本发明属于基因工程
技术领域：
，尤其涉及一种编码car基因的mrna、组合mrna、构建方法、car-t细胞和应用。
背景技术：
：随着全球环境因素的不断恶化，无论发达国家还是发展中国家，恶性肿瘤的发病率和死亡率均呈上升趋势，恶性肿瘤已成为人类生命健康的最大危害。由于传统治疗方法的固有缺陷以及全球生物高新技术的火热发展，人们对新疗法的出现期待已久。而生物治疗作为刚崭露头角的全新治疗理念，其特有优点包括选择性靶向杀伤肿瘤细胞，对正常细胞无毒性作用。生物疗法治疗癌症能够明显改善患者临床症状，提高生活质量，延长生存期。生物疗法治疗癌症能激活机体免疫功能，有望根除肿瘤细胞，生物治疗能够增强手术成功率，必将为癌症患者所广泛接受。car-t疗法是一种以嵌合型抗原受体为基础的细胞免疫治疗方案。其通过体外基因转移技术，将编码嵌合抗原受体(car)的基因序列转导入t细胞中，生成可以结合靶抗原的肿瘤特异性t细胞。近几年，car-t疗法在治疗恶性血液肿瘤中取得的成果是有目共睹的，如针对难治性/复发性急性b淋巴母细胞白血病的kymriah和针对难治性/复发性非何杰金氏淋巴瘤的yescarta去年已在美国上市，但会有至少50％以上的复发率，且car-t细胞治疗实体瘤效果不佳，主要原因是缺乏合适的靶抗原、car-t细胞在体内持续时间短、免疫逃逸、免疫抑制肿瘤微环境等。因此，目标抗原的选择对于car的特异性、有效性以及基因改造的t细胞自身安全性来讲都至关重要。技术实现要素：有鉴于此，本发明的目的在于提供一种编码car基因的mrna、组合mrna、构建方法、car-t细胞和应用，采用本发明提供的编码car基因的mrna和mrna组合构建得到的car-t细胞，能够表达car基因，能够杀伤肿瘤细胞。为了实现上述发明目的，本发明提供了以下技术方案：本发明提供了一种编码car基因的mrna，所述mrna的核苷酸序列选自seqidno.1～4中的任一种。本发明还提供了一种防治肿瘤的组合mrna，包括上述技术方案所述的编码car基因的mrna和编码细胞因子的mrna，所述编码细胞因子的mrna的核苷酸序列选自seqidno.5～9中的一种或几种。优选的，包括编码car基因的mrna和编码细胞因子的mrna，所述编码car基因的mrna的核苷酸序列如seqidno.4所示，所述编码细胞因子的mrna的核苷酸序列如seqidno.5所示。优选的，包括编码car基因的mrna和编码细胞因子的mrna，所述编码car基因的mrna的核苷酸序列如seqidno.4所示，所述编码细胞因子的mrna的核苷酸序列如seqidno.6所示。优选的，包括编码car基因的mrna和编码细胞因子的mrna，所述编码car基因的mrna的核苷酸序列如seqidno.4所示，所述编码细胞因子的mrna的核苷酸序列如seqidno.7和8所示。优选的，包括编码car基因的mrna和编码细胞因子的mrna，所述编码car基因的mrna的核苷酸序列如seqidno.4所示，所述编码细胞因子的mrna的核苷酸序列如seqidno.9所示。本发明还提供了一种car-t细胞的构建方法，包括以下步骤：将上述技术方案所述的编码car基因的mrna或上述技术方案所述的组合mrna转染细胞，得到car-t细胞。本发明还提供了一种基于上述技术方案所述的构建方法构建得到的car-t细胞。本发明还提供了上述技术方案所述的car-t细胞在制备防治肿瘤的药物中的应用。优选的，所述肿瘤包括人淋巴瘤。本发明提供了一种编码car基因的mrna、组合mrna、构建方法、car-t细胞和应用，所述mrna的核苷酸序列选自seqidno.1～4中的任一种；所述的编码car基因的mrna和编码细胞因子的mrna，所述编码细胞因子的mrna的核苷酸序列选自seqidno.5～9中的一种或几种。本发明以编码car基因的mrna或组合mrna为抗原，不仅便于保存和运输，还可以在原位将t细胞转化成为car-t细胞，相比传统体外car-t制备在流程上大大简化，成本大幅下降。以信使rna作为携带嵌合抗原受体的载体，转入患者t细胞后，将t细胞激活，并装上定位导航装置car(肿瘤嵌合抗原受体)，将t细胞这个普通"战士"改造成"超级战士"，即car-t细胞，他利用其"定位导航装置"car，专门识别体内肿瘤细胞，并通过免疫作用释放大量的多种效应因子，它们能高效地杀灭肿瘤细胞，从而达到治疗恶性肿瘤的目的。本发明实施例的结果显示：采用本发明提供的编码car基因的mrna、组合mrna构建得到的car-t细胞能够表达car基因，对肿瘤具有杀伤作用。附图说明图1为car-t细胞中car的表达率；图2为car-t细胞杀伤肿瘤细胞结果；图3为car-t细胞被靶细胞激活后外泌ifnγ的结果；图4为car-t细胞被靶细胞激活后cd4和cd8细胞分泌ifnγ的结果。