CN108504689B - 慢病毒载体及其递送外源rna的方法 - Google Patents

慢病毒载体及其递送外源rna的方法 Download PDF

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CN108504689B
CN108504689B CN201810533437.XA CN201810533437A CN108504689B CN 108504689 B CN108504689 B CN 108504689B CN 201810533437 A CN201810533437 A CN 201810533437A CN 108504689 B CN108504689 B CN 108504689B
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蔡宇伽
凌思凯
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Shanghai Bendao Gene Technology Co ltd
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Abstract

本发明公开了一种慢病毒载体及其递送外源RNA的方法；利用RNA结合蛋白和其所识别的RNA序列相互结合的原理，将RNA结合蛋白整合至慢病毒GagPol长链蛋白的骨架，并将其所识别的RNA序列连接到外源目的RNA,从而使目的RNA在慢病毒颗粒的组装过程中被包装进慢病毒颗粒。外源目的RNA可以是mRNA、gRNA或者具有其它功能的RNA。利用本发明的方法，通过慢病毒颗粒的保护，RNA变得更加稳定；借助慢病毒颗粒高效感染细胞的能力，目的RNA可高效进入细胞表达基因编辑酶、肿瘤或者病毒抗原、细胞重编程因子、嵌合抗原受体等。本发明可用于基因编辑、基因治疗、细胞治疗、免疫治疗、再生医学以及基础生物学等领域。

Description

慢病毒载体及其递送外源RNA的方法
技术领域
本发明属于生物技术领域，具体涉及一种慢病毒载体及其递送外源RNA的方法。
背景技术
慢病毒载体是从HIV-1改造而来的、失去自我复制能力的病毒载体。可以高效感染细胞，是生物学研究和基因治疗常用的载体。目前，慢病毒载体可以分为第一代、第二代和第三代，代数越高，安全性越好。
RNA由核糖核苷酸通过磷酸二酯键形成的线性长链分子。RNA可以分为编码和非编码RNA。编码RNA通过编码蛋白质发挥功能；非编码RNA不编码蛋白质，可以直接发挥生物学功能。
RNA在疫苗、基因治疗、基因编辑、细胞重编程等领域具有重要的应用潜力。但其应用受到以下几个因素的限制，(1)RNA稳定较差，极易被环境中的核酸酶降解；(2)RNA自身无法进入细胞，需要一套有效的载体系统；(3)现有RNA递送技术很难直接用于体内。
目前，RNA的递送方案有电转、化学材料形成的纳米颗粒、仙台病毒以及第二代慢病毒载体改造而来的慢病毒颗粒。其中，第二代慢病毒载体改造而来的慢病毒颗粒(Prel,A.,et al.,Highly efficient in vitro and in vivo delivery of functional RNAsusing new versatile MS2-chimeric retrovirus-like particles.Mol Ther MethodsClin Dev,2015.2:p.15039.)；该方案通过(1)将MS2衣壳蛋白(RNA结合蛋白)整合至慢病毒核衣壳(nucleocapsid,NC)蛋白内部，(2)将MS2衣壳蛋白所识别的茎环结构放至目的RNA的表达框中，实现将目的RNA包装进慢病毒颗粒。然而第二代慢病毒载体保留有较多的HIV基因,文献显示第二代慢病毒载体可以在体内生成具有复制能力的HIV病毒(Skrdlant,L.M.,et al.,Detection of Replication Competent Lentivirus Using a qPCR Assay forVSV-G.Mol Ther Methods Clin Dev,2018.8:p.1-7.)。因此，出于安全性考虑，第二代慢病毒载体已经不再被用于基因治疗。
发明内容
本发明的目的在于克服上述现有技术问题，提供一种慢病毒载体及其递送外源RNA的方法。本发明解决的是RNA往细胞内的递送问题，包括RNA的体外和体内递送。本发明可以递送Cas9mRNA和gRNA用于基因编辑和基因治疗；可以递送肿瘤或者病毒抗原mRNA用于免疫治疗；可以递送细胞重编程因子mRNA制造多能干细胞、改造细胞功能；可以递送嵌合抗原受体mRNA用于细胞免疫治疗。
本发明将目的RNA包装进慢病毒载体，利用慢病毒保护和递送RNA。本发明的原理为：利用RNA结合蛋白与其所识别的RNA茎环结构的相互作用，将携带该种可识别的RNA序列的外源目的RNA包装进慢病毒颗粒。
本发明的核心特点是：将RNA结合蛋白整合至第三代慢病毒GagPol长链蛋白的N-端；将RNA结合蛋白所识别的茎环结构置于外源目的RNA表达框。
具体而言，本发明的目的是通过以下技术方案来实现的：
