CN116622752A - 一种适于表达mRNA的质粒载体及其制备方法与应用 - Google Patents
一种适于表达mRNA的质粒载体及其制备方法与应用 Download PDFInfo
- Publication number
- CN116622752A CN116622752A CN202310511022.3A CN202310511022A CN116622752A CN 116622752 A CN116622752 A CN 116622752A CN 202310511022 A CN202310511022 A CN 202310511022A CN 116622752 A CN116622752 A CN 116622752A
- Authority
- CN
- China
- Prior art keywords
- plasmid vector
- mrna
- sequence
- utr
- expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108020004999 messenger RNA Proteins 0.000 title claims abstract description 52
- 239000013600 plasmid vector Substances 0.000 title claims abstract description 50
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 239000013612 plasmid Substances 0.000 claims abstract description 18
- 238000010367 cloning Methods 0.000 claims abstract description 12
- 108700026244 Open Reading Frames Proteins 0.000 claims abstract description 9
- 239000003814 drug Substances 0.000 claims abstract description 9
- 108091026898 Leader sequence (mRNA) Proteins 0.000 claims abstract description 7
- 108091036066 Three prime untranslated region Proteins 0.000 claims abstract description 7
- 238000003776 cleavage reaction Methods 0.000 claims description 11
- 239000012634 fragment Substances 0.000 claims description 11
- 230000007017 scission Effects 0.000 claims description 11
- 239000002773 nucleotide Substances 0.000 claims description 10
- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- 108700008625 Reporter Genes Proteins 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 6
- 108020005345 3' Untranslated Regions Proteins 0.000 claims description 5
- 108020003589 5' Untranslated Regions Proteins 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 3
- 229960000723 ampicillin Drugs 0.000 claims description 3
- -1 adenine nucleoside triphosphates Chemical class 0.000 claims description 2
- OIRDTQYFTABQOQ-UHFFFAOYSA-N ara-adenosine Natural products Nc1ncnc2n(cnc12)C1OC(CO)C(O)C1O OIRDTQYFTABQOQ-UHFFFAOYSA-N 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- 239000001226 triphosphate Substances 0.000 claims description 2
- 235000011178 triphosphate Nutrition 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 abstract description 16
- 102000004190 Enzymes Human genes 0.000 abstract description 16
