WO2022077330A1 - 一种新型g基因及其在高效逆向跨单突触中的应用 - Google Patents

一种新型g基因及其在高效逆向跨单突触中的应用 Download PDF

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WO2022077330A1
WO2022077330A1 PCT/CN2020/121122 CN2020121122W WO2022077330A1 WO 2022077330 A1 WO2022077330 A1 WO 2022077330A1 CN 2020121122 W CN2020121122 W CN 2020121122W WO 2022077330 A1 WO2022077330 A1 WO 2022077330A1
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gene
rabies virus
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recombinant adeno
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贾凡
徐富强
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中国科学院深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
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    • C07K14/145—Rhabdoviridae, e.g. rabies virus, Duvenhage virus, Mokola virus or vesicular stomatitis virus
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

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  • the invention belongs to the field of biotechnology, and in particular relates to a novel rabies virus G gene and its application in high-efficiency reverse trans-single-synapse.
  • the human brain is one of the most complex systems in nature, and neural networks are the basis for the functioning of the brain.
  • the normal connection of the neural network enables the human body to produce normal physiological activities, such as cognition, learning, memory and fear.
  • Abnormal neural networks often lead to the emergence of neurological diseases, such as Alzheimer's disease, Parkinson's disease, depression, etc., but there is no effective means to treat these neurological diseases.
  • the normal physiological activities and pathogenic mechanisms are not clear, the main reason is that people lack the information of brain neural network connections. Therefore, it is of great significance to develop a high-precision brain functional connection map for the study of brain neural circuits, which is of great significance for understanding human physiological activities and pathogenic mechanisms.
  • the Chinese government attaches great importance to the research of brain science.
  • Rabies virus is a member of the genus Lyssavirus of the family Rhabdoviridae, which infects nerve cells and is transported in neural networks. Its genome is negative-strand RNA, about 12kb in length, encoding five proteins, namely N, P, M, G and L. Among them, the G protein, as the outer membrane protein, is packaged into complete virus particles, but it has no effect on the replication of the virus. The transmission direction of rabies virus between neurons is reverse, that is, it can only be transmitted from the postsynaptic membrane to the presynaptic membrane. In 2007, Ian R.
  • Wickersham took the rabies virus vaccine strain SAD B19 as the object, deleted the G gene, and inserted the eGFP gene into the position of the G gene to express the G protein, which is compatible with the N, P, M and L proteins in the system. Together, we constructed a visualized recombinant rabies virus and achieved in vivo labeling of neurons in the mouse brain.
  • Edward M. Callaway and Liqun Luo clearly pointed out that the output or input signals marked by current neural circuit tracing tools only represent a small part of the real connections. Although rabies virus-based retrograde transsynaptic tools are recognized and used, the transsynaptic efficiency is low, making rabies virus only partially useful for retrograde labeling of upstream networks.
  • the present invention proposes a novel rabies virus G gene and its application in efficient reverse trans-single-synapse.
  • the present invention first rearranges the rabies virus G gene sequence to obtain a novel rabies virus G gene, Its nucleotide sequence is shown in SEQ ID NO.1; and then a recombinant adeno-associated virus expressing the novel rabies virus G gene is prepared.
  • the present invention provides a novel rabies virus G gene, the nucleotide sequence of which is shown in SEQ ID NO.1:
  • the sequence of the new G gene obtained after rearrangement is different from the original sequence of the G gene and the oG gene with the highest expression efficiency reported in the literature, and is a completely new sequence.
  • the novel rabies virus G gene can enhance the expression level of the rabies virus G protein and efficiently assist the rabies virus to cross the single synapse in the reverse direction.
  • the present invention also provides a preparation method of recombinant adeno-associated virus, comprising the following steps:
  • the step (1) includes the step of obtaining the codon pair bias value of the whole gene.
  • SEQ ID NO.2 and SEQ ID NO.1 are respectively inserted into the carrier in sequence to obtain a cloned plasmid, and the cloned plasmid and the packaging plasmid of the adeno-associated virus are co-transfected into cells, The cells were collected and lysed and purified to obtain a recombinant adeno-associated virus expressing the novel rabies virus G gene.
  • SEQ ID NO.2 is the Syn promoter sequence, which can enhance the expression level of the novel rabies virus G gene in the present invention.
  • SEQ ID NO.1 is the novel rabies virus G gene sequence of the present invention, and the novel rabies virus G gene obtained by the present invention is named ooG.
  • the vector is pdsAAV-CBAp-eGFP, and the cell is HEK293 cell.
  • the present invention also provides a recombinant adeno-associated virus, which is prepared by the preparation method.
  • the present invention provides a method for labeling a neural circuit, comprising the steps of: injecting the recombinant adeno-associated virus and rabies virus expressing rabies virus N, P, M and L proteins into the mammalian hippocampus brain region respectively, and after infection
  • the animals were anesthetized, perfused with normal saline, and then fixed with paraformaldehyde, and then the brain tissue was placed in a sucrose solution; the brain tissue was embedded and frozen and sliced; observed with a fluorescence microscope.
  • the mammal is any one of a mouse, a rabbit, a monkey, and a human.
  • the application object is not limited to mice, but can also be used for neural circuit labeling in monkeys, rabbits and humans.
  • the present invention also provides the application of the novel rabies virus G gene in the preparation of a rabies virus reverse transsynaptic tracing tool.
  • the present invention also provides the application of the novel rabies virus G gene in neural circuit marking.
  • the present invention achieves the following technical effects:
  • the present invention has prepared a highly efficient recombinant adeno-associated virus that assists rabies virus in the reverse synapse, which has high G protein expression efficiency, can better assist rabies virus in the reverse synapse, and facilitates related research.
  • the present invention provides a method for improving gene design for assisting trans-synaptic virus efficiency, which is convenient for improving the expression efficiency of the virus and plays an important role in carrying out related research.
  • the present invention provides a G protein sequence that efficiently assists the reverse trans-synapse of rabies virus, which is convenient for carrying out related research.
  • the present invention is important for basic research (such as pathogenic mechanism, replication mechanism, etc.) and applied research (such as neural circuit markers, cell gene therapy, oncolytic virus, new vaccines and diagnostic reagents, etc.) of rabies virus. practical significance and extensive application value.
  • Analyzing the structure of neural circuits is the basis of brain research.
  • a good tool for labeling neural circuits is of great significance for analyzing the structures of neural circuits.
  • Efficient expression of the G protein that assists the reverse trans-synapse of rabies virus can improve the efficiency of the reverse trans-synapse of rabies virus, and can be used as a neural circuit labeling tool.
  • Figure 1 shows the comparison between the codon pair preference value of ooG and the codon pair preference value of oG in the context of the whole mouse genome.
  • FIG. 2 is a schematic diagram of the construction of a recombinant adeno-associated virus that efficiently assists rabies virus in reverse transsynapses; the promoter is Syn; the G protein gene of rabies virus is an optimized new sequence, named ooG, which is the expression efficiency ratio The original protein as well as the oG proteins reported in the literature are better G proteins.
  • Figure 3 is a schematic diagram of the application of a highly efficient rabies virus-assisted reverse transsynaptic recombinant adeno-associated virus in neural circuit labeling.
  • the present invention provides a preparation method of recombinant adeno-associated virus, which is characterized in that comprising the following steps:
  • SEQ ID NO.2 is the Syn promoter sequence, which can drive the transcription of the novel rabies virus G gene in the present invention.
  • SEQ ID NO.1 is the novel rabies virus G gene sequence of the present invention, and the novel rabies virus G gene obtained by the present invention is named ooG, which has the ability to express efficiently.
  • the strategy used to obtain the new gene was to use the original virus G protein chimera of different strains, and to use the method of codon usage frequency to sequence the sequence. optimize.
  • the novel rabies virus G gene obtained by the present invention is named ooG, and the strategy for obtaining the new sequence is different from the strategy for obtaining the oG in the literature.
  • the method of the present invention calculates the codons by using the method established by Coleman JR according to the whole genome sequence information GRCm38 of mice.
  • the preference value of the pair is obtained, and the codon pair preference value of the whole gene of the mouse is obtained, and then the sequence rearrangement is carried out for the oG gene to obtain.
  • the present invention also provides the application steps of the recombinant adeno-associated virus in neural circuit labeling: taking the recombinant adeno-associated virus and rabies virus expressing rabies virus N, P, M and L proteins and injecting them into mammalian hippocampus respectively
  • the animals were anesthetized after infection, perfused with normal saline, and then fixed with paraformaldehyde, and then the brain tissue was placed in sucrose solution; the brain tissue was embedded and frozen and then sliced; observed with a fluorescence microscope.
  • the mammal is any one of mouse, rabbit, monkey and human.
  • the application object is not limited to mice, but can also be used for neural circuit labeling in monkeys, rabbits and humans.
  • Example 1 Obtaining the ooG gene sequence in the recombinant adeno-associated virus that efficiently assists rabies virus in reverse transsynapses
  • codon pair preference value refers to: the virus and the host
  • the codon pairs of its genome-encoded proteins have species-dependent preferences, for example, the codon pair combinations of amino acid pairs A-E are GCTGAA, GCTGAG, GCCGAA, GCCGAG, GCAGAA, GCAGAG, GCGGAA and GCGGAG, the frequency of these codon pairs used in different hosts varies greatly, that is, a certain codon pair has a preference in a certain species.
  • Example 2 Preparation of recombinant adeno-associated virus with high efficiency to help rabies virus reverse synapse
  • the Syn promoter (see SEQ ID NO. 2 for the sequence) and ooG (see SEQ ID NO. 1 for the sequence) were synthesized by means of whole gene synthesis, and the Syn promoter and ooG were inserted into the double-stranded AAV core as shown in Figure 2.
  • Plasmid pdsAAV-CBAp-eGFP Choen Ling et al., Hum Gene Ther. 2014
  • the cloned plasmid was obtained and named pscAAV-hsynP-DIO-ooG-bGHpA.
  • the primers for amplifying the corresponding sequences are as follows:
  • the pdsAAV-CBAp-eGFP vector was digested with KpnI and HindIII, and then SEQ ID NO.2 and SEQ ID NO.1 were sequentially inserted into the vector by homologous recombination, and the clone was obtained and named pscAAV-hsynP-DIO- ooG-bGHpA.
  • the primers used in the PCR of the present invention are all synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and the constructed clones are all verified by sequencing in Sangon Bioengineering (Shanghai) Co., Ltd.
  • Plasmid transfection of HEK293 cells to package rAAV HEK293 cells were digested with trypsin one day in advance and then plated into a 15cm culture dish for adherent culture. The cell confluence was about 80% during transfection.
  • the three plasmids required for packaging rAAV namely pAAV-Helper plasmid, serotype plasmid pAAV-RC, and core expression plasmid pscAAV-hsynP-DIO-ooG-bGHpA, were used to transfect HEK293 cells with calcium phosphate or PEI transfection reagents. After 72 hours, the culture supernatant and cells were collected.
  • the liquid from the 40% iodixanol gradient layer was sucked with a needle, dialyzed with PBS overnight, and then centrifuged and concentrated to a final volume of about 1 mL with an Amicon ultra-4 (100KD cutoff) retention column.
  • Fluorescence quantitative PCR to detect the titer of rAAV use specific primers to perform fluorescent quantitative PCR to detect the DNA copy number of rAAV virions, that is, the number of rAAV VG (indicated by Viral genomes), converted into the content of rAAV per unit volume, That is, the titer of rAAV is expressed as VG/mL (Viral genomes/mL).
  • VG/mL Virtual genomes/mL
  • Example 3 