第一方面，本发明涉及一种慢病毒载体，所述慢病毒载体是通过将含有慢病毒载体基因组序列的质粒转染入病毒生产细胞；收集上清，浓缩，制备而得；
所述慢病毒载体基因组序列分别位于表达膜蛋白的质粒、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒和含RNA结合蛋白所识别的RNA茎环结构的质粒上。
优选的，所述病毒生产细胞包括293T、293FT、HEK293。
优选的，所述表达膜蛋白的质粒包括VSV-G、CD4识别蛋白、CD8识别蛋白、RD114、狒狒内源逆转录病毒(baboon endogenous retrovirus)膜蛋白所改造来的膜蛋白。
优选的，所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒是将RNA结合蛋白整合至第三代慢病毒GagPol长链蛋白的N-端。
优选的，所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒中，GagPol长链蛋白的密码子序列如SEQ ID NO.1所示。
优选的，所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒中，所述RNA结合蛋白为MS2衣壳蛋白；所述MS2衣壳蛋白的密码子序列如SEQ ID NO.2所示。
优选的，所述含RNA结合蛋白所识别的RNA茎环结构的质粒中，所述RNA结合蛋白为MS2衣壳蛋白；所述MS2衣壳蛋白所识别的RNA序列如SEQ ID NO.3所示。
优选的，所述浓缩选用高速离心或者HPLC方法进行浓缩。
第二方面，本发明还涉及一种利用本发明的慢病毒载体递送外源目的RNA的方法。
优选的，所述外源目的RNA为mRNA、gRNA、其它功能的RNA中的一种或几种。
优选的，所述方法包括如下步骤：
S1、将所述表达膜蛋白的质粒、所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒和表达带RNA结合蛋白所识别的RNA茎环结构的外源目的RNA的质粒共转染病毒生产细胞；
S2、收集含病毒颗粒的上清，浓缩，得到含外源目的RNA的慢病毒颗粒。
优选的，所述表达带RNA结合蛋白所识别的RNA茎环结构的外源目的RNA的质粒是通过将RNA结合蛋白所识别的RNA序列与外源目的RNA序列融合而得。
更优选的，所述表达带RNA结合蛋白所识别的RNA茎环结构的外源目的RNA的质粒是通过将MS2衣壳蛋白所识别的RNA序列与目的RNA序列融合而得到。
进一步优选的，所述MS2衣壳蛋白所识别的RNA序列如SEQ ID NO.3所示。
优选的，所述浓缩选用高速离心或者HPLC方法进行浓缩。
优选的，所述病毒生产细胞包括293T、293FT、HEK293。
优选的，所述表达膜蛋白的质粒包括VSV-G、CD4识别蛋白、CD8识别蛋白、RD114、狒狒内源逆转录病毒(baboon endogenous retrovirus)膜蛋白所改造来的膜蛋白。
优选的，所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒是通过将MS2衣壳蛋白与慢病毒GagPol长链蛋白融合而得到。
更优选的，所述GagPol长链蛋白的密码子序列如SEQ ID NO.1所示；所述MS2衣壳蛋白的密码子序列如SEQ ID NO.2所示。
第三方面，本发明还涉及一种本发明的慢病毒载体在递送Cas9mRNA和gRNA用于实现基因编辑和基因治疗中的用途。
第四方面，本发明还涉及一种本发明的慢病毒载体在携带表达肿瘤抗原和病毒抗原的mRNA用于疫苗中的用途。
第五方面，本发明还涉及一种本发明的慢病毒载体在表达细胞重编程因子用于制造多能干细胞、改造细胞功能中的用途。
第六方面，本发明还涉及一种本发明的慢病毒载体在递送嵌合抗原受体(chimeric antigen receptor)mRNA用于细胞免疫治疗中的用途。
与现有技术相比，本发明具有如下有益效果：
(1)以第三代慢病毒技术为基础，在慢病毒GagPol长链蛋白中去掉了tat和rev等HIV基因，减小了病毒颗粒包装过程中，病毒基因组重组产生具有复制能力的HIV的可能性，极大提高了安全性。
(2)优化了慢病毒GagPol长链蛋白的密码子，使其可以在人源细胞系高效表达，不再需要表达HIV蛋白REV来提高慢病毒颗粒的产量。
(3)将RNA结合蛋白置于慢病毒GagPol长链蛋白的N-端，减小了外源蛋白对病毒颗粒正常形态的负面影响。
(4)本发明具有包装和携带长链RNA的能力，比如可以携带Cas9mRNA(约4.2kb)和碱基编辑酶(约5.1kb)等，极大提高了应用价值。
(5)本发明可以递送Cas9mRNA和gRNA用于实现基因编辑和基因治疗。
(6)本发明可以携带表达肿瘤抗原和病毒抗原的mRNA用于疫苗。
(7)本发明可以用于表达细胞重编程因子和嵌合抗原受体，用于制造多能干细胞、改造细胞功能。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1为慢病毒载体递送GFP mRNA实验；
图2为慢病毒载体递送Cas9mRNA实现基因编辑实验。
