CN116463380A - A recombinant adeno-associated virus vector composition, exogenous gene expression system and method - Google Patents
A recombinant adeno-associated virus vector composition, exogenous gene expression system and method Download PDFInfo
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- CN116463380A CN116463380A CN202310328671.XA CN202310328671A CN116463380A CN 116463380 A CN116463380 A CN 116463380A CN 202310328671 A CN202310328671 A CN 202310328671A CN 116463380 A CN116463380 A CN 116463380A
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Abstract
Description
技术领域technical field
本发明属于生物技术领域，具体涉及一种重组腺相关病毒载体组合物、外源基因表达系统及方法。The invention belongs to the field of biotechnology, and in particular relates to a recombinant adeno-associated virus vector composition, exogenous gene expression system and method.
背景技术Background technique
近年来，神经科学与基因治疗是生命科学的两大重点研究领域，病毒载体工具在这两个领域中扮演着重要的角色。在神经科学中，病毒载体可被用于标记或操控神经细胞，从而探究神经网络的结构与功能；在基因治疗领域，病毒载体常被用于基因编辑或基因递送，从而发展遗传疾病的治疗方法。因此，不断丰富现有病毒工具库对科学研究和疾病治疗均具有重要意义。重组腺相关病毒(recombinant adeno-associated virus,rAAV)载体具有基因组结构简单、感染细胞范围广、免疫原性低、表达外源基因时间长等特点，并且已经被广泛用于科学研究。In recent years, neuroscience and gene therapy are two key research areas of life science, and viral vector tools play an important role in these two fields. In neuroscience, viral vectors can be used to label or manipulate nerve cells to explore the structure and function of neural networks; in the field of gene therapy, viral vectors are often used for gene editing or gene delivery to develop treatments for genetic diseases. Therefore, continuously enriching the existing virus tool library is of great significance to both scientific research and disease treatment. Recombinant adeno-associated virus (rAAV) vectors have the characteristics of simple genome structure, wide range of infected cells, low immunogenicity, and long-term expression of foreign genes, and have been widely used in scientific research.
科学研究中常用的rAAV载体基因组中往往需要携带启动子、目的基因、调控序列等多种必需元件，这些元件像电路一样串联成完整的基因表达盒从而在细胞内表达外源基因。使用不同的启动子和调控序列可以实现目的基因的组织细胞特异性表达以及表达强度的控制。目前常用的单个rAAV载体基因组中具有固定的启动子、目的基因和调控序列，需要通过重新构建基因组的方式改变其中的单个或多个基因电路元件，这限制了该载体工具的灵活性；限于单个rAAV基因组的容量，基因电路元件的选择也更为局限。例如，在神经科学等领域，随着单细胞测序、空间转录组等技术的发展，细胞亚类被进一步细化，亚类细胞的标记和操控需要组合使用不同的特异性启动子、增强子等元件，但受限于现有的单个rAAV基因组组织结构，达到这种不同组合的目的需要设计并构建大量不同的rAAV基因组，且能选择的元件的长度范围较局限。目前虽然有通过两个rAAV反式剪接表达大基因片段的方法，但该方法的困难在于需要对每个目的基因寻找拆分位点，不适用于所有基因片段，且该方法下的两种rAAV是专为单个目的基因设计，不具有与其他rAAV组合的灵活性和拓展性。此外，相关研究往往聚焦于表达更大的目的基因片段而忽视了使用更大的启动子、更大的调控序列以靶向更特异类型、具有更多特征的细胞的需求。The rAAV vector genome commonly used in scientific research often needs to carry a variety of essential elements such as promoters, target genes, and regulatory sequences. These elements are connected in series like a circuit to form a complete gene expression cassette to express foreign genes in cells. Different promoters and regulatory sequences can be used to achieve tissue cell-specific expression and control of expression intensity of target genes. The currently commonly used single rAAV vector genome has fixed promoters, target genes and regulatory sequences, and needs to change single or multiple gene circuit elements in it by reconstructing the genome, which limits the flexibility of the vector tool; limited to the capacity of a single rAAV genome, the selection of gene circuit elements is also more limited. For example, in neuroscience and other fields, with the development of technologies such as single-cell sequencing and spatial transcriptomics, cell subclasses have been further refined. The labeling and manipulation of subclass cells requires the combination of different specific promoters, enhancers and other elements, but limited by the existing single rAAV genome organization structure. To achieve the purpose of this different combination requires the design and construction of a large number of different rAAV genomes, and the length range of the elements that can be selected is relatively limited. Although there is currently a method for expressing large gene fragments through trans-splicing of two rAAVs, the difficulty of this method is that it is necessary to find a split site for each target gene, which is not applicable to all gene fragments, and the two rAAVs under this method are designed for a single target gene and do not have the flexibility and scalability to combine with other rAAVs. In addition, related research often focuses on the expression of larger target gene fragments and ignores the need to use larger promoters and larger regulatory sequences to target more specific types of cells with more characteristics.
鉴于生物学研究中灵活组合更多种类的基因元件、靶向具有更多特征的细胞的需求，亟需发展一套更易于设计和使用的、具有特殊基因组组织结构的rAAV载体工具。In view of the need to flexibly combine more types of genetic elements and target cells with more characteristics in biological research, it is urgent to develop a set of rAAV vector tools that are easier to design and use and have special genome organization structures.
发明内容Contents of the invention
为了解决现有技术中的问题，本发明提供了一种重组腺相关病毒载体组合物、外源基因表达系统及方法。In order to solve the problems in the prior art, the present invention provides a recombinant adeno-associated virus vector composition, exogenous gene expression system and method.
本发明的具体技术方案如下：Concrete technical scheme of the present invention is as follows:
本发明第一方面提供一种重组腺相关病毒载体组合物，其包括重组腺相关病毒载体rAAV-L与重组腺相关病毒载体rAAV-R；所述rAAV-L基因组包含重组酶识别序列I和至少一个启动子序列，其中一个启动子序列记为启动子序列I，启动子序列I位于重组酶识别序列I的一侧，重组酶识别序列I位于启动子序列I的下游；所述rAAV-R基因组包含重组酶识别序列II和至少一个目的基因序列，其中一个目的基因序列记为目的基因序列I，目的基因序列I位于重组酶识别序列II的一侧，重组酶识别序列II位于目的基因序列I的上游；所述重组酶识别序列I和重组酶识别序列II为同一种重组酶的识别序列且方向相同。The first aspect of the present invention provides a recombinant adeno-associated virus vector composition, which includes a recombinant adeno-associated virus vector rAAV-L and a recombinant adeno-associated virus vector rAAV-R; the rAAV-L genome comprises a recombinase recognition sequence I and at least one promoter sequence, wherein one promoter sequence is denoted as a promoter sequence I, the promoter sequence I is located on one side of the recombinase recognition sequence I, and the recombinase recognition sequence I is located downstream of the promoter sequence I; the rAAV-R genome comprises a recombinase recognition sequence II and at least one target gene sequence, wherein The sequence is recorded as the target gene sequence I, and the target gene sequence I is located on one side of the recombinase recognition sequence II, and the recombinase recognition sequence II is located upstream of the target gene sequence I; the recombinase recognition sequence I and the recombinase recognition sequence II are recognition sequences of the same recombinase and have the same direction.