图5为car-t细胞被靶细胞激活后cd4和cd8细胞分泌ifnγ的结果。图6为car-t细胞被靶细胞激活后cd4和cd8细胞分泌ifnγ的结果。图7为注射car基因mrna后原位构建car-t细胞的结果。图8为car-t细胞体内杀伤靶细胞的结果。具体实施方式本发明提供了一种编码car基因的mrna，所述mrna的核苷酸序列选自seqidno.1～4中的任一种。本发明还提供了一种防治肿瘤的组合mrna，包括上述技术方案所述的编码car基因的mrna和编码细胞因子的mrna，所述编码细胞因子的mrna的核苷酸序列选自seqidno.5～9中的一种或几种。在本发明中，核苷酸序列如seqidno.5所示的mrna编码il-2细胞因子，核苷酸序列如seqidno.6所示的mrna编码il-10细胞因子，核苷酸序列如seqidno.7所示的mrna编码il12a细胞因子，核苷酸序列如seqidno.8所示的mrna编码il12b细胞因子，核苷酸序列如seqidno.9所示的mrna编码ccl-19细胞因子。在本发明中，所述编码il-2细胞因子的mrna的核苷酸序列如seqidno.5所示。在本发明中，所述编码il-10细胞因子的mrna的核苷酸序列如seqidno.6所示。在本发明中，编码il12a细胞因子的mrna的核苷酸序列如seqidno.7所示。在本发明中，编码il12b细胞因子的mrna的核苷酸序列如seqidno.8所示。在本发明中，所述编码ccl-19细胞因子的mrna的核苷酸序列如seqidno.9所示。在本发明中，所述的mrna组合包括编码car基因的mrna和编码细胞因子的mrna，所述编码car基因的mrna的核苷酸序列优选如seqidno.4所示，所述编码细胞因子的mrna的核苷酸序列优选如seqidno.5所示。在本发明中，所述的mrna组合包括编码car基因的mrna和编码细胞因子的mrna，所述编码car基因的mrna的核苷酸序列优选如seqidno.4所示，所述编码细胞因子的mrna的核苷酸序列优选如seqidno.6所示。在本发明中，所述的mrna组合包括编码car基因的mrna和编码细胞因子的mrna，所述编码car基因的mrna的核苷酸序列优选如seqidno.4所示，所述编码细胞因子的mrna的核苷酸序列优选选自seqidno.7和seqidno.8两种。seqidno.7和seqidno.8分别编码il12的a链和b链，二者结合在一起才能发挥作用，因此本质上还是一个car基因和一个细胞因子基因组合。在本发明中，所述的mrna组合包括编码car基因的mrna和编码细胞因子的mrna，所述编码car基因的mrna的核苷酸序列优选如seqidno.4所示，所述编码细胞因子的mrna的核苷酸序列优选如seqidno.9所示。本发明还提供了一种car-t细胞的构建方法，包括以下步骤：将上述技术方案所述的编码car基因的mrna或上述技术方案所述的mrna组合转染细胞，得到car-t细胞。在本发明中，所述细胞优选为外周血单个核细胞、h9(人t淋巴细胞系)或jurkat细胞。本发明还提供了一种基于上述技术方案所述的构建方法构建得到的car-t细胞。本发明还提供了上述技术方案所述的car-t细胞在制备防治肿瘤的药物中的应用。本发明对所述药物的剂型没有特殊限定，采用car-t细胞在医学上可接受的剂型即可。在本发明中，所述肿瘤优选包括人霍奇金淋巴瘤、外周t细胞淋巴瘤、弥漫性大b细胞淋巴瘤、淋巴母细胞淋巴瘤和间变性大细胞淋巴瘤中的一种或几种，注射方式优选为生理盐水溶解，静脉注射。下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。实施例1制备car-t细胞从健康捐献者的外周血中分离出pbmc细胞，在加入il2(50u/ml)的aim-v培养基中培养，将car基因mrna和细胞因子mrna按照seq1、seq2ky、seq3ye、seq4、seq4 5、seq4 6、seq4 7 8和seq4 9分别转染细胞，在细胞培养结束后，取样后分装至取样管中，进行质控检测。编码car基因的mrna，核苷酸序列如seqidno.1所示，简称seq1；编码car基因的mrna，核苷酸序列如seqidno.2所示，简称seq2ky；编码car基因的mrna，核苷酸序列如seqidno.3所示，简称seq3ye；编码car基因的mrna，核苷酸序列如seqidno.4所示，简称seq4；防治肿瘤的mrna组合，包括编码car基因的mrna和编码细胞因子的mrna，编码car基因的mrna的核苷酸序列如seqidno.4所示，编码细胞因子的mrna核苷酸序列如seqidno.5所示，简称seq4 5；防治肿瘤的mrna组合，包括编码car基因的mrna和编码细胞因子的mrna，编码car基因的mrna的核苷酸序列如seqidno.4所示，编码细胞因子的mrna核苷酸序列如seqidno.6所示，以下简称4 6；防治肿瘤的mrna组合，包括编码car基因的mrna和编码细胞因子的mrna，编码car基因的mrna的核苷酸序列如seqidno.4所示，编码细胞因子的mrna核苷酸序列选自seqidno.7和8，以下简称4 7 8；防治肿瘤的mrna组合，包括编码car基因的mrna和编码细胞因子的mrna，编码car基因的mrna的核苷酸序列如seqidno.4所示，编码细胞因子的mrna核苷酸序列如seqidno.9所示，以下简称4 9。1、pbmc细胞准备：根据使用时间确定传代密度和体积，传代于细胞培养瓶内，保证使用时细胞处于对数生长期。细胞消化计数：取生长状态良好的细胞，去掉培养基，以10mlpbs清洗细胞后，加入适量0.25％胰酶(t75瓶加1ml0.25％胰酶，t175瓶加3ml0.25％胰酶)消化5分钟，然后加入含10％fbs的dmem培养基(t75瓶用9ml培养基，t175瓶用17ml培养基)中和胰酶，吹打细胞并转至50ml离心管，反复吹打混匀，然后取约0.3ml的细胞悬液，适当稀释后计数。2、细胞稀释：取适量细胞悬液，用含10％fbs的aim-v培养基稀释到5×105个/ml，吹打混匀。3、细胞接种：取2ml细胞悬液加到6孔板内。每个样品需要准备2孔平行细胞，对照组样品需要准备1孔细胞，空白对照1孔。将6孔板放入37±1℃、5±0.5％co2培养箱培养过夜。4、转染：4.1接种完细胞后约24小时，观察6孔板内的细胞状态，汇合度在90％。在生物安全柜内，配制所需体积的90％aim-v 10％fbs培养基。转染前30min弃掉孔板的培养基，每孔加入1ml新鲜培养基(90％aim-v 10％fbs)。4.2配制转染体系：取200μlopti-mem，加入10μgseq1、seq2ky、seq3ye、seq4、seq4 5(质量比1:1)、seq4 6(质量比1:1)、seq4 7 8(质量比1:1:1)和seq4 9(质量比1:1)或阴性对照gfp-mrna，用枪头轻轻吹打混匀，再加入60μlpei(浓度1mg/ml)，立即置于漩涡振荡器上振荡10次，每次1s，充分混匀，静置10min。4.3将配制好的转染体系，直接均匀滴加进入培养的细胞中，再前后左右摇匀，使得转染体系均匀分布于细胞上。流式细胞术1、单细胞悬液制备：取生长状态良好的细胞，去掉培养基，以10mlpbs清洗细胞后，加入适量0.25％胰酶消化5分钟，然后加入含10％fbs的dmem培养基中和胰酶，吹打细胞并转至50ml离心管，反复吹打混匀，然后将细胞悬液稀释后计数。2、固定：通过离心收集细胞并吸除上清液。用0.5～1ml1xpbs中重悬细胞。添加甲醛并使其终浓度为4％。在室温下固定15分钟。用足够的1xpbs离心洗涤。将上清液丢弃在合适的废液缸中。在0.5～1ml1xpbs中重悬细胞。4、免疫染色：在100μl稀释一抗(1：2000)中重悬细胞。在室温下孵育1小时。通过离心分离，用孵育缓冲液洗涤。丢弃上清液。在100μl稀释的荧光染料标记的二抗(1：5000)中重悬细胞。在室温下孵育30分钟。上机检测。将制得的car-t细胞通过流式细胞术进行car表达率的检测，结果如图1和表1所示。表1制得的car-t细胞中car的表达率细胞名称表达率seq135.29％seq2ky38.02％seq3ye37.00％seq441.78％seq4 538.08％seq4 644.53％seq4 7 845.31％seq4 946.54从图1和表1中可以得出，本发明提供的mrna剂型能够有效地对t细胞进行转染，使其成为car-t细胞。实施例2对外周血制成的car-t细胞进行体外杀实验，具体实验步骤如下：第一步：car-t细胞制备如实施例1。第二步：calcein-am标记靶细胞1)将calcein-am用dmso稀释成lmg/ml；2)将人淋巴瘤细胞系daudi用全培养基重悬成1×106/ml的密度；3)加入15ul的calcein-am,37℃、5％c02培养30min，每lomin轻轻混匀；4)1500rpm离心，去上清，用培养基重悬，重复两遍；第三步：杀伤1)将标记好的人淋巴瘤细胞系daudi按照5000-50000个/ml的密度重悬，取100ul加入到96孔板中；2)按照适当的比例加入car-t细胞100μl，使car-t细胞与癌细胞的数目比分别为50:1、25:1、12.5:1、6:1和3:1，每组3个平行；同时，有单独的a组6个平行，只有靶细胞(阴性对照)：有单独的b组6个平行，只有靶细胞 2％tritonx-100(阳性对照)；第四步：1)37℃、5％co2培养4小时后，离心，取75μl上清，转移到一个新的培养板上；2)通过分光光度计检测样品，并记录afu数据；3)计算细胞裂解的百分比：[(实验组吸光度-阴性对照吸光度)/(阳性对照组吸光度-阴性对照组吸光度)]×l00％。