- 238000005520 cutting process Methods 0.000 abstract description 10
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 239000013598 vector Substances 0.000 abstract description 5
- 239000013613 expression plasmid Substances 0.000 abstract description 3
- 239000002547 new drug Substances 0.000 abstract description 2
- 108700021021 mRNA Vaccine Proteins 0.000 description 11
- 229940126582 mRNA vaccine Drugs 0.000 description 11
- 241000588724 Escherichia coli Species 0.000 description 5
- 238000011161 development Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 230000003362 replicative effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 108700001237 Nucleic Acid-Based Vaccines Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 229940023146 nucleic acid vaccine Drugs 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108010041986 DNA Vaccines Proteins 0.000 description 1
- 101001009007 Homo sapiens Hemoglobin subunit alpha Proteins 0.000 description 1
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 229940031567 attenuated vaccine Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940022399 cancer vaccine Drugs 0.000 description 1
- 238000009566 cancer vaccine Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 108010026228 mRNA guanylyltransferase Proteins 0.000 description 1
- 229940038694 mRNA-based vaccine Drugs 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 229940126583 recombinant protein vaccine Drugs 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229940031626 subunit vaccine Drugs 0.000 description 1
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940023147 viral vector vaccine Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种适于表达mRNA的质粒载体及其制备方法与应用,属于生物新药领域,本发明提供的适于表达mRNA的质粒载体以pUC18为骨架质粒,其序列与SEQ ID No.1具有至少85%的序列相似性,包含成药序列和多克隆位点,成药序列包括:5‑帽结构、5’‑UTR、开放阅读框架、3’‑聚腺苷酸序列和3’‑UTR。本发明提供的适于表达mRNA的质粒载体相比于传统表达mRNA的质粒具有多个酶切位点,具有更广的适用性。同时,使用本发明提供的适于表达mRNA的质粒载体表达表达的mRNA的效率高、稳定性强。对mRNA药物的生产具有重要意义。
Description
技术领域
本发明涉及生物新药领域,具体而言,涉及一种适于表达mRNA的质粒载体及其制备方法与应用,更具体地,涉及一种由pUC18质粒改进制得的质粒载体及其制备方法与应用。
背景技术
20世纪70年代以来,全球疫苗的研发进入快速发展阶段,从最开始的第一代传统疫苗包括灭活疫苗、减毒疫苗等,发展到第二代疫苗包括由微生物的天然成分及其产物制成的亚单位疫苗和将能激发免疫应答的成分基因重组而产生的重组蛋白疫苗,再到目前最新第三代以mRNA疫苗、DNA疫苗、重组病毒载体疫苗为代表的核酸疫苗。
与传统疫苗相比,核酸疫苗尤其是mRNA疫苗主要有以下核心优势:可以表达任意种类,包括而不限于天然抗体、病毒蛋蛋、可同时表达多种蛋白、作用机制明确、生产周期短、生产环境要求低、一条生产线可生产所有产品。因此,mRNA疫苗有潜力解决传染病和癌症疫苗开发中的许多挑战。