具体实施方式
下面结合实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干调整和改进。这些都属于本发明的保护范围。
实施例1、慢病毒颗粒的制备
步骤1：将表达膜蛋白的质粒、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒、表达带RNA结合蛋白所识别的RNA茎环结构的外源目的RNA的质粒共转染病毒生产细胞(293T)。其中，
1)表达膜蛋白的质粒包括VSV-G、CD4识别蛋白、CD8识别蛋白、RD114、狒狒内源逆转录病毒(baboon endogenous retrovirus)膜蛋白所改造来的膜蛋白。本实施例中选用VSV-G。
2)病毒生产细胞包括293T、293FT、HEK293等。本实施例中选用293T。
3)本实施例中表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒是通过将MS2衣壳蛋白与慢病毒GagPol长链蛋白融合而得到。
GagPol长链蛋白的密码子序列如下(SEQ ID NO.1)：
gccagggccagcgtgctgagcggcggcgagctggacaggtgggagaagatcaggctgaggcccggcggcaagaagaagtataagctgaagcacatcgtgtgggccagcagggagctggagaggttcgccgtgaaccccggcctgctggagaccagcgagggctgcaggcagatcctgggccagctgcagcccagcctgcagaccggcagcgaggagctgaggagcctgtacaacaccgtggccaccctgtactgcgtgcaccagaggatcgagatcaaggacaccaaggaggccctggacaagatcgaggaggagcagaacaagtccaagaagaaggcccagcaggccgccgccgacaccggccacagcagccaggtgagccagaactaccccatcgtgcagaacatccagggccagatggtgcaccaggccatcagccccaggaccctgaacgcctgggtgaaggtggtggaggagaaggccttcagccccgaggtgatccccatgttcagcgccctgagcgagggagccaccccccaggacctgaacaccatgctgaacaccgtgggcggccaccaggccgccatgcagatgctgaaggagaccatcaacgaggaggccgccgagtgggacagggtgcaccccgtgcacgccggccccatcgcccccggccagatgagggagccccgcggcagcgacatcgccggcaccaccagcaccctgcaggagcagatcggctggatgaccaacaacccccccatccccgtgggcgaaatctacaagaggtggatcatcctgggcctgaacaagatcgtgaggatgtacagccccaccagcatcctggatatcaggcagggccccaaagagcccttcagggactacgtggacaggttctacaagaccctgcgcgccgagcaggccagccaggaggtgaagaactggatgaccgagaccctgctggtgcagaacgccaaccccgactgcaagaccatcctgaaggccctgggacccgccgccaccctggaggagatgatgaccgcctgccagggcgtgggcggccccggccacaaggccagggtgctggccgaggccatgagccaggtgaccaacaccgccaccatcatgatgcagaggggcaacttcaggaaccagaggaagatggtgaagtgcttcaactgcggcaaggagggccacaccgccaggaactgccgcgcccccaggaagaagggctgctggaagtgcggcaaggagggccaccagatgaaggactgcaccgagaggcaggctaattttttagggaagatctggccttcctacaagggaaggccagggaattttcttcagagcagaccagagccaacagccccaccatttcttcagagcagaccagagccaacagccccaccagaagagagcttcaggtctggggtagagacaacaactccccctcagaagcaggagccgatagacaaggaactgtatcctttaacttccctcagatcactctttggcaacgacccctcgtcacaataaagatcggtggccagctgaaggaggccctgctggacaccggcgccgacgacaccgtgctggaggagatgagcctgcccggcaggtggaagcccaagatgatcggcggcatcggcggcttcatcaaggtgaggcagtacgaccagatcctgatcgagatctgcggccacaaggccatcggcaccgtgctggtgggacctacacctgtgaacatcatcggcaggaacctgctgacccagatcggctgcaccctgaacttccccatcagccccatcgagaccgtgcccgtgaagctgaagcccggcatggacggccctaaggtgaagcagtggcccctgaccgaggagaagatcaaggccctggtggagatctgcaccgagatggagaaggagggcaagatcagcaagatcggccccgagaacccctacaacacccccgtgttcgccatcaagaagaaggacagcaccaagtggaggaagctggtggacttcagggagctgaacaagaggacccaggacttctgggaggtgcagctgggcatcccccaccccgccggcctgaagaagaagaagagcgtgaccgtgctggacgtgggcgacgcctacttcagcgtgcccctggacgaggacttcaggaagtataccgccttcaccatccccagcatcaacaacgagacccccggcatccgctaccagtacaacgtgctgccccagggctggaagggcagccccgccatcttccagagcagcatgacaaagatcctggagcccttcaagaagcagaaccccgacatcgtgatctatcagtacatggacgacctgtacgtgggcagcgacctggagatcggccagcacaggaccaagatcgaggagctgaggcagcacctgctgaggtggggcctgaccacccccgacaagaagcaccagaaggagcccccattcctgtggatgggctacgagctgcaccccgacaagtggaccgtgcagcccatcgtgctgcccgagaaggacagctggaccgtgaacgacattcagaagctggtgggcaagctgaactgggccagccagatctaccccggcatcaaggtgaggcagctgtgcaagctgctgaggggcacaaaggctctgaccgaggtgatccccctgaccgaggaggccgagctggagctggccgagaacagggagatcctgaaggagcccgtgcacggcgtgtactacgaccccagcaaggacctgatcgccgagatccagaagcagggccagggccagtggacctaccagatctaccaggagcccttcaagaacctgaagaccggcaagtacgcccgcatgcgcggcgcccacaccaacgacgtgaagcagctgaccgaggccgtgcagaagatcaccaccgagagcatcgtgatctggggcaagactcctaagttcaagctgcccatccagaaggagacctgggagacctggtggaccgagtactggcaggccacctggattcccgagtgggagttcgtgaacacccctcccctggtgaagctgtggtatcagctggagaaggagcccatcgtgggcgccgagaccttctacgtggacggcgccgccaacagggagaccaagctgggcaaggccggctacgtgaccaacaagggccgccagaaggtggtgcccctgaccaacaccaccaaccagaagaccgagctgcaggctatctacctggccctgcaggactcaggcctggaggtgaacatcgtgaccgacagccagtacgccctgggcatcatccaggcccagcccgacaagagcgagagcgagctggtgaaccagatcatcgagcagctgatcaagaaggagaaggtgtacctggcctgggtgcccgcccacaagggcatcggcggcaacgagcaggtggacaagctggtgagcgccggcatcaggaagatcctgttcctggacggcatcgacaaggcccaggacgagcacgagaagtaccacagcaactggagggctatggctagcgacttcaacctgcctcccgtggtggctaaggagatcgtggccagctgcgacaagtgccagctgaagggcgaggccatgcacggccaggtggactgcagccccggcatctggcagctggtttgcacccacctggagggcaaggtgatcctggtggccgtgcacgtggcctccggctacatcgaggccgaggtgatccccgccgagaccggccaggagaccgcctacttcctgctgaagctggccggccgctggcccgtgaagaccatccacaccgacaacggcagcaacttcaccagcgccaccgtgaaggccgcctgctggtgggccggcatcaagcaggagttcggcatcccctacaacccccagtctcagggcgtggtggagagcatgaacaaggagctgaagaagatcatcggccaggtgagggaccaggccgagcacctgaagaccgccgtgcagatggccgtgttcatccacaacttcaagaggaagggcggcatcggcggctacagcgccggcgagaggatcgtggacatcatcgccaccgacatccagaccaaggagctgcagaagcagatcaccaagatccagaacttcagggtgtactacagggacagcaggaaccctctgtggaagggccccgccaagctgctgtggaagggcgagggcgccgtggtgatccaggacaacagcgacatcaaggtggtgcccaggaggaaggccaagatcatcagggactacggcaagcagatggccggcgacgactgcgtggcctccaggcaggacgaggactgaSEQ ID NO.1。
MS2衣壳蛋白的密码子如下(SEQ ID NO.2)，其编码的蛋白可与上述SEQ IDNO.1序列编码的GagPol长链蛋白相连，比如置于该GagPol长链蛋白的N-端。
atggcctctaattttactcaatttgtgcttgtcgataatggggggacgggagatgtgaccgttgcccctagcaatttcgcaaatggcgttgcagaatggatctctagcaacagcagaagccaagcgtacaaagtaacgtgttccgttcgccaaagctccgcccaaaaacggaagtatacaataaaggttgaggtgccgaaagtagccactcaaacagttggtggggtagaattgcccgtagcggcatggcggtcatatctcaatatggaactcactatcccaatcttcgccacgaatagcgattgtgagctgatagttaaggctatgcaaggtcttctcaaagatggaaaccctattccatctgctatcgccgccaacagcgggatatac SEQ ID NO.2。
4)表达带RNA结合蛋白所识别的RNA茎环结构的外源目的RNA的质粒是通过将MS2衣壳蛋白所识别的RNA序列与目的RNA序列融合而得到。
其中，MS2衣壳蛋白所识别的RNA序列如下(SEQ ID NO.3)，该序列可以以单次或者多次重复的方式与外源目的RNA链接。
acatgaggatcacccatgt SEQ ID NO.3。
5)上述3)和4)所涉及的用于制备携带外源目的RNA的慢病毒颗粒所需要的质粒通过分子克隆方法得到。
步骤2：收集含病毒颗粒的上清，通过高速离心或者HPLC等方法进行浓缩，得到高滴度的含外源目的RNA的慢病毒颗粒。
实施例2、利用慢病毒载体递送GFP mRNA。
具体步骤同实施例1，外源目的RNA选用GFP mRNA。GFP为一种发荧光的报告基因蛋白；本实施例具体为慢病毒载体递送GFP mRNA至293T细胞。
由图1可知，293T细胞感染携带GFP mRNA的慢病毒颗粒48小时后，流式细胞术分析GFP阳性细胞高达99.8％。
实施例3、利用慢病毒载体递送Cas9mRNA以及靶向AAVS1位点的gRNA
具体步骤同实施例1，外源目的RNA选用Cas9mRNA以及靶向AAVS1位点的gRNA。本实施例具体为慢病毒载体递送Cas9mRNA和靶向人类基因组AAVS1位点的gRNA至293T细胞。
如图2所示，293T细胞感染携带Cas9mRNA以及靶向AAVS1位点的gRNA的慢病毒颗粒72小时后，PCR扩增AAVS1位点并测序。TIDE分析基因敲除效率达34.1％。
综上，利用本发明的方法，通过慢病毒颗粒的保护，RNA变得更加稳定；同时借助慢病毒颗粒高效感染细胞的能力，目的RNA可高效进入细胞表达基因编辑酶、肿瘤或者病毒抗原、细胞重编程因子、嵌合抗原受体等。因此，本发明可以递送Cas9mRNA和gRNA用于基因编辑和基因治疗；可以递送肿瘤或者病毒抗原mRNA用于免疫治疗；可以递送细胞重编程因子mRNA制造多能干细胞、改造细胞功能；可以递送嵌合抗原受体mRNA用于细胞免疫治疗。
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变形或修改，这并不影响本发明的实质内容。
SEQUENCE LISTING
<110> 上海交通大学
<120> 慢病毒载体及其递送外源RNA的方法
<130> DAG33015
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 4337
<212> DNA
<213> GagPol长链蛋白的密码子
<400> 1
gccagggcca gcgtgctgag cggcggcgag ctggacaggt gggagaagat caggctgagg 60
cccggcggca agaagaagta taagctgaag cacatcgtgt gggccagcag ggagctggag 120
aggttcgccg tgaaccccgg cctgctggag accagcgagg gctgcaggca gatcctgggc 180