得到的结果如图2、表2所示。表2本发明制备的car-t细胞体外杀伤癌细胞结果统计(细胞裂解百分比)从图2可以看出，制备的car-t细胞具有人淋巴瘤细胞杀伤能力。实施例3检测转染car基因后，在靶细胞的刺激下t细胞释放免疫因子情况。将实施例1制备的car-t细胞和人淋巴瘤细胞系daudi共培养，24小时后收取上清和细胞沉淀。上清使用人ifnγ检测试剂盒(bd)进行elisa实验检测car-t细胞在靶细胞刺激后释放的ifnγ含量。细胞沉淀采用流式细胞术进行内源细胞免疫因子检测，所用抗体为allophycocyanin(apc)-cy7-conjugatedmabtohumancd8,percp-cy5.5–conjugatedmabtohumancd4,v450-conjugatedmabtohumanifng,pe-cy7–conjugatedantitumornecrosisfactor(tnf)mab,andflfluoresceinisothiocyanate(fitc)-conjugatedmabtohumanil2(bdbiosciences)。结果如图3～6及表3、4、5所示。表3car-t细胞在靶细胞刺激后外泌的免疫因子ifnγ含量分组ifnγ(pg/ml)分组ifnγ(pg/ml)对照105seq4 5411seq1310seq4 6422seq2336seq4 7 8440seq3391seq4 9400seq4382表4受靶细胞激活后cd4细胞中表达ifnγ、tnfα和il2的阳性细胞比例(％)对照seq1seq2seq3seq4seq4 5seq4 6seq4 7 8seq4 9infγ0.6527.553025.53234.637.137.542.5tnfα0.351.51.31.151.51.61.351.41.55il23.6571.57779.278.878.58181.386.4表5受靶细胞激活后cd8细胞中表达ifnγ、tnfα和il2的阳性细胞比例(％)对照seq1seq2seq3seq4seq4 5seq4 6seq4 7 8seq4 9infγ0.52.63.051.420252.32.41.81.915tnfα0.651512.51212.251413.1514.414.35il20.7520.523.51918.315.21611.513由图3～6可知，mrna介导的car-t细胞在靶细胞的刺激下，可分泌免疫因子ifnγ、tnfα和il2，介导细胞免疫。实施例4对实施例1制备得到的car-t细胞验证其安全性和有效性。首先利用肿瘤模型小鼠验证实施例1构建的car-t细胞的有效性，具体步骤如下：一、淋巴瘤小鼠模型的构建：1.细胞系：人淋巴瘤细胞系daudi；daudi细胞是人淋巴瘤细胞系，可以通过静脉注射的方式构建小鼠的人淋巴细胞性白血病白血病模型。其cd19表达为阳性，可以作为本发明所述car-t细胞的靶细胞。2.细胞培养daudi细胞培养(1640 20％fbs)计数并测定活率，离心后用生理盐水重悬，调整其活细胞浓度为3×108个/ml，总数达1.8×109个。3.细胞系接种用生理盐水重悬daudi细胞，调整其活细胞浓度为3×108个/ml。通过尾静脉注射的方式进行接种。4.一周后，体内原位构建car-t细胞，将合成的car基因mrna和细胞因子mrna按照以下组合seq1、seq2ky、seq3ye、seq4、seq4 5(质量比1:1)、seq4 6(质量比1:1)、seq4 7 8(质量比1:1:1)和seq4 9(质量比1:1)各1mg对小鼠进行脾脏注射，不同时间点通过流式细胞术检测小鼠血液中的car-t细胞数目和癌症细胞数目，见表6和表7。结果如图7和图8所示。表6注射mrna原位构建car-t细胞结果分组car-t比例分组car-t比例对照0.4％seq4 519.1％seq18.6％seq4 621.1％seq211.3％seq4 7 825.75seq313.9％seq4 920.9％seq414.45％表7接受mrna介导的car-t治疗的小鼠体内癌细胞数目变化(log10/μl)由图7～图8可知，注射car-tmrna能有效地进行原位car-t细胞构建，同时对血液中地淋巴瘤细胞进行有效的杀灭。以上所述仅是本发明的优选实施方式，应当指出，对于本
技术领域：