mRNA疫苗的核心原理是将相关转录本传递到宿主细胞的细胞质中,随后在细胞内表达翻译蛋白即抗原,使其位于细胞膜内或分泌到细胞膜外,进而呈现在主要组织相容性复合物上即MHCⅠ类和MHCⅡ类,然后被CD8+和CD4+T细胞识别从而启动适应性免疫应答。目前,临床中正在研究的mRNA疫苗主要分为两种:非复制性mRNA疫苗(NRM)和病毒衍生的自扩增mRNA疫苗(SAM)。传统的基于mRNA的疫苗主要包括编码目标抗原序列的开放阅读框(openreading frame,ORF),以及5′和3′未翻译区(UTRs)、帽子结构(Cap)和多聚腺苷酸尾巴(Poly-A)。与使用自扩增的mRNA疫苗相比,一方面非复制mRNA疫苗的优势在于其结构简单,mRNA体积小,相比之下在自扩增mRNA疫苗ORF中插入目标抗原转录本的大小限制更大;另外一方面,非复制mRNA疫苗ORF中没有任何额外的编码蛋白序列,而这些编码蛋白如复制机制产生的复制酶可能会诱导非预期的免疫反应,理论上可能会限制其技术平台在同一人体中的重复使用。
在常规的体外合成mRNA时,为保证质粒大小合适,一般载体如申请号为CN202010259281.8的中国发明专利所公开的质粒载体中仅包含单一的酶切位点,然而,当目标mRNA序列中含有相应酶切位点时,需要对序列进行改造,这增加了mRNA后续功能失活的风险,且单一酶的选择少,不易优化工艺和成本。因此有必要开发一种具有多酶切位点的用于生产mRNA的新型质粒载体。
发明内容
本发明所要解决的问题是如何在保证mRNA质量的同时增加mRNA表达质粒载体中的酶切位点。
为解决上述问题,本发明第一方面提供一种适于表达mRNA的质粒载体,所述质粒载体的序列与SEQ ID No.1具有至少85%的序列相似性,所述质粒载体包含成药序列和多克隆位点,所述成药序列包括:5-帽结构、5’-UTR、开放阅读框架、3’-聚腺苷酸序列和3’-UTR所述质粒载体以pUC18为骨架质粒。本发明提供的质粒在载体内安装有5-帽结构,相比于传统mRNA需要在体外进行加帽的操作而言,简化了传统mRNA制备中的加帽操作,同时,本发明提供的质粒载体相比于现有的用于表达RNA的质粒载体,酶切位点更多,有效提升了实验效率。
优选地,所述5’-UTR包含10~200个核苷酸,所述5’-UTR包括一段KOZAK序列,所述KOZAK序列为:5’-GGCTAGCGCCGCCACC-3’。
优选地,所述3’-聚腺苷酸序列包含60~150个腺嘌呤核苷三磷酸。
优选地,所述3’-UTR为血红蛋白HBA2的3’-UTR序列。
优选地,所述开放阅读框中具有报告基因,所述报告基因为常规报告基因,所述报告基因前后具有相同或不同的酶切位点。
优选地,所述多克隆位点包括Apal、Xhol、Notl、EcoRI、Spel、BamHI、SacI、Kpnl、HindIII。本发明提供的适于表达mRNA的质粒载体添加多种酶切位点,使线性化酶的选择更加多样化,在试剂订购时,更容易找到酶切温度、buffer相同的试剂进行双酶切验证,当目的序列与载体含有相同酶切位点时,能够通过回避相同酶切位点,选择其他酶切位点而不是对mRNA序列进行修改来进行试验,减少了试验失败风险,当市场上某一酶的价格过高或者技术不成熟、拥有专利保护时,可以选择其他酶来进行替代,使试验拥有更多选择。
优选地,所述质粒载体包含2个T7启动子和氨苄青霉素抗性基因。
本发明第二方面提供一种前述适于表达mRNA的质粒载体的构建方法,包括以下步骤:
S1:合成酶切位点对应的核苷酸链;
S2:使用Hind III和EcoR I双酶切pUC18骨干质粒载体;
S3:分离步骤S2中的酶切片段,得到两个不同大小的片段;
S4:将步骤S1中制得的核苷酸链片段与步骤S3中分离的较大的片段进行连接,获得的质粒载体。
进一步地,本发明第三方面提供一种前述适于表达mRNA的质粒载体的应用,具体地,将所述质粒载体用于RNA药物的制备。
本发明具备的有益效果:通过对多克隆位点和特殊原件的优化和改造,本发明设计了一种用于mRNA药物的质粒载体,针对载体序列的抗性基因、多克隆酶切位点;mRNA成药部分序列的T7启动子、5’UTR、3'UTR、polyA等进行了特殊设计,实现载体平台的创建,可通过功能区目的基因序列的替换,以即插即用的方式实现mRNA药物的快速设计和开发。
附图说明
图1为本发明具体实施方式提供的质粒的质粒图谱;
图2为本发明具体实施方式中将mRNA注射入小鼠体内后的荧光照相结果图。
具体实施方式
为使本发明的上述目的、特征和优点能够更为明显易懂,下面对本发明的具体实施例做详细的说明。需要说明的是,以下各实施例仅用于说明本发明的实施方法和典型参数,而不用于限定本发明所述的参数范围,由此引申出的合理变化,仍处于本发明权利要求的保护范围内。
需要说明的是,在本文中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开。
除非另有定义,本文中使用的所有术语、符号和其他科学术语旨在具有与本发明所属领域技术人员通常所理解的相同含义。在一些情况下,本文处于阐明或便于引用目的对具有常规理解含义的术语加以限定,本文中此类限定不应理解为表示与本领域常规理解具有显著差异。