cagctgcagc ccagcctgca gaccggcagc gaggagctga ggagcctgta caacaccgtg 240
gccaccctgt actgcgtgca ccagaggatc gagatcaagg acaccaagga ggccctggac 300
aagatcgagg aggagcagaa caagtccaag aagaaggccc agcaggccgc cgccgacacc 360
ggccacagca gccaggtgag ccagaactac cccatcgtgc agaacatcca gggccagatg 420
gtgcaccagg ccatcagccc caggaccctg aacgcctggg tgaaggtggt ggaggagaag 480
gccttcagcc ccgaggtgat ccccatgttc agcgccctga gcgagggagc caccccccag 540
gacctgaaca ccatgctgaa caccgtgggc ggccaccagg ccgccatgca gatgctgaag 600
gagaccatca acgaggaggc cgccgagtgg gacagggtgc accccgtgca cgccggcccc 660
atcgcccccg gccagatgag ggagccccgc ggcagcgaca tcgccggcac caccagcacc 720
ctgcaggagc agatcggctg gatgaccaac aaccccccca tccccgtggg cgaaatctac 780
aagaggtgga tcatcctggg cctgaacaag atcgtgagga tgtacagccc caccagcatc 840
ctggatatca ggcagggccc caaagagccc ttcagggact acgtggacag gttctacaag 900
accctgcgcg ccgagcaggc cagccaggag gtgaagaact ggatgaccga gaccctgctg 960
gtgcagaacg ccaaccccga ctgcaagacc atcctgaagg ccctgggacc cgccgccacc 1020
ctggaggaga tgatgaccgc ctgccagggc gtgggcggcc ccggccacaa ggccagggtg 1080
ctggccgagg ccatgagcca ggtgaccaac accgccacca tcatgatgca gaggggcaac 1140
ttcaggaacc agaggaagat ggtgaagtgc ttcaactgcg gcaaggaggg ccacaccgcc 1200
aggaactgcc gcgcccccag gaagaagggc tgctggaagt gcggcaagga gggccaccag 1260
atgaaggact gcaccgagag gcaggctaat tttttaggga agatctggcc ttcctacaag 1320
ggaaggccag ggaattttct tcagagcaga ccagagccaa cagccccacc atttcttcag 1380
agcagaccag agccaacagc cccaccagaa gagagcttca ggtctggggt agagacaaca 1440
actccccctc agaagcagga gccgatagac aaggaactgt atcctttaac ttccctcaga 1500
tcactctttg gcaacgaccc ctcgtcacaa taaagatcgg tggccagctg aaggaggccc 1560
tgctggacac cggcgccgac gacaccgtgc tggaggagat gagcctgccc ggcaggtgga 1620
agcccaagat gatcggcggc atcggcggct tcatcaaggt gaggcagtac gaccagatcc 1680
tgatcgagat ctgcggccac aaggccatcg gcaccgtgct ggtgggacct acacctgtga 1740
acatcatcgg caggaacctg ctgacccaga tcggctgcac cctgaacttc cccatcagcc 1800
ccatcgagac cgtgcccgtg aagctgaagc ccggcatgga cggccctaag gtgaagcagt 1860
ggcccctgac cgaggagaag atcaaggccc tggtggagat ctgcaccgag atggagaagg 1920
agggcaagat cagcaagatc ggccccgaga acccctacaa cacccccgtg ttcgccatca 1980
agaagaagga cagcaccaag tggaggaagc tggtggactt cagggagctg aacaagagga 2040
