagga taggagggat ggccccttat 1200
gaattattag cacaacaaga atccttaaga atacaagatt atttttctgc aataccacaa 1260
aaattgcaag cacagtggat ttattataaa gatcaaaaag ataagaaatg gaaaggacca 1320
atgagagtag aatactgggg acagggatca gtattattaa aggatgaaga gaagggatat 1380
tttcttataa tcggtactag tgctccggtg cccgtcagtg ggcagagcgc acatcgccca 1440
cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc 1500
gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 1560
gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 1620
ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg 1680
gcccttgcgt gccttgaatt acttccacgc ccctggctgc agtacgtgat tcttgatccc 1740
gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg 1800
cctcgtctgc ttgagttgag gcctggcttg ggcgctgggg ccgccgcgtg cgaatctggt 1860
ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat 1920
gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc 1980
acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca 2040
catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc 2100
aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg 2160
cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc 2220
ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac 2280
ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt 2340
accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag 2400
gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag 2460
ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat 2520
cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt 2580
cgtgatctag agccaccatg ctgctgctgg tcacttctct gctgctgtgc gaactgcccc 2640
accccgcctt tctgctgatt cccgacatcc agatgacaca gactacatcc tccctgtctg 2700
cctctctggg agacagagtc accatcagtt gcagggcaag tcaggacatt agtaaatatt 2760
taaattggta tcagcagaaa ccagatggaa ctgttaaact cctgatctac catacatcaa 2820
gattacactc aggagtccca tcaaggttca gtggcagtgg gtctggaaca gattattctc 2880
tcaccattag caacctggag caagaagata ttgccactta cttttgccaa cagggtaata 2940
cgcttccgta cacgttcgga ggggggacca agctggagat cacaggtggc ggtggctcgg 3000
gcggtggtgg gtcgggtggc ggcggatctg aggtgaaact gcaggagtca ggacctggcc 3060
tggtggcgcc ctcacagagc ctgtccgtca catgcactgt ctcaggggtc tcattacccg 3120
actatggtgt aagctggatt cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa 3180
tatggggtag tgaaaccaca tactataatt cagctctcaa atccagactg accatcatca 3240
aggacaactc caagagccaa gttttcttaa aaatgaacag tctgcaaact gatgacacag 3300
ccatttacta ctgtgccaaa cattattact acggtggtag ctatgctatg gactactggg 3360
gccaaggaac ctcagtcacc gtctcctcag aacagaaact gatttccgag gaagatctgt 3420
tcgtccccgt gttcctgcct gccaagccaa caactacccc tgctccacga ccacctactc 3480
cagcacctac catcgcaagt cagcccctgt cactgcgacc tgaggcttgc cggccagcag 3540
ctggaggagc agtgcacacc cgaggcctgg acttcgcatg cgatatctac atttgggcac 3600
cactggctgg aacctgtggg gtcctgctgc tgagcctggt catcaccctg tattgtaacc 3660
acagaaatag gagcaaacgc tcccgactgc tgcattccga ctacatgaac atgacacctc 3720
ggagaccagg ccccactaga aagcattacc agccatatgc cccacccagg gatttcgcag 3780
cctatcggag ccggttcagc gtcgtgaaaa gggggcgcaa gaaactgctg tacatcttca 3840
agcagccttt tatgcgccca gtgcagacaa ctcaggagga agacggatgc tcttgtcggt 3900
tcccagagga ggaggaagga ggctgcgagc tgagagtgaa gttcagccgg agcgccgatg 3960
caccagcata tcagcaggga cagaatcagc tgtacaacga gctgaatctg ggcaggcgcg 4020
aggaatatga cgtgctggat aagcgacgag gacgggaccc cgaaatggga ggaaaaccca 4080
gaaggaagaa ccctcaggag gggctgtata atgaactgca gaaagacaag atggctgagg 4140
catacagcga aattggaatg aaaggagagc gccgacgggg gaagggacac gatgggctgt 4200
accagggact gtcaaccgcc actaaagata cctacgacgc actgcacatg caggctctgc 4260
ccccaagaga attcgaagga tccgcggccg ctgagggcag aggaagtctt ctaacatgcg 4320
gtgacgtgga ggagaatccc ggcccttccg ggatgaccga gtacaagccc acggtgcgcc 4380
tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact 4440
accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc 4500
aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg 4560
gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg 4620
agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg 4680
aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg 4740
tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg 4800
cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct 4860
tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca 4920
cctggtgcat gacccgcaag cccggtgcct gaatctaggt cgacaatcaa cctctggatt 4980
acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 5040
gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 5100
cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 5160
aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 5220
ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 5280
tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 5340
ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct 5400
ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 5460
cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tctccgcctt cgccctcaga 5520
cgagtcggat ctccctttgg gccgcctccc cgcctggtac cgatgacaga gttagaagat 5580
cgcttcagga agctatttgg cacgacttct acaacgggag acagcacagt agattctgaa 5640
gatgaacctc ctaaaaaaga aaaaagggtg gactgggatg agtattggaa ccctgaaatc 5700
gatgaacgct gacgtcatca acccgctcca aggaatcgcg ggcccagtgt cactaggcgg 5760
gaacacccag cgcgcgtgcg ccctggcagg aagatggctg tgagggacag gggagtggcg 5820
ccctgcaata tttgcatgtc gctatgtgtt ctgggaaatc accataaacg tgaaatgtct 5880
ttggatttgg gaatcttata agttctgtat gagaccactt ggatccgata tcagatctca 5940
attgaagctt ttttgaattc tagcgtccat aagcattctt tctttcgata gcttccagtg 6000
ctttgtgaaa cttcgaggag tctctttgtt gaggactttt gagttctccc ttgaggctcc 6060
cacagataca ataaatattt gagattgaac cctgtcgagt atctgtgtaa tcttttttac 6120
ctgtgaggtc tcggaatccg ggccgagaac ttcgcagcga gctcattgta ccgcgaactt 6180
gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 6240
agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 6300
tgtctggctc tagctatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat 6360
ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc 6420
cagaagtagt gaggaggctt ttttggaggc ctagactttt gcagagacgg cccaaattcg 6480
taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 6540
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 6600
ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 6660
taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 6720
tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 6780
aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 6840
aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 6900
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 6960
acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 7020
ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 7080
tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 7140
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 7200
gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 7260
agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 7320
tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 7380
agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 7440
tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 7500
acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 7560
tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 7620
agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 7680
tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 7740
acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 7800
tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 7860
ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 7920
agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 7980
tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 8040
acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 8100
agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 8160
actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 8220
tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 8280
gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 8340
ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 8400
tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 8460
aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 8520
tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 8580
tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 8640
gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg 8700
ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg 8760
gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg 8820
tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta 8880
ctgagagtgc ac 8892
<210> 4
<211> 285
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 60
gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 120
gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 180
ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 240
tccgtcacat gcactgtctc aggggtctca ttacccgact atggt 285
Claims (3)
1.一种整合microRNA和CAR功能的载体构建方法，其特征在于：包括以下步骤：
(1)构建含有microRNA(以hsa-miR-301a为例)模拟物(pre-miR-301a)和阻遏物(sponge-miR-301a)的载体，并由H1启动子驱动。构建含有CAR(以CD19-CAR为例)由EF1α驱动并包括：CD19的单链抗体可变区(single chain variable fragment,scFv)基因片段序列，CD8来源的连接和穿膜片段序列，传递信号的效应分子CD3ζ序列和胞内信号分子CD28和4-1BB片段序列组成；
(2)采用合成、酶切、连接和转化的方法，分别将hsa-miR-301a(模拟物或阻遏物)和CD19-CAR结构序列连接到含有H1和EF1α的第三代FIV载体上(名称为：pFIV-miR-301a-CD19CAR)，将连接产物转入大肠杆菌感受态DH5α中进行大量扩增并提取质粒；
(3)共转染质粒pFIV-miR-301a-CD19CAR和FIV慢病毒包装质粒(pFIV-34N和pVSV-G质粒)于293T细胞，收集293T细胞上清，并制备慢病毒颗粒，感染Jurkat细胞。采用荧光定量PCR检测miR-301a表达，流式细胞技术检测myc-tag标签来显示CD19-CAR在细胞膜的表达水平。
2.根据权利要求1所述的一种整合microRNA和CAR功能的载体构建方法，其特征在于：所述步骤(1)中的合成的hsa-miR-301a的模拟物(阻遏物)两条单链的反应条件为：37℃条件下30分钟，95℃条件下2分钟，放置室温。
3.根据权利要求1所述的一种整合microRNA和CAR功能的载体构建方法，其特征在于：所述步骤(3)中的慢病毒感染Jurkat细胞的时间为48小时。
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