如背景技术所述,当前市售适于RNA表达的载体一般仅含有一组酶切位点,这使得本领域技术人员在进行含有相应酶切位点RNA表达时,必须对目标序列进行修改,而对序列的修改可能造成RNA表达下降等一系列问题,有鉴于此,本发明的具体实施方式旨在提供一种适于表达RNA的质粒载体。
在本发明具体实施方式提供的适于表达mRNA的质粒载体与SEQ ID No.1具有至少85%的序列相似性,其质粒图谱如图1所示,该质粒载体以pUC18为骨架质粒,通过对抗性基因、启动子、多克隆位点进行优化替换制得,在该质粒载体中,包含多种多克隆位点,且该质粒适于表达mRNA,为便于理解,本发明具体实施方式提供的质粒载体命名为PUCX1。
本发明具体实施方式中提供的适于表达mRNA的质粒载体包括2个T7启动子、多克隆位点和成药序列,其中,多克隆位点包括Apal、Xhol、Notl、EcoRI、Spel、BamHI、SacI、Kpnl、HindIII。较多的多克隆位点确保了mRNA不用修改本身序列即可进行表达。成药序列具体包括:5-帽结构、5’-UTR、开放阅读框架、3’-聚腺苷酸序列和3’-UTR,5’-UTR为一段包含KOZAK的含有10~200个核苷酸的序列,3’-UTR为含有60~150个核苷酸的血红蛋白HBA2的3’-UTR序列,开放阅读框中具有常规报告基因,同时在常规报告基因的两段具有不同的酶切位点。
实施例1
本实施例提供一种适于表达mRNA的质粒并向其搭载eGFP,命名为PUCX1,首先,按照靶标序列,分别合成EcoRI、SpeI、BamHI、SacI、KpnI、HindIII酶切位点对应的核苷酸链,得到约140bp长度的片段,使用Hind III和EcoR I双酶切pUC18骨干质粒载体,通过电泳验证,得到2200bp和400bp的两个片段,将合成的核苷酸链与2200bp的大片段相连,获得适于表达mRNA的质粒载体。
向前述获得的适于表达mRNA的质粒载体中插入eGFP核酸片段获得重组质粒,将重组质粒通过热激转化入JM108大肠杆菌工程菌中,获得重组大肠杆菌,使用含有氨苄青霉素的LB平板筛选转化成功的重组大肠杆菌,挑选转化成功的克隆,使用液体培养基对其进行发酵培养扩增质粒获得发酵液。
收集发酵液,离心收集大肠杆菌,对大肠杆菌进行裂解、质粒提取、纯化并酶切获得线性化质粒,对线性化质粒进行IVT反应,获得mRNA,对mRNA进行包封,获得mRNA样品。
对小鼠进行肌肉注射mRNA样品,并对小鼠进行荧光照相检测,观察小鼠体内的荧光显色反应。实验结果如图2所示,从中可以看出,在mRNA注射入小鼠体内1h后,mRNA即可到达病灶,同时,使用本发明提供的适于表达mRNA的质粒载体表达的mRNA在小鼠体内可以保持48h不被降解,稳定性高。
虽然本公开披露如上,但本公开的保护范围并非仅限于此。本领域技术人员,在不脱离本公开的精神和范围的前提下,可进行各种变更与修改,这些变更与修改均将落入本发明的保护范围。
Claims (9)
1.一种适于表达mRNA的质粒载体,其特征在于,所述适于表达mRNA的质粒载体的序列与SEQ ID No.1具有至少85%的序列相似性,所述质粒载体包含成药序列和多克隆位点,所述成药序列包括:5-帽结构、5’-UTR、开放阅读框架、3’-聚腺苷酸序列和3’-UTR,所述适于表达mRNA的质粒载体以pUC18为骨架质粒。
2.如权利要求1所述的适于表达mRNA的质粒载体,其特征在于,所述5’-UTR具有10~200个核苷酸,所述5’-UTR包括一段KOZAK序列,所述KOZAK序列为:5’-GGCTAGCGCCGCCACC-3’。
3.如权利要求1所述的适于表达mRNA的质粒载体,其特征在于,所述3’-聚腺苷酸序列包含60~150个腺嘌呤核苷三磷酸。
4.如权利要求1所述的适于表达mRNA的质粒载体,其特征在于,所述3’-UTR含有血红蛋白HBA2的3’-UTR序列。
5.如权利要求1所述的适于表达mRNA的质粒载体,其特征在于,所述开放阅读框中具有报告基因,所述报告基因为常规报告基因,所述报告基因前后具有相同或不同的酶切位点。
6.如权利要求1所述的适于表达mRNA的质粒载体,其特征在于,所述多克隆位点包括Apal、Xhol、Notl、EcoRI、Spel、BamHI、SacI、Kpnl、HindIII。
7.如权利要求6所述的适于表达mRNA的质粒载体,其特征在于,所述质粒载体包含2个T7启动子和氨苄青霉素抗性基因。
8.一种权利要求1~7任一所述的适于表达mRNA的质粒载体的构建方法,其特征在于,包括以下步骤:
S1:合成酶切位点对应的核苷酸链;
S2:使用Hind III和EcoR I双酶切pUC18骨干质粒载体;
S3:分离步骤S2中的酶切片段,得到两个不同大小的片段;
S4:将步骤S1中制得的核苷酸链片段与步骤S3中分离的较大的片段进行连接,获得的质粒载体。
9.一种权利要求1~7任一所述适于表达mRNA的质粒载体的应用,其特征在于,将所述质粒载体用于RNA药物的制备。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310511022.3A CN116622752A (zh) | 2023-05-09 | 2023-05-09 | 一种适于表达mRNA的质粒载体及其制备方法与应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310511022.3A CN116622752A (zh) | 2023-05-09 | 2023-05-09 | 一种适于表达mRNA的质粒载体及其制备方法与应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116622752A true CN116622752A (zh) | 2023-08-22 |