cccaggactt ctgggaggtg cagctgggca tcccccaccc cgccggcctg aagaagaaga 2100
agagcgtgac cgtgctggac gtgggcgacg cctacttcag cgtgcccctg gacgaggact 2160
tcaggaagta taccgccttc accatcccca gcatcaacaa cgagaccccc ggcatccgct 2220
accagtacaa cgtgctgccc cagggctgga agggcagccc cgccatcttc cagagcagca 2280
tgacaaagat cctggagccc ttcaagaagc agaaccccga catcgtgatc tatcagtaca 2340
tggacgacct gtacgtgggc agcgacctgg agatcggcca gcacaggacc aagatcgagg 2400
agctgaggca gcacctgctg aggtggggcc tgaccacccc cgacaagaag caccagaagg 2460
agcccccatt cctgtggatg ggctacgagc tgcaccccga caagtggacc gtgcagccca 2520
tcgtgctgcc cgagaaggac agctggaccg tgaacgacat tcagaagctg gtgggcaagc 2580
tgaactgggc cagccagatc taccccggca tcaaggtgag gcagctgtgc aagctgctga 2640
ggggcacaaa ggctctgacc gaggtgatcc ccctgaccga ggaggccgag ctggagctgg 2700
ccgagaacag ggagatcctg aaggagcccg tgcacggcgt gtactacgac cccagcaagg 2760
acctgatcgc cgagatccag aagcagggcc agggccagtg gacctaccag atctaccagg 2820
agcccttcaa gaacctgaag accggcaagt acgcccgcat gcgcggcgcc cacaccaacg 2880
acgtgaagca gctgaccgag gccgtgcaga agatcaccac cgagagcatc gtgatctggg 2940
gcaagactcc taagttcaag ctgcccatcc agaaggagac ctgggagacc tggtggaccg 3000
agtactggca ggccacctgg attcccgagt gggagttcgt gaacacccct cccctggtga 3060
agctgtggta tcagctggag aaggagccca tcgtgggcgc cgagaccttc tacgtggacg 3120
gcgccgccaa cagggagacc aagctgggca aggccggcta cgtgaccaac aagggccgcc 3180
agaaggtggt gcccctgacc aacaccacca accagaagac cgagctgcag gctatctacc 3240
tggccctgca ggactcaggc ctggaggtga acatcgtgac cgacagccag tacgccctgg 3300
gcatcatcca ggcccagccc gacaagagcg agagcgagct ggtgaaccag atcatcgagc 3360
agctgatcaa gaaggagaag gtgtacctgg cctgggtgcc cgcccacaag ggcatcggcg 3420
gcaacgagca ggtggacaag ctggtgagcg ccggcatcag gaagatcctg ttcctggacg 3480
gcatcgacaa ggcccaggac gagcacgaga agtaccacag caactggagg gctatggcta 3540
gcgacttcaa cctgcctccc gtggtggcta aggagatcgt ggccagctgc gacaagtgcc 3600
agctgaaggg cgaggccatg cacggccagg tggactgcag ccccggcatc tggcagctgg 3660
tttgcaccca cctggagggc aaggtgatcc tggtggccgt gcacgtggcc tccggctaca 3720
tcgaggccga ggtgatcccc gccgagaccg gccaggagac cgcctacttc ctgctgaagc 3780
tggccggccg ctggcccgtg aagaccatcc acaccgacaa cggcagcaac ttcaccagcg 3840
ccaccgtgaa ggccgcctgc tggtgggccg gcatcaagca ggagttcggc atcccctaca 3900