进一步地，所述rAAV-L基因组包含的另一个启动子序列记为启动子序列II，启动子序列II位于重组酶识别序列I的另一侧，重组酶识别序列I位于启动子序列II的下游；所述rAAV-R基因组包含的另一个目的基因序列记为目的基因序列II，目的基因序列II位于重组酶识别序列II的另一侧，重组酶识别序列II位于目的基因序列II的上游。Further, the other promoter sequence contained in the rAAV-L genome is marked as promoter sequence II, and the promoter sequence II is located on the other side of the recombinase recognition sequence I, and the recombinase recognition sequence I is located downstream of the promoter sequence II; the other target gene sequence contained in the rAAV-R genome is marked as the target gene sequence II, the target gene sequence II is located on the other side of the recombinase recognition sequence II, and the recombinase recognition sequence II is located upstream of the target gene sequence II.
进一步地，所述rAAV-L基因组还包含目的基因序列和/或调控序列，所述目的基因序列的转录被所述rAAV-L基因组的任一启动子启动。Further, the rAAV-L genome further comprises a target gene sequence and/or a regulatory sequence, and the transcription of the target gene sequence is initiated by any promoter of the rAAV-L genome.
进一步地，所述rAAV-R基因组还包含启动子序列和/或调控序列，所述启动子序列启动所述rAAV-R基因组的任一目的基因序列的转录。Further, the rAAV-R genome further comprises a promoter sequence and/or a regulatory sequence, and the promoter sequence initiates the transcription of any target gene sequence of the rAAV-R genome.
进一步地，所述重组酶识别序列包括但不限于loxP、lox66、lox71、lox2272、lox5171、loxm2、JT15、JTZ17、JT510、FRT、F3、F5、FRT-LE、FRT-RE、mFRT71、Rox、VloxP、Vlox2272、SloxP、Vox、att；Further, the recombinase recognition sequence includes but is not limited to loxP, lox66, lox71, lox2272, lox5171, loxm2, JT15, JTZ17, JT510, FRT, F3, F5, FRT-LE, FRT-RE, mFRT71, Rox, VloxP, Vlox2272, SloxP, Vox, att;
所述启动子序列选自CAG、CaMKIIα、CAR、CBA、CD68、c-fos、ChAT、CMV、CR、Ef1α、E-SARE、GAD67、GFAP、GFAP104、gfaABC1D、Grm6、hGRK1、hSyn、hUbC、LP1B、L7/Pcp2、MBP、MCK、mDlX、mOXT、mTH、nEF、Nestin、NPY、Nrl、PGK、PV、RAM、RK、ROH、RPE65、SFRP2、SST、TBG、TCAP、TH、Thy1、TPH2、TRE、TRPV1、UAS和Vgat中的一种或以上；The promoter sequence is selected from CAG, CaMKIIα, CAR, CBA, CD68, c-fos, ChAT, CMV, CR, Ef1α, E-SARE, GAD67, GFAP, GFAP104, gfaABC1D, Grm6, hGRK1, hSyn, hUbC, LP1B, L7/Pcp2, MBP, MCK, mDlX, mOXT, mTH, nEF, One or more of Nestin, NPY, Nrl, PGK, PV, RAM, RK, ROH, RPE65, SFRP2, SST, TBG, TCAP, TH, Thy1, TPH2, TRE, TRPV1, UAS and Vgat;
所述目的基因序列选自用于研究组织器官结构与功能、过表达基因产物、操控神经系统、基因治疗相关的蛋白的核苷酸编码序列和/或功能RNA产物的基因等；所述蛋白优选为荧光蛋白、激活神经元蛋白、抑制神经元蛋白、钙离子信号探针蛋白、小分子信号探针蛋白、介导凋亡蛋白、疾病相关突变蛋白、生理条件下的正常蛋白、细胞因子、抗病毒因子、病毒感染辅助受体、重组酶和基因编辑工具蛋白中一种或多种；所述功能性RNA选自小RNA、小干扰RNA、小发卡RNA、小向导RNA、细胞器定位RNA和用于RNA测序或原位杂交分析的Barcode RNA中的一种或多种；The target gene sequence is selected from genes used to study the structure and function of tissues and organs, overexpressing gene products, manipulating the nervous system, nucleotide coding sequences and/or functional RNA products of proteins related to gene therapy; the protein is preferably one or more of fluorescent proteins, neuronal activating proteins, neuronal inhibitory proteins, calcium ion signal probe proteins, small molecule signal probe proteins, apoptosis-mediating proteins, disease-related mutant proteins, normal proteins under physiological conditions, cytokines, antiviral factors, viral infection co-receptors, recombinases and gene editing tool proteins; the functional RNA is selected from small RNA, small interference One or more of RNA, small hairpin RNA, small guide RNA, organelle-localized RNA, and Barcode RNA for RNA sequencing or in situ hybridization analysis;
所述调控序列包括转录和/或转录后调控序列；The regulatory sequences include transcriptional and/or post-transcriptional regulatory sequences;
优选地，所述调控序列选自WPRE、oPRE、cw3sl、SV40 polyA、hGHpolyA、bGH polyA、rbGlob polyA等中的一种或以上。Preferably, the regulatory sequence is selected from one or more of WPRE, oPRE, cw3sl, SV40 polyA, hGH polyA, bGH polyA, rbGlob polyA and the like.
进一步地，所述重组腺相关病毒载体rAAV-L与重组腺相关病毒载体rAAV-R具有相同或不同的血清型，且具有感染组织或器官中同一细胞的能力。Further, the recombinant adeno-associated virus vector rAAV-L and the recombinant adeno-associated virus vector rAAV-R have the same or different serotypes, and have the ability to infect the same cell in a tissue or organ.
进一步地，所述血清型包括AAV1、AAV2、AAV2-Retro、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV9-Retro、AAV10、AAV11、AAV12、AAV13、AAV-DJ、AAV-PHP.eB、AAV-myo及其衍生血清型。Further, the serotypes include AAV1, AAV2, AAV2-Retro, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9-Retro, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV-PHP.eB, AAV-myo and derivative serotypes thereof.
本发明第二方面提供一种重组腺相关病毒载体库，其包括所述的重组腺相关病毒载体组合物。The second aspect of the present invention provides a recombinant adeno-associated virus vector library, which includes the recombinant adeno-associated virus vector composition.
本发明第三方面提供一种外源基因的表达系统，其包括所述的重组腺相关病毒载体组合物。The third aspect of the present invention provides a foreign gene expression system, which includes the recombinant adeno-associated virus vector composition.
进一步地，所述表达系统还包括重组酶导入载体，所述重组酶与所述的重组腺相关病毒载体组合物携带的重组酶识别序列对应；Further, the expression system also includes a recombinase introduction vector, and the recombinase corresponds to the recombinase recognition sequence carried by the recombinant adeno-associated virus vector composition;
优选地，重组酶与重组酶识别序列的对应使用的方式包括但不限于：Cre-loxP/lox66/lox71/lox2272/lox5171/loxm2/JT15/JTZ17/JT510、Vika-Vox、ΦC31-attB/attP、Flp-FRT/FRT3/FRT5/FRT-LE/LRT-RE/mFRT71、Dre-Rox、VCre-VLoxP/VLox2272、SCre/SLoxP；Preferably, the corresponding use of the recombinase and the recombinase recognition sequence includes but is not limited to: Cre-loxP/lox66/lox71/lox2272/lox5171/loxm2/JT15/JTZ17/JT510, Vika-Vox, ΦC31-attB/attP, Flp-FRT/FRT3/FRT5/FRT-LE/LRT-RE/mFRT71, Dre -Rox, VCre-VLoxP/VLox2272, SCre/SLoxP;
优选地，所述重组酶导入载体选自携带重组酶基因的表达质粒、携带重组酶基因的病毒载体、其他导入重组酶基因或蛋白的化学或生物载体中的一种或多种；Preferably, the recombinase-introducing vector is selected from one or more of expression plasmids carrying recombinase genes, viral vectors carrying recombinase genes, and other chemical or biological vectors for introducing recombinase genes or proteins;
优选地，所述重组酶包括但不限于Cre、Flp、Dre、VCre、SCre、Vika等酪氨酸重组酶及其衍生蛋白，ΦC31、Bxb1、R4等丝氨酸重组酶及其衍生蛋白。Preferably, the recombinases include but are not limited to Cre, Flp, Dre, VCre, SCre, Vika and other tyrosine recombinases and their derivative proteins, ΦC31, Bxb1, R4 and other serine recombinases and their derivative proteins.