的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。序列表<110>深圳市瑞吉生物科技有限公司<120>一种编码car基因的mrna、组合mrna、构建方法、car-t细胞和应用<160>9<170>siposequencelisting1.0<210>1<211>2184<212>rna<213>人工序列(artificialsequence)<400>1cugauguuccuaguacugccucugauauuccuagugcuauaacuaauguuucuacugcug60cuguuucuguaggucuacugugucugauguaggagggacagacggagagacccucugucu120cagugguagucaacgucccguucaguccuguagagauucauaaacuuaaccauagucguc180uuuggucuaccuugacaauuugaggacuagaugguauguaguucuaaugugaguccucag240gguaguuccaagucaccgucacccagaccuugucuaauaagagagugguaaucguuggac300cucguucuucuauaacggugaaugaaaacgguugucccauuaugcgaaggcaugugcaag360ccuccccccugauucaaccuuuauuguccgagguggagaccuaggccguucgggccuaga420ccgcucccuaggugguucccgcuccacuuugacguccucaguccuggaccggaccaccgc480gggagugucucggacaggcaguguacgugacagaguccccagaguaaugggcugauacca540cauucgaccuaagcggucggaggugcuuucccagaccucaccgacccucauuauacccca600ucacuuugguguaugauauuaagucgagaguuuaggucugacugguaguaguuccuguug660agguucucgguucaaaagaauuuuuacuugucagacguuugacuacugugucgguaaaug720augacacgguuuguaauaaugaugccaccaucgauacgauaccugaugaccccaguuccu780uggagucaguggcagaggagucgccggcguccauggugguguugcgggcgaggagccggu840ggcugcggucgcgguugauaacgcucagucggagagucagacgcuggacuccgaacagcu900ggucgucggccuccgcgucacgugugcucccccgaccugaagcggacacuaucuucugga960ggaagauucgggaaaacccacgaccaccaccaaccaccucaggaccgaacgauaucgaac1020gaucauugucaccggaaauaauaaaagacccacucccgaucggacucucacuucaagucg1080uccucgcgucugcgggggcgcauggucgucccggucuuggucgagauauugcucgaguua1140gauccugcuucucuccucaugcuacaaaaccuguucucugcaccggcccugggacucuac1200cccccuuucggcgucucuuccuucuugggaguccuuccggacauguuacuugacgucuuu1260cuauucuaccgccuccggaugucacucuaacccuacuuuccgcucgcggccuccccguuc1320cccgugcuaccggaaauggucccagagucaugucggugguuccuguggaugcugcgggaa1380guguacguccgggacgggggagcgcgaucgcggugcuugaagagagacaauuucguucgu1440ccgcugcaccuucuuuuggggccagggcacucguucccgcuccucgacaaguggccccac1500cacggguaggaccagcucgaccugccgcugcauuugccgguguucaagucgcacaggccg1560cucccgcucccgcuacgguggaugccguucgacugggacuucaaguagacgugguggccg1620uucgacgggcacgggaccgggugggagcacuggugggacuggaugccgcacgucacgaag1680ucggcgauggggcugguguacuucgucgugcugaagaaguucaggcgguacgggcuuccg1740augcagguccucgcgugguagaagaaguuccugcugccguugauguucugggcgcggcuc1800cacuucaagcucccgcugugggaccacuuggcguagcucgacuucccguagcugaaguuc1860cuccugccguuguaggaccccguguucgaccucauguugauguugucgguguugcagaua1920uaguaccggcuguucgucuucuugccguaguuccacuugaaguucuaggcgguguuguag1980cuccugccgucgcacgucgagcggcuggugauggucgucuuguggggguagccgcugccg2040gggcacgacgacgggcuguuggugauggacucgugggucaggcgggacucguuucugggg2100uugcucuucgcgcuaguguaccaggacgaccucaagcacuggcggcggcccuagugagag2160ccguaccugcucgacauguucauu2184<210>2<211>2310<212>rna<213>人工序列(artificialsequence)<400>2cugauguuccuaguacugccucugauauuccuagugcuauaacuaauguuucuacugcug60cuguuucuguaggucuacugugucugauguaggagggacagacggagagacccucugucu120cagugguagucaacgucccguucaguccuguagagauucauaaacuuaaccauagucguc180uuuggucuaccuugacaauuugaggacuagaugguauguaguucuaaugugaguccucag240gguaguuccaagucaccgucacccagaccuugucuaauaagagagugguaaucguuggac300cucguucuucuauaacggugaaugaaaacgguugucccauuaugcgaaggcaugugcaag360ccuccccccugauucaaccuuuauuguccgagguggagaccuaggccguucgggccuaga420ccgcucccuaggugguucccgcuccacuuugacguccucaguccuggaccggaccaccgc480gggagugucucggacaggcaguguacgugacagaguccccagaguaaugggcugauacca540cauucgaccuaagcggucggaggugcuuucccagaccucaccgacccucauuauacccca600ucacuuugguguaugauauuaagucgagaguuuaggucugacugguaguaguuccuguug660agguucucgguucaaaagaauuuuuacuugucagacguuugacuacugugucgguaaaug720augacacgguuuguaauaaugaugccaccaucgauacgauaccugaugaccccaguuccu780uggagucaguggcagaggagucgccggcguccauggugguguugcgggcgaggagccggu840ggcugcggucgcgguugauaacgcucagucggagagucagacgcuggacuccgaacagcu900ggucgucggccuccgcgucacgugugcucccccgaccugaagcggacacuaucuucugga960ggaagauucgggaaaacccacgaccaccaccaaccaccucaggaccgaacgauaucgaac1020gaucauugucaccggaaauaauaaaagacccacuccuuugccccgucuuucuuugaggac1080auauauaaguuuguugguaaauacucuggucauguuugaugaguucuccuucuaccgaca1140ucgacggcuaaaggucuucuucuucuuccuccuacacuugaccgaucggacucucacuuc1200aagucguccucgcgucugcgggggcgcauggucgucccggucuuggucgagauauugcuc1260gaguuagauccugcuucucuccucaugcuacaaaaccuguucucugcaccggcccuggga1320cucuaccccccuuucggcgucucuuccuucuugggaguccuuccggacauguuacuugac1380gucuuucuauucuaccgccuccggaugucacucuaacccuacuuuccgcucgcggccucc1440ccguuccccgugcuaccggaaauggucccagagucaugucggugguuccuguggaugcug1500cgggaaguguacguccgggacgggggagcgcgaucgcggugcuugaagagagacaauuuc1560guucguccgcugcaccuucuuuuggggccagggcacucguucccgcuccucgacaagugg1620ccccaccacggguaggaccagcucgaccugccgcugcauuugccgguguucaagucgcac1680aggccgcucccgcucccgcuacgguggaugccguucgacugggacuucaaguagacgugg1740uggccguucgacgggcacgggaccgggugggagcacuggugggacuggaugccgcacguc1800acgaagucggcgauggggcugguguacuucgucgugcugaagaaguucaggcgguacggg1860cuuccgaugcagguccucgcgugguagaagaaguuccugcugccguugauguucugggcg1920cggcuccacuucaagcucccgcugugggaccacuuggcguagcucgacuucccguagcug1980aaguuccuccugccguuguaggaccccguguucgaccucauguugauguugucgguguug2040cagauauaguaccggcuguucgucuucuugccguaguuccacuugaaguucuaggcggug2100uuguagcuccugccgucgcacgucgagcggcuggugauggucgucuuguggggguagccg2160cugccggggcacgacgacgggcuguuggugauggacucgugggucaggcgggacucguuu2220cugggguugcucuucgcgcuaguguaccaggacgaccucaagcacuggcggcggcccuag2280u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2 3
技术特征：1.一种编码car基因的mrna，其特征在于，所述mrna的核苷酸序列选自seqidno.1～4中的任一种。
2.一种防治肿瘤的组合mrna，其特征在于，包括权利要求1所述的编码car基因的mrna和编码细胞因子的mrna，所述编码细胞因子的mrna的核苷酸序列选自seqidno.5～9中的一种或几种。
3.根据权利要求2所述的组合mrna，其特征在于，所述编码car基因的mrna的核苷酸序列如seqidno.4所示，所述编码细胞因子的mrna的核苷酸序列如seqidno.5所示。
4.根据权利要求2所述的组合mrna，其特征在于，所述编码car基因的mrna的核苷酸序列如seqidno.4所示，所述编码细胞因子的mrna的核苷酸序列如seqidno.6所示。
5.根据权利要求2所述的组合mrna，其特征在于，所述编码car基因的mrna的核苷酸序列如seqidno.4所示，所述编码细胞因子的mrna的核苷酸序列选自seqidno.7和seqidno.8两种。
6.根据权利要求2所述的组合mrna，其特征在于，所述编码car基因的mrna的核苷酸序列如seqidno.4所示，所述编码细胞因子的mrna的核苷酸序列如seqidno.9所示。
7.一种car-t细胞的构建方法，其特征在于，包括以下步骤：
将权利要求1所述的编码car基因的mrna或权利要求2～6任一项所述的组合mrna转染细胞，得到car-t细胞。
8.一种基于权利要求7所述的构建方法构建得到的car-t细胞。
9.权利要求8所述的car-t细胞在制备防治肿瘤的药物中的应用。
10.根据权利要求9所述的应用，其特征在于，所述肿瘤包括人霍奇金淋巴瘤、外周t细胞淋巴瘤、弥漫性大b细胞淋巴瘤、淋巴母细胞淋巴瘤和间变性大细胞淋巴瘤中的一种或几种。
技术总结本发明提供了一种编码CAR基因的mRNA、组合mRNA、构建方法、CAR‑T细胞和应用，属于基因工程技术领域，所述mRNA的核苷酸序列选自SEQ ID No.1～4中的任一种；所述的编码CAR基因的mRNA和编码细胞因子的mRNA，所述编码细胞因子的mRNA的核苷酸序列选自SEQ ID No.5～9中的一种或几种。采用本发明提供的编码CAR基因的mRNA、组合mRNA构建得到的CAR‑T细胞能够表达CAR基因，对肿瘤具有杀伤作用。
技术研发人员：张苗苗;洪丹;胡迅
受保护的技术使用者：深圳市瑞吉生物科技有限公司
技术研发日：2020.12.07
技术公布日：2021.03.12