acccccagtc tcagggcgtg gtggagagca tgaacaagga gctgaagaag atcatcggcc 3960
aggtgaggga ccaggccgag cacctgaaga ccgccgtgca gatggccgtg ttcatccaca 4020
acttcaagag gaagggcggc atcggcggct acagcgccgg cgagaggatc gtggacatca 4080
tcgccaccga catccagacc aaggagctgc agaagcagat caccaagatc cagaacttca 4140
gggtgtacta cagggacagc aggaaccctc tgtggaaggg ccccgccaag ctgctgtgga 4200
agggcgaggg cgccgtggtg atccaggaca acagcgacat caaggtggtg cccaggagga 4260
aggccaagat catcagggac tacggcaagc agatggccgg cgacgactgc gtggcctcca 4320
ggcaggacga ggactga 4337
<210> 2
<211> 390
<212> DNA
<213> MS2衣壳蛋白的密码子
<400> 2
atggcctcta attttactca atttgtgctt gtcgataatg gggggacggg agatgtgacc 60
gttgccccta gcaatttcgc aaatggcgtt gcagaatgga tctctagcaa cagcagaagc 120
caagcgtaca aagtaacgtg ttccgttcgc caaagctccg cccaaaaacg gaagtataca 180
ataaaggttg aggtgccgaa agtagccact caaacagttg gtggggtaga attgcccgta 240
gcggcatggc ggtcatatct caatatggaa ctcactatcc caatcttcgc cacgaatagc 300
gattgtgagc tgatagttaa ggctatgcaa ggtcttctca aagatggaaa ccctattcca 360
tctgctatcg ccgccaacag cgggatatac 390
<210> 3
<211> 19
<212> DNA
<213> MS2衣壳蛋白识别的RNA
<400> 3
acatgaggat cacccatgt 19

Claims (6)

1.一种慢病毒载体，其特征在于，所述慢病毒载体是通过将含有慢病毒载体基因组序列的质粒转染入病毒生产细胞；收集上清，浓缩，制备而得；
所述慢病毒载体基因组序列分别位于表达膜蛋白的质粒、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒和含RNA结合蛋白所识别的RNA茎环结构的质粒上；
所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒是将RNA结合蛋白整合至第三代慢病毒GagPol长链蛋白的N-端；所述表达膜蛋白的质粒为VSV-G；
所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒中，GagPol长链蛋白的密码子序列如SEQ ID NO.1所示；
所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒中，所述RNA结合蛋白为MS2衣壳蛋白；所述MS2衣壳蛋白的密码子序列如SEQ ID NO.2所示。
2.根据权利要求1所述的慢病毒载体，其特征在于，所述病毒生产细胞包括293T、293FT、HEK293。
3.根据权利要求1所述的慢病毒载体，其特征在于，所述含RNA结合蛋白所识别的RNA茎环结构的质粒中，RNA结合蛋白所识别的RNA序列如SEQ ID NO.3所示。
4.一种非疾病诊断治疗目的的利用如权利要求1～3中任一项所述的慢病毒载体递送外源目的RNA的方法。
5.根据权利要求4所述的非疾病诊断治疗目的的利用慢病毒载体递送外源目的RNA的方法，其特征在于，所述方法包括如下步骤：
S1、将所述表达膜蛋白的质粒、所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒和表达带RNA结合蛋白所识别的RNA茎环结构的外源目的RNA的质粒共转染病毒生产细胞；
S2、收集含病毒颗粒的上清，浓缩，得到含外源目的RNA的慢病毒颗粒。
6.一种如权利要求1～3中任一项所述的慢病毒载体在制备递送Cas9 mRNA和gRNA用于实现基因编辑和基因治疗药物中的用途，或是在携带表达肿瘤抗原和病毒抗原的mRNA用于制备疫苗中的用途，或是在表达细胞重编程因子用于制造多能干细胞中的用途，或是在表达细胞重编程因子用于改造细胞功能中的用途，或是在递送嵌合抗原受体mRNA用于制备细胞免疫治疗药物中的用途。
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