本发明第四方面提供一种表达外源基因的方法，所述方法为如下(1)-(3)中的一种：The fourth aspect of the present invention provides a method for expressing an exogenous gene, the method being one of the following (1)-(3):
(1)将所述的重组腺相关病毒载体组合物注射至目标组织或器官中，所述组织或器官的部分或全部细胞表达重组酶基因；(1) injecting the recombinant adeno-associated virus vector composition into the target tissue or organ, some or all cells of the tissue or organ express the recombinase gene;
(2)将所述的重组腺相关病毒载体组合物注射至目标组织或器官中，所述重组腺相关病毒载体rAAV-L或rAAV-R表达重组酶基因；(2) injecting the recombinant adeno-associated virus vector composition into the target tissue or organ, and the recombinant adeno-associated virus vector rAAV-L or rAAV-R expresses a recombinase gene;
(3)将所述的重组腺相关病毒载体组合物注射至目标组织或器官中，并通过其他途径导入重组酶。(3) Inject the recombinant adeno-associated virus vector composition into the target tissue or organ, and introduce the recombinase through other ways.
进一步地，方法(3)中导入重组酶的途径为将携带重组酶基因的表达质粒、携带重组酶基因的病毒载体、其他导入重组酶基因或蛋白的化学或生物载体中的一种导入至目标组织或器官；Further, the way to introduce the recombinase in method (3) is to introduce one of the expression plasmid carrying the recombinase gene, the viral vector carrying the recombinase gene, and other chemical or biological vectors for introducing the recombinase gene or protein into the target tissue or organ;
进一步地，所述组织选自神经组织、肌肉、上皮组织或结缔组织。Further, the tissue is selected from nerve tissue, muscle, epithelial tissue or connective tissue.
优选地，神经组织选自中枢神经系统及其神经环路相关组织。Preferably, the nervous tissue is selected from the central nervous system and its neural circuit-related tissues.
进一步地，所述器官选自膀胱、眼、耳、鼻、口腔、舌、咽、喉、犁鼻器、唾液腺、肝、肾、脾、心、肠道、胃、胰腺、肺、气管、血管、淋巴管、淋巴结、四肢、垂体、甲状腺、甲状旁腺、胰岛、肾上腺或生殖器。Further, the organ is selected from bladder, eye, ear, nose, oral cavity, tongue, pharynx, larynx, vomeronasal organ, salivary gland, liver, kidney, spleen, heart, intestinal tract, stomach, pancreas, lung, trachea, blood vessel, lymphatic vessel, lymph node, limbs, pituitary gland, thyroid gland, parathyroid gland, pancreatic islets, adrenal gland or genitalia.
本发明第五方面提供所述的重组腺相关病毒载体组合物、所述的重组腺相关病毒载体库、所述的外源基因的表达系统、所述的方法在生理机制研究、疾病造模、细胞调控、基因治疗或神经环路标记中的应用。rAAV-L与rAAV-R在重组酶作用下发生分子间重组后可构成完整的基因表达盒并表达外源目的基因，从而可用于观察目标组织或器官，或进行生理机制研究、疾病造模、细胞调控、基因治疗或神经环路标记等具体的研究。The fifth aspect of the present invention provides the recombinant adeno-associated virus vector composition, the recombinant adeno-associated virus vector library, the expression system of the foreign gene, and the application of the method in physiological mechanism research, disease modeling, cell regulation, gene therapy or neural circuit labeling. After rAAV-L and rAAV-R undergo intermolecular recombination under the action of recombinases, they can form a complete gene expression cassette and express exogenous target genes, which can be used to observe target tissues or organs, or to conduct specific studies such as physiological mechanism research, disease modeling, cell regulation, gene therapy, or neural circuit markers.
本发明中rAAV-L与rAAV-R各代表一类rAAV，rAAV-L与rAAV-R基因组分别可装载多种不同的元件，rAAV-L与rAAV-R在重组酶作用下发生分子间重组，从而将rAAV-L与rAAV-R中的启动子序列、目的基因序列和调控序列连接形成完整的表达盒结构，进而表达外源目的基因，该策略由于避免了拆分单个元件，而是将基因表达盒的不同元件分别装载到两个rAAV基因组中，便于尝试已有元件、新发现元件之间的组合效果(例如，新发现一种特异性启动子，仅需构建一种含该启动子的rAAV-L即可尝试与所有重组序列对应的已有rAAV-R的组合效果)，兼具了组合灵活性和更大的元件选择空间。本发明仅利用重组酶识别两个rAAV基因组上的特定序列并介导两个基因组之间重组连接，无需mRNA剪接等其他细胞内过程，避免了细胞毒性产生，更广泛适用于各种目的基因的表达。本发明可以在具有重组酶的细胞中实现细胞特异的目的基因表达，在神经科学、基因治疗等领域的相关研究中具有巨大的应用价值和前景。In the present invention, rAAV-L and rAAV-R each represent a class of rAAV. The genomes of rAAV-L and rAAV-R can be loaded with various elements, and rAAV-L and rAAV-R undergo intermolecular recombination under the action of recombinases, so that the promoter sequence, target gene sequence and regulatory sequence in rAAV-L and rAAV-R are connected to form a complete expression cassette structure, and then the exogenous target gene is expressed. Loaded into two rAAV genomes, it is convenient to try the combination effect between existing elements and newly discovered elements (for example, if a specific promoter is newly discovered, only one rAAV-L containing the promoter can be constructed to try the combination effect of the existing rAAV-R corresponding to all recombinant sequences), which has both combination flexibility and greater space for element selection. The present invention only uses recombinase to recognize specific sequences on two rAAV genomes and mediates recombination connection between the two genomes, does not need other intracellular processes such as mRNA splicing, avoids cytotoxicity, and is more widely applicable to the expression of various target genes. The invention can realize cell-specific target gene expression in cells with recombinase, and has great application value and prospect in related researches in neuroscience, gene therapy and other fields.
与现有方法相比，本发明具有以下优点：Compared with existing methods, the present invention has the following advantages:
1.本发明中rAAV载体将完整的表达盒分成两部分并构建rAAV-L与rAAV-R库，可通过挑选识别序列对应的任一rAAV-L与任一rAAV-R组合，可在含有重组酶的细胞中构成具有不同特性的表达盒，灵活改变基因表达盒元件，从而达到灵活调整外源基因表达的目的，在一定程度上替代现有的DIO(Double-floxed inversive open-reading-frame)等依赖于重组酶分子开关的策略实现细胞特异靶向的表达。基于此方法，可以实现灵活装载不同的启动子、调控序列及基因片段，达到更便捷地调整表达盒特性、更特异地标记和操控细胞等目的。1. In the present invention, the rAAV vector divides the complete expression cassette into two parts and constructs rAAV-L and rAAV-R libraries. By selecting any rAAV-L and any rAAV-R combination corresponding to the recognition sequence, expression cassettes with different characteristics can be formed in cells containing recombinases, and the elements of the gene expression cassette can be flexibly changed, so as to achieve the purpose of flexibly adjusting the expression of exogenous genes, and replace the existing DIO (Double-floxed inversive open-reading-frame) to a certain extent. The strategy of recombinase molecular switch achieves cell-specific targeted expression. Based on this method, different promoters, regulatory sequences, and gene fragments can be loaded flexibly, so as to more conveniently adjust the characteristics of the expression cassette, and more specifically mark and manipulate cells.
2.本发明具有易拓展性，发现的新元件可以扩充至rAAV库中，构建一种新rAAV即可产生多种组合方式。2. The present invention is easy to expand, and the new elements discovered can be expanded into the rAAV library, and multiple combinations can be produced by constructing a new rAAV.
3.本发明中rAAV载体基因组可使用更大的启动子、调控序列从而提高靶向特异性和效率，单个启动子和调控序列片段大小理论上最大可达单个rAAV的全部容量大小(4.7kb)，这是其他方法所不具备的。3. In the present invention, the rAAV vector genome can use larger promoters and regulatory sequences to improve targeting specificity and efficiency. The size of a single promoter and regulatory sequence fragments can theoretically reach the full capacity of a single rAAV (4.7kb), which is not available in other methods.
4.本发明中各rAAV载体使用通用的重组序列，不利用目的基因自身重组和mRNA剪接等手段，降低研发成本且避免因为剪接错误产生毒性产物。4. Each rAAV vector in the present invention uses a general recombination sequence, does not use means such as target gene recombination and mRNA splicing, reduces research and development costs and avoids the production of toxic products due to splicing errors.
5.本发明中rAAV载体基因组利用重组酶进行重组，具有微量高效性。5. In the present invention, the rAAV vector genome is recombined with recombinase, which has trace efficiency.
6.本发明可应用在特异性表达重组酶的转基因细胞、转基因动物上，从而达到选择性地表达基因的目的。6. The present invention can be applied to transgenic cells and transgenic animals specifically expressing recombinase, so as to achieve the purpose of selectively expressing genes.
7.本发明在研究组织器官结构与功能、表达蛋白和功能RNA、操控神经系统、基因治疗等相关研究中具有应用价值。7. The present invention has application value in research on the structure and function of tissues and organs, expression of protein and functional RNA, manipulation of nervous system, gene therapy and other related research.
附图说明Description of drawings
图1为核心质粒pAAV-CAG-lox71、pAAV-CAG-(ATG+)lox71、pAAV-lox66-ChR2-eGFP、pAAV-lox66-(ATG-)ChR2-eGFP、pAAV-pA-lox66-ChR2-eGFP、pAAV-pA-lox66-(ATG-)ChR2-eGFP的质粒图谱。Fig. 1 is the core plasmid pAAV-CAG-lox71, pAAV-CAG-(ATG + ) lox71, pAAV-lox66-ChR2-eGFP, pAAV-lox66-(ATG - ) ChR2-eGFP, pAAV-pA-lox66-ChR2-eGFP, pAAV-pA-lox66-(ATG - ) ChR2-eGFP Plasmid map.
图2为原始质粒的质粒图谱。Figure 2 is the plasmid map of the original plasmid.
图3为核心质粒L1、L2的构建流程图。Fig. 3 is a flowchart of the construction of core plasmids L1 and L2.
图4为核心质粒R1、R2、R3、R4的构建流程图，其中“替换175”表示的是175位T碱基突变为A碱基。Fig. 4 is a flow chart of the construction of core plasmids R1, R2, R3, R4, wherein "replacement 175" means that the T base at position 175 is mutated into an A base.
图5为rAAV-L、rAAV-R的基因组结构示例及其组合使用的方式示意图。Fig. 5 is a schematic diagram showing examples of genome structures of rAAV-L and rAAV-R and their combined use.
图6为选择rAAV库中的rAAV-L1、rAAV-L2、rAAV-R1、rAAV-R2、rAAV-R3、rAAV-R4单独或组合使用并注射至vGluT2-ires-Cre基因修饰品系小鼠或C57BL/6品系小鼠的LH脑区的结果展示图。Scale bars:250μm。Figure 6 is a graph showing the results of selecting rAAV-L1, rAAV-L2, rAAV-R1, rAAV-R2, rAAV-R3, and rAAV-R4 from the rAAV library to be used alone or in combination and injected into the LH brain region of vGluT2-ires-Cre genetically modified mice or C57BL/6 strain mice. Scale bars: 250μm.
具体实施方式Detailed ways
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and understandable, the specific implementation of the present invention will be described in detail below in conjunction with the accompanying drawings, but it should not be construed as limiting the scope of the present invention.
本发明所述技术方案，如未特别说明，均为rAAV载体领域的常规技术。The technical solutions described in the present invention are all conventional techniques in the field of rAAV vectors unless otherwise specified.
本发明提供一种重组腺相关病毒载体组合物，其包括重组腺相关病毒载体rAAV-L与重组腺相关病毒载体rAAV-R；所述rAAV-L基因组包含重组酶识别序列I和至少一个启动子序列，其中一个启动子序列记为启动子序列I，启动子序列I位于重组酶识别序列I的一侧，重组酶识别序列I位于启动子序列I的下游；所述rAAV-R基因组包含重组酶识别序列II和至少一个目的基因序列，其中一个目的基因序列记为目的基因序列I，目的基因序列I位于重组酶识别序列II的一侧，重组酶识别序列II位于目的基因序列I的上游；所述重组酶识别序列I和重组酶识别序列II为同一种重组酶的识别序列且方向相同。rAAV-L基因组结构为：-启动子序列I(顺向)-重组酶识别序列I-；rAAV-R基因组结构为：-重组酶识别序列II-目的基因序列I(顺向)-。The present invention provides a recombinant adeno-associated virus vector composition, which comprises a recombinant adeno-associated virus vector rAAV-L and a recombinant adeno-associated virus vector rAAV-R; the rAAV-L genome comprises a recombinase recognition sequence I and at least one promoter sequence, wherein one promoter sequence is marked as a promoter sequence I, the promoter sequence I is located on one side of the recombinase recognition sequence I, and the recombinase recognition sequence I is located downstream of the promoter sequence I; the rAAV-R genome comprises a recombinase recognition sequence II and at least one target gene sequence, wherein one target gene sequence is marked as For the target gene sequence I, the target gene sequence I is located on one side of the recombinase recognition sequence II, and the recombinase recognition sequence II is located upstream of the target gene sequence I; the recombinase recognition sequence I and the recombinase recognition sequence II are recognition sequences of the same recombinase and have the same direction. The rAAV-L genome structure is: -promoter sequence I (forward)-recombinase recognition sequence I-; the rAAV-R genome structure is: -recombinase recognition sequence II-target gene sequence I (forward)-.
在一个具体的实施方案中，所述rAAV-L基因组包含的另一个启动子序列记为启动子序列II，启动子序列II位于重组酶识别序列I的另一侧，重组酶识别序列I位于启动子序列II的下游；所述rAAV-R基因组包含的另一个目的基因序列记为目的基因序列II，目的基因序列II位于重组酶识别序列II的另一侧，重组酶识别序列II位于目的基因序列II的上游。rAAV-L基因组结构为：-启动子序列I(顺向)-重组酶识别序列I-启动子序列II(逆向)-；rAAV-R基因组结构为：-目的基因序列II(逆向)-重组酶识别序列II-目的基因序列I(顺向)-。In a specific embodiment, the other promoter sequence contained in the rAAV-L genome is marked as promoter sequence II, the promoter sequence II is located on the other side of the recombinase recognition sequence I, and the recombinase recognition sequence I is located downstream of the promoter sequence II; the other target gene sequence contained in the rAAV-R genome is marked as the target gene sequence II, the target gene sequence II is located on the other side of the recombinase recognition sequence II, and the recombinase recognition sequence II is located upstream of the target gene sequence II. The genome structure of rAAV-L is:-promoter sequence I (forward)-recombinase recognition sequence I-promoter sequence II (reverse)-; the genome structure of rAAV-R is:-target gene sequence II (reverse)-recombinase recognition sequence II-target gene sequence I (forward)-.
在一个具体的实施方案中，所述rAAV-L基因组还包含目的基因序列和/或调控序列，所述目的基因序列的转录被所述rAAV-L基因组的任一启动子启动。In a specific embodiment, the rAAV-L genome further comprises a target gene sequence and/or a regulatory sequence, and the transcription of the target gene sequence is initiated by any promoter of the rAAV-L genome.
在一个具体的实施方案中，所述rAAV-R基因组还包含启动子序列和/或调控序列，所述启动子序列启动所述rAAV-R基因组的任一目的基因序列的转录。In a specific embodiment, the rAAV-R genome further comprises a promoter sequence and/or a regulatory sequence, and the promoter sequence initiates the transcription of any target gene sequence of the rAAV-R genome.
在一个具体的实施方案中，重组腺相关病毒载体rAAV-L包含一个启动子序列。In a specific embodiment, the recombinant adeno-associated virus vector rAAV-L comprises a promoter sequence.
在一个具体的实施方案中，重组腺相关病毒载体rAAV-L包含一个启动子序列、调控序列、和/或一个或多个目的基因序列。In a specific embodiment, the recombinant adeno-associated virus vector rAAV-L comprises a promoter sequence, a regulatory sequence, and/or one or more target gene sequences.
在一个具体的实施方案中，重组腺相关病毒载体rAAV-R包含一个或多个目的基因序列、调控序列、和/或启动子序列。In a specific embodiment, the recombinant adeno-associated virus vector rAAV-R comprises one or more target gene sequences, regulatory sequences, and/or promoter sequences.
在一个具体的实施方案中，重组腺相关病毒载体rAAV-L包含两个启动子序列，两个启动子序列位于重组酶识别位点的两侧，重组酶识别位点位于两个启动子序列的下游。In a specific embodiment, the recombinant adeno-associated virus vector rAAV-L comprises two promoter sequences, the two promoter sequences are located on both sides of the recombinase recognition site, and the recombinase recognition site is located downstream of the two promoter sequences.
在一个具体的实施方案中，重组腺相关病毒载体rAAV-L包含两个启动子序列，两个启动子序列位于重组酶识别位点的两侧，重组酶识别位点位于两个启动子序列的下游，其还包含调控序列，和/或一个或多个目的基因序列。In a specific embodiment, the recombinant adeno-associated virus vector rAAV-L comprises two promoter sequences, and the two promoter sequences are located on both sides of the recombinase recognition site, and the recombinase recognition site is located downstream of the two promoter sequences, which also includes regulatory sequences, and/or one or more target gene sequences.
在一个具体的实施方案中，重组腺相关病毒载体组合物包括rAAV-L与rAAV-R，rAAV-L基因组包含重组酶识别序列I和一个启动子序列I，启动子序列I位于重组酶识别序列I的一侧，重组酶识别序列I位于启动子序列I的下游；rAAV-R基因组包含重组酶识别序列II和至少一个目的基因序列，其中一个目的基因序列记为目的基因序列I，目的基因序列I位于重组酶识别序列II的一侧，重组酶识别序列II位于目的基因序列I的上游；重组酶识别序列I和重组酶识别序列II为同一种重组酶的识别序列且方向相同。即rAAV-L基因组结构为：-启动子序列I(顺向)-重组酶识别序列I-；rAAV-R基因组结构为：-重组酶识别序列II-目的基因序列I(顺向)-。作为优选方案，rAAV-L与rAAV-R基因组还包含调控序列。In a specific embodiment, the recombinant adeno-associated virus vector composition includes rAAV-L and rAAV-R, the rAAV-L genome comprises a recombinase recognition sequence I and a promoter sequence I, the promoter sequence I is located on one side of the recombinase recognition sequence I, and the recombinase recognition sequence I is located downstream of the promoter sequence I; The sequence II is located upstream of the target gene sequence I; the recombinase recognition sequence I and the recombinase recognition sequence II are recognition sequences of the same recombinase and have the same direction. That is, the rAAV-L genome structure is: -promoter sequence I (forward)-recombinase recognition sequence I-; the rAAV-R genome structure is: -recombinase recognition sequence II-target gene sequence I (forward)-. As a preferred solution, the rAAV-L and rAAV-R genomes also contain regulatory sequences.
在一个具体的实施方案中，重组腺相关病毒载体组合物包括rAAV-L与rAAV-R，rAAV-L基因组包含重组酶识别序列I和2个启动子序列(启动子序列I和启动子序列II)，启动子序列I和启动子序列II位于重组酶识别序列I的两侧，重组酶识别位点I位于两个启动子序列的下游；rAAV-R基因组包含重组酶识别序列II和至少一个目的基因序列，其中一个目的基因序列记为目的基因序列I，一个目的基因序列记为目的基因序列II，目的基因序列I和目的基因序列II位于重组酶识别序列II的两侧，重组酶识别序列II位于目的基因序列I和目的基因序列II的上游。即rAAV-L基因组结构为：-启动子序列I(顺向)-重组酶识别序列I-启动子序列II(逆向)-；rAAV-R基因组结构为：-目的基因序列II(逆向)-重组酶识别序列II-目的基因序列I(顺向)-。作为优选方案，rAAV-L与rAAV-R基因组还包含调控序列。In a specific embodiment, the recombinant adeno-associated virus vector composition includes rAAV-L and rAAV-R, and the rAAV-L genome comprises a recombinase recognition sequence I and two promoter sequences (promoter sequence I and promoter sequence II), the promoter sequence I and the promoter sequence II are located on both sides of the recombinase recognition sequence I, and the recombinase recognition site I is located downstream of the two promoter sequences; , a target gene sequence is recorded as the target gene sequence II, the target gene sequence I and the target gene sequence II are located on both sides of the recombinase recognition sequence II, and the recombinase recognition sequence II is located upstream of the target gene sequence I and the target gene sequence II. That is, the rAAV-L genome structure is:-promoter sequence I (forward)-recombinase recognition sequence I-promoter sequence II (reverse)-; rAAV-R genome structure is:-target gene sequence II (reverse)-recombinase recognition sequence II-target gene sequence I (forward)-. As a preferred solution, the rAAV-L and rAAV-R genomes also contain regulatory sequences.
在一个具体实施方案中，构建重组腺相关病毒载体库，其包括rAAV-L库和rAAV-R库，将基因表达盒的不同元件分别装载到rAAV-L和rAAV-R基因组中形成。例如，rAAV-L库中的rAAV具有不同的启动子序列、调控序列，rAAV-R库中的rAAV具有不同的目的基因序列、调控序列。并且可根据研究进展随时拓展rAAV库，例如，新发现一种特异性启动子，仅需构建一种含该启动子的rAAV-L即可尝试与所有重组序列对应的已有rAAV-R的组合效果。rAAV-L与对应的rAAV-R组合使用，在重组酶作用下发生分子间重组后可构成完整的基因表达盒并表达外源目的基因。In a specific embodiment, a recombinant adeno-associated virus vector library is constructed, which includes an rAAV-L library and an rAAV-R library, and different elements of the gene expression cassette are respectively loaded into the rAAV-L and rAAV-R genomes to form. For example, the rAAV in the rAAV-L library has different promoter sequences and regulatory sequences, and the rAAV in the rAAV-R library has different target gene sequences and regulatory sequences. And the rAAV library can be expanded at any time according to the research progress. For example, if a specific promoter is newly discovered, only one rAAV-L containing the promoter can be constructed to try the combination effect of the existing rAAV-R corresponding to all recombinant sequences. When rAAV-L is used in combination with the corresponding rAAV-R, a complete gene expression cassette can be formed and an exogenous target gene can be expressed after intermolecular recombination occurs under the action of recombinase.
在一个具体实施方案中，提供一种表达外源基因的方法，可通过如下(1)-(3)中的一种方法实现：In a specific embodiment, a method for expressing a foreign gene is provided, which can be achieved by one of the following methods (1)-(3):
(1)将所述的重组腺相关病毒载体组合物注射至目标组织或器官中，所述组织或器官的部分或全部细胞表达重组酶基因；(1) injecting the recombinant adeno-associated virus vector composition into the target tissue or organ, some or all cells of the tissue or organ express the recombinase gene;
(2)将所述的重组腺相关病毒载体组合物注射至目标组织或器官中，所述重组腺相关病毒载体rAAV-L或rAAV-R表达重组酶基因；(2) injecting the recombinant adeno-associated virus vector composition into the target tissue or organ, and the recombinant adeno-associated virus vector rAAV-L or rAAV-R expresses a recombinase gene;
(3)将所述的重组腺相关病毒载体组合物注射至目标组织或器官中，并通过其他途径导入重组酶。可通过将携带重组酶基因的表达质粒、携带重组酶基因的病毒载体、其他导入重组酶基因或蛋白的化学或生物载体中的一种导入至目标组织或器官实现重组酶的导入。(3) Inject the recombinant adeno-associated virus vector composition into the target tissue or organ, and introduce the recombinase through other ways. The introduction of the recombinase can be achieved by introducing one of the expression plasmid carrying the recombinase gene, the virus vector carrying the recombinase gene, and other chemical or biological vectors for introducing the recombinase gene or protein into the target tissue or organ.
rAAV-L与rAAV-R在重组酶作用下发生分子间重组后可构成完整的基因表达盒并表达外源目的基因，从而可用于观察目标组织或器官，或进行生理机制研究、疾病造模、细胞调控、基因治疗或神经环路标记等具体的研究。After rAAV-L and rAAV-R undergo intermolecular recombination under the action of recombinases, they can form a complete gene expression cassette and express exogenous target genes, which can be used to observe target tissues or organs, or to conduct specific studies such as physiological mechanism research, disease modeling, cell regulation, gene therapy, or neural circuit markers.
本发明实施方案中，重组酶识别序列包括但不限于loxP、lox66、lox71、lox2272、lox5171、loxm2、JT15、JTZ17、JT510、FRT、F3、F5、FRT-LE、FRT-RE、mFRT71、Rox、VloxP、Vlox2272、SloxP、Vox、att。In an embodiment of the present invention, the recombinase recognition sequence includes but is not limited to loxP, lox66, lox71, lox2272, lox5171, loxm2, JT15, JTZ17, JT510, FRT, F3, F5, FRT-LE, FRT-RE, mFRT71, Rox, VloxP, Vlox2272, SloxP, Vox, att.
启动子序列选自CAG、CaMKIIα、CAR、CBA、CD68、c-fos、ChAT、CMV、CR、Ef1α、E-SARE、GAD67、GFAP、GFAP104、gfaABC1D、Grm6、hGRK1、hSyn、hUbC、LP1B、L7/Pcp2、MBP、MCK、mDlX、mOXT、mTH、nEF、Nestin、NPY、Nrl、PGK、PV、RAM、RK、ROH、RPE65、SFRP2、SST、TBG、TCAP、TH、Thy1、TPH2、TRE、TRPV1、UAS和Vgat中的一种或以上。Promoter sequence selected from CAG, CaMKIIα, CAR, CBA, CD68, c-fos, ChAT, CMV, CR, Ef1α, E-SARE, GAD67, GFAP, GFAP104, gfaABC1D, Grm6, hGRK1, hSyn, hUbC, LP1B, L7/Pcp2, MBP, MCK, mDlX, mOXT, mTH, nEF, N One or more of estin, NPY, Nrl, PGK, PV, RAM, RK, ROH, RPE65, SFRP2, SST, TBG, TCAP, TH, Thy1, TPH2, TRE, TRPV1, UAS and Vgat.
目的基因序列选自用于研究组织器官结构与功能、过表达基因产物、操控神经系统、基因治疗相关的蛋白的核苷酸编码序列和/或功能RNA产物的基因等。所述蛋白优选为荧光蛋白、激活神经元蛋白、抑制神经元蛋白、钙离子信号探针蛋白、小分子信号探针蛋白、介导凋亡蛋白、疾病相关突变蛋白、生理条件下的正常蛋白、细胞因子、抗病毒因子、病毒感染辅助受体、重组酶和基因编辑工具蛋白中一种或多种；所述功能性RNA选自小RNA、小干扰RNA、小发卡RNA、小向导RNA、细胞器定位RNA和用于RNA测序或原位杂交分析的Barcode RNA中的一种或多种。The target gene sequence is selected from genes used for studying the structure and function of tissues and organs, overexpressing gene products, manipulating the nervous system, nucleotide coding sequences of proteins related to gene therapy and/or functional RNA products, etc. The protein is preferably one or more of fluorescent protein, neuron-activating protein, neuron-inhibiting protein, calcium ion signal probe protein, small molecule signal probe protein, apoptosis-mediated protein, disease-associated mutant protein, normal protein under physiological conditions, cytokine, antiviral factor, virus infection co-receptor, recombinase, and gene editing tool protein; the functional RNA is selected from one or more of small RNA, small interfering RNA, small hairpin RNA, small guide RNA, organelle-localized RNA, and Barcode RNA for RNA sequencing or in situ hybridization analysis.
调控序列包括转录和/或转录后调控序列。所述调控序列选自WPRE、oPRE、cw3sl、SV40polyA、hGH polyA、bGH polyA、rbGlob polyA等中的一种或以上。Regulatory sequences include transcriptional and/or post-transcriptional regulatory sequences. The regulatory sequence is selected from one or more of WPRE, oPRE, cw3sl, SV40 polyA, hGH polyA, bGH polyA, rbGlob polyA and the like.
rAAV-L与rAAV-R具有相同或不同的血清型，且具有感染组织或器官中同一细胞的能力。所述血清型包括AAV1、AAV2、AAV2-Retro、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV9-Retro、AAV10、AAV11、AAV12、AAV13、AAV-DJ、AAV-PHP.eB、AAV-myo及其衍生血清型。rAAV-L and rAAV-R have the same or different serotypes and have the ability to infect the same cells in tissues or organs. The serotypes include AAV1, AAV2, AAV2-Retro, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9-Retro, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV-PHP.eB, AAV-myo and derivative serotypes thereof.
下面结合具体实施例说明本发明技术方案。The technical solutions of the present invention will be described below in conjunction with specific embodiments.
实施例1：rAAV库中部分载体的设计与制备Example 1: Design and preparation of some vectors in the rAAV library
构建装载rAAV核心元件的质粒。质粒分别为：Construction of plasmids loaded with rAAV core elements. The plasmids are:
L1：pAAV-CAG-lox71，核苷酸序列如SEQ ID NO.1所示，质粒图谱如图1，其中含有启动子CAG和lox71重组酶识别序列；L1: pAAV-CAG-lox71, the nucleotide sequence is shown in SEQ ID NO.1, and the plasmid map is shown in Figure 1, which contains the promoter CAG and lox71 recombinase recognition sequence;
L2：pAAV-CAG-(ATG+)lox71，核苷酸序列如SEQ ID NO.2所示，质粒图谱如图1，其中含有启动子CAG和lox71重组酶识别序列，lox71前加入了起始密码子和Kozak序列；L2: pAAV-CAG-(ATG + )lox71, the nucleotide sequence is shown in SEQ ID NO.2, and the plasmid map is shown in Figure 1, which contains the promoter CAG and lox71 recombinase recognition sequence, and the start codon and Kozak sequence are added before lox71;
R1：pAAV-lox66-ChR2-eGFP，核苷酸序列如SEQ ID NO.3所示，质粒图谱如图1，其中含有lox66重组酶识别序列、ChR2-eGFP融合蛋白及尾端调控序列WPRE和polyA；R1: pAAV-lox66-ChR2-eGFP, the nucleotide sequence is shown in SEQ ID NO.3, and the plasmid map is shown in Figure 1, which contains the lox66 recombinase recognition sequence, ChR2-eGFP fusion protein, and tail regulatory sequences WPRE and polyA;
R2：pAAV-lox66-(ATG-)ChR2-eGFP，核苷酸序列如SEQ ID NO.4所示，质粒图谱如图1，其中含有lox66重组酶识别序列、ChR2-eGFP融合蛋白及尾端调控序列WPRE和polyA，融合蛋白起始端的起始密码子和Kozak序列被删除；R2: pAAV-lox66-(ATG - )ChR2-eGFP, the nucleotide sequence is shown in SEQ ID NO.4, and the plasmid map is shown in Figure 1, which contains the lox66 recombinase recognition sequence, ChR2-eGFP fusion protein and tail regulatory sequences WPRE and polyA, and the start codon and Kozak sequence at the beginning of the fusion protein are deleted;
R3：pAAV-pA-lox66-ChR2-eGFP，核苷酸序列如SEQ ID NO.5所示，质粒图谱如图1，其中含有polyA调控序列、lox66重组酶识别序列、ChR2-eGFP融合蛋白及尾端调控序列WPRE和polyA；R3: pAAV-pA-lox66-ChR2-eGFP, the nucleotide sequence is shown in SEQ ID NO.5, and the plasmid map is shown in Figure 1, which contains polyA regulatory sequence, lox66 recombinase recognition sequence, ChR2-eGFP fusion protein and tail regulatory sequences WPRE and polyA;
R4：pAAV-pA-lox66-(ATG-)ChR2-eGFP，核苷酸序列如SEQ ID NO.6所示，质粒图谱如图1，其中含有polyA调控序列、lox66重组酶识别序列、ChR2-eGFP融合蛋白及尾端调控序列WPRE和polyA，融合蛋白起始端的起始密码子和Kozak序列被删除。R4: pAAV-pA-lox66-(ATG - )ChR2-eGFP, the nucleotide sequence is shown in SEQ ID NO.6, and the plasmid map is shown in Figure 1, which contains polyA regulatory sequence, lox66 recombinase recognition sequence, ChR2-eGFP fusion protein and tail regulatory sequences WPRE and polyA, and the start codon and Kozak sequence at the beginning of the fusion protein are deleted.
本发明中的核心质粒是基于实验室已有或购买的原始质粒进行构建，质粒图谱如图2所示，通过分子克隆手段重构质粒得到，其中的重组酶识别序列lox66、lox71通过设计特殊引物，并使用引物退火-酶连的方法引入质粒中。核心质粒L1、L2的构建流程如图3，核心质粒R1、R2、R3、R4的构建流程如图4。其中原始质粒AAV-CAG-hChR2_tdTomato为图3中的Addgene#28015。The core plasmid in the present invention is constructed based on the existing or purchased original plasmid in the laboratory. The plasmid map is shown in Figure 2. It is obtained by reconstructing the plasmid by means of molecular cloning. The recombinase recognition sequences lox66 and lox71 are introduced into the plasmid by designing special primers and using the method of primer annealing-enzyme ligation. The construction process of core plasmids L1 and L2 is shown in Figure 3, and the construction process of core plasmids R1, R2, R3, and R4 is shown in Figure 4. The original plasmid AAV-CAG-hChR2_tdTomato is Addgene #28015 in Fig. 3 .
lox66、lox71的引物序列如下：The primer sequences of lox66 and lox71 are as follows:
Lox71F:AATTCTACCGTTCGTATAGCATACATTATACGAAGTTATGC；SEQ ID NO.7Lox71F:AATTCTACCGTTCGTATAGCATACATTATACGAAGTTATGC; SEQ ID NO.7
Lox71R:GGCCGCATAACTTCGTATAATGTATGCTATACGAACGGTAG；SEQ ID NO.8Lox71R: GGCCGCATAACTTCGTATAATGTATGCTATACGAACGGTAG; SEQ ID NO.8
Lox66F:CTAGTATAACTTCGTATAGCATACATTATACGAACGGTAG；SEQ ID NO.9Lox66F:CTAGTATAACTTCGTATAGCATACATTATACGAACGGTAG; SEQ ID NO.9
Lox66R:GATCCTACCGTTCGTATAATGTATGCTATACGAAGTTATA；SEQ ID NO.10Lox66R: GATCCTACCGTTCGTATAATGTATGCTATACGAAGTTATA; SEQ ID NO. 10
核心质粒L1、L2、R1、R2、R3、R4涉及到的引物序列如下：The primer sequences involved in core plasmids L1, L2, R1, R2, R3, and R4 are as follows:
引物1F：TCTTCTTTTTCCTACAGGGATCCGCAACGTGCTGGTTATT；SEQ ID NO.11Primer 1F: TCTTCTTTTTTCCTACAGGGATCCGCAACGTGCTGGTTATT; SEQ ID NO.11
引物1R：TGCGGCCGCACTAGTTCGGTCCGTCTCCCCCTGAACCTGAAAC；SEQ ID NO.12Primer 1R: TGCGGCCGCACTAGTTCGGTCCGTCTCCCCCCTGAACCTGAAAC; SEQ ID NO.12
引物2F：GGCAAAGAATTCGCCACCATGGGTACCGTTCGTATAGCAT；SEQ ID NO.13Primer 2F: GGCAAAGAATTCGCCACCATGGGTACCGTTCGTATAGCAT; SEQ ID NO.13
引物2R：TGCGGCCGCACTAGTTCGGTCCGTCTCCCCCTGAACCTGAAAC；SEQ ID NO.14Primer 2R: TGCGGCCGCACTAGTTCGGTCCGTCTCCCCCCTGAACCTGAAAC; SEQ ID NO.14
引物3F：CCTCTAGAGCCACCATGGACTATGGCGGCGCT；SEQ ID NO.15Primer 3F: CCTCTAGAGCCACCATGGACTATGGCGGCGCT; SEQ ID NO.15
引物3R：CTTGTACAGCTCGTCCATG；SEQ ID NO.16Primer 3R: CTTGTACAGCTCGTCCATG; SEQ ID NO.16
引物4F：AGAGGTTGATTATCGATAAGCT；SEQ ID NO.17Primer 4F: AGAGGTTGATTATCGATAAGCT; SEQ ID NO.17
引物4R：GGTAGGATCCTCTAGAGACTATGGCGGCGCTTTGT；SEQ ID NO.18Primer 4R: GGTAGGATCCTCTTAGAGACTATGGCGGCGCTTTGT; SEQ ID NO.18
引物5F：CTAGGGGTTCCTGCGGCCGCATCGAGCAGACAT；SEQ ID NO.19Primer 5F: CTAGGGGTTCCTGCGGCCGCATCGAGCAGACAT; SEQ ID NO.19
引物5R：TCTAGTCAATAATCAATGTCAACTTGTTTATTGCAGCTTATAATGGTT；SEQ ID NO.20Primer 5R: TCTAGTCAATAATCAATGTCAACTTGTTTATTGCAGCTTATAATGGTT; SEQ ID NO. 20
制备rAAV载体采用三质粒包装系统。针对核心质粒L1、L2、R1、R2、R3、R4，分别将每种核心质粒与腺病毒辅助质粒pAd-Helper、AAV衣壳质粒pAAV-RC2/9三种质粒按相同质粒分子数共转染HEK-293T细胞。转染72小时后收集细胞培养物，用碘克沙醇密度梯度离心法进行浓缩和纯化，最后用SYBR Green qPCR法检测rAAV滴度，最终获得六种rAAV。rAAV分别为：The rAAV vector was prepared using a three-plasmid packaging system. For the core plasmids L1, L2, R1, R2, R3, and R4, each core plasmid was co-transfected into HEK-293T cells with the adenovirus helper plasmid pAd-Helper and the AAV capsid plasmid pAAV-RC2/9, respectively, according to the same number of plasmid molecules. Cell cultures were collected 72 hours after transfection, concentrated and purified by iodixanol density gradient centrifugation, and finally the titer of rAAV was detected by SYBR Green qPCR, and finally six kinds of rAAV were obtained. rAAV are:
rAAV-L1：rAAV9-CAG-lox71，病毒的滴度为6.3×1011VG/mL；rAAV-L1: rAAV9-CAG-lox71, the virus titer is 6.3×10 11 VG/mL;
rAAV-L2：rAAV9-CAG-(ATG+)lox71，病毒的滴度为2.8×1012VG/mL；rAAV-L2: rAAV9-CAG-(ATG + )lox71, the virus titer is 2.8×10 12 VG/mL;
rAAV-R1：rAAV9-lox66-ChR2-eGFP，病毒的滴度为1.5×1013VG/mL；rAAV-R1: rAAV9-lox66-ChR2-eGFP, the virus titer is 1.5×10 13 VG/mL;
rAAV-R2：rAAV9-lox66-(ATG-)ChR2-eGFP，病毒的滴度为5.5×1012VG/mL；rAAV-R2: rAAV9-lox66-(ATG - )ChR2-eGFP, the virus titer is 5.5×10 12 VG/mL;
rAAV-R3：rAAV9-pA-lox66-ChR2-eGFP，病毒的滴度为8.5×1012VG/mL；rAAV-R3: rAAV9-pA-lox66-ChR2-eGFP, the virus titer is 8.5×10 12 VG/mL;
rAAV-R4：rAAV9-pA-lox66-(ATG-)ChR2-eGFP，病毒的滴度为1.2×1013VG/mL。rAAV-R4: rAAV9-pA-lox66-(ATG − )ChR2-eGFP, the virus titer is 1.2×10 13 VG/mL.
实施例2：rAAV载体组合效果的活体测试Embodiment 2: In vivo test of rAAV vector combination effect
图5为rAAV-L、rAAV-R的基因组结构示例及其组合使用的方式示意图。将rAAV-L和rAAV-R组合使用，组一为rAAV-L1+rAAV-R1，组二为rAAV-L1+rAAV-R3，组三为rAAV-L2+rAAV-R2，组四为rAAV-L2+rAAV-R4。Fig. 5 is a schematic diagram showing examples of genome structures of rAAV-L and rAAV-R and their combined use. Use rAAV-L and rAAV-R in combination, group 1 is rAAV-L1+rAAV-R1, group 2 is rAAV-L1+rAAV-R3, group 3 is rAAV-L2+rAAV-R2, group 4 is rAAV-L2+rAAV-R4.
针对每一组病毒，将两种病毒按体积1:1混合后，通过脑立体定位注射法注射300nL病毒混合溶液于vGluT2-Cre转基因小鼠的LH脑区。vGluT2-Cre转基因小鼠的谷氨酸能神经元中表达Cre重组酶，当两种病毒感染同一谷氨酸能神经元时其基因组会发生交叉反应，从而表达ChR2-eGFP融合蛋白。For each group of viruses, the two viruses were mixed at a volume ratio of 1:1, and then 300 nL of the mixed virus solution was injected into the LH brain region of vGluT2-Cre transgenic mice by brain stereotaxic injection. The glutamatergic neurons of vGluT2-Cre transgenic mice express Cre recombinase, and when the two viruses infect the same glutamatergic neurons, their genomes will cross-react, thereby expressing the ChR2-eGFP fusion protein.
作为没有重组酶的对照，将两种病毒按体积1:1混合后，通过脑立体定位注射法注射300nL病毒混合溶液于C57BL/6小鼠的LH脑区。As a control without recombinase, the two viruses were mixed 1:1 by volume, and 300 nL of the virus mixed solution was injected into the LH brain region of C57BL/6 mice by stereotaxic injection.
作为没有rAAV-L的对照，通过脑立体定位注射法分别注射300nL rAAV-R1、rAAV-R2、rAAV-R3、rAAV-R4病毒溶液于vGluT2-Cre转基因小鼠的LH脑区。As a control without rAAV-L, 300 nL of rAAV-R1, rAAV-R2, rAAV-R3, and rAAV-R4 virus solutions were injected into the LH brain region of vGluT2-Cre transgenic mice by stereotaxic injection.
三周后通过心脏灌流取小鼠脑，经过多聚甲醛固定、蔗糖溶液脱水后，使用冷冻切片机切成40μm厚度的切片用于成像。Three weeks later, mouse brains were obtained by cardiac perfusion, fixed with paraformaldehyde and dehydrated with sucrose solution, and cut into 40 μm thick slices for imaging using a cryostat.
活体检测结果如图6所示，对于四组病毒，在vGluT2-Cre转基因小鼠的LH脑区内均有较明显的荧光蛋白表达，而在C57BL/6小鼠的LH脑区内荧光明显较少。在单独注射rAAV-R1、rAAV-R2、rAAV-R3、rAAV-R4的vGluT2-Cre转基因小鼠的LH脑区中，仅有rAAV-R1出现了较明显的荧光，这可能是由于高滴度病毒注射量过大导致的泄露表达。The in vivo detection results are shown in Figure 6. For the four groups of viruses, there is obvious expression of fluorescent protein in the LH brain region of vGluT2-Cre transgenic mice, while the fluorescence in the LH brain region of C57BL/6 mice is significantly less. In the LH brain region of vGluT2-Cre transgenic mice injected with rAAV-R1, rAAV-R2, rAAV-R3, and rAAV-R4 alone, only rAAV-R1 showed obvious fluorescence, which may be due to the leakage expression caused by the excessive injection of high titer virus.
以上结果表明，作为示例的rAAV-L和rAAV-R可以在重组酶的作用下发生重组反应并且表达目的基因，且当使用合适的策略时，可以较好防止目的基因的泄露表达。因此，构建rAAV-L库与rAAV-R库并灵活组合使用是一种可行的策略。The above results show that rAAV-L and rAAV-R as examples can undergo a recombination reaction under the action of recombinase and express the target gene, and when using an appropriate strategy, the leaky expression of the target gene can be better prevented. Therefore, it is a feasible strategy to construct rAAV-L library and rAAV-R library and use them flexibly in combination.
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