Family
ID=87620493
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310511022.3A Pending CN116622752A (zh) | 2023-05-09 | 2023-05-09 | 一种适于表达mRNA的质粒载体及其制备方法与应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116622752A (zh) |
-
2023
- 2023-05-09 CN CN202310511022.3A patent/CN116622752A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Gan et al. | A combined cell‐free transcription‐translation system from Saccharomyces cerevisiae for rapid and robust protein synthe | |
Li et al. | RNA sequence determinants of a coupled termination-reinitiation strategy for downstream open reading frame translation in Helminthosporium victoriae virus 190S and other victoriviruses (Family Totiviridae) | |
EP4257677A1 (en) | Stable lentivirus packaging cell line and preparation method therefor | |
WO2023227124A1 (zh) | 一种构建mRNA体外转录模板的骨架 | |
Deaner et al. | Modular ligation extension of guide RNA operons (LEGO) for multiplexed dCas9 regulation of metabolic pathways in Saccharomyces cerevisiae | |
CN116004696B (zh) | 可与IRES组合的3ˊUTR加茎环结构基因及其应用、mRNA表达系统 | |
CN110981968B (zh) | 含有狂犬病病毒g蛋白的融合蛋白及其制备方法、应用和疫苗 | |
CN117230062A (zh) | 人工优化设计的5`utr序列提高外源基因的翻译表达 | |
CN106636162A (zh) | 基于人rna聚合酶i系统的肠道病毒68型微复制子体系及其构建方法 | |
CN112126644B (zh) | 一种非依赖帽结构下进行细胞外转录的mRNA、呈递细胞及应用 | |
CN108410902B (zh) | 一种新型酿酒酵母表达体系及其构建方法 | |
Thean et al. | To plate or to simply unfreeze, that is the question for optimal plasmid extraction | |
CN116622752A (zh) | 一种适于表达mRNA的质粒载体及其制备方法与应用 | |
CN111269301A (zh) | 香蕉转录因子MaARF12、MaARF24及其在抑制MaSBE2.3表达上的应用 | |
CN116286931A (zh) | 用于富养罗尔斯通氏菌快速基因编辑的双质粒系统及应用 | |
CN106566842A (zh) | 一种蛋白或多肽的植物表达载体及其应用 | |
CN111808858A (zh) | 一种siRNA序列及其靶标在提高PEDV毒价中的应用 | |
JP2022528252A (ja) | 環状rnaを使用したタンパク質翻訳およびその応用 | |
CN117925714B (zh) | 一种高效稳定的mRNA转录载体构建方法及其应用 | |
CN118086288B (zh) | 多顺反子的自放大rna及制备方法 | |
CN118256506B (zh) | 人工设计的5'utr结构增强目的基因的表达 | |
US7179457B2 (en) | Modified nodavirus RNA for gene delivery | |
CN114480469B (zh) | 装载茯苓内源序列的基因编辑载体、编辑系统及应用 | |
CN112553239B (zh) | 基于非天然氨基酸的基因组重排调控系统和方法 | |
CN118581155A (zh) | 一种基于CRISPR/Cas12a的多靶点编辑糖基化酶基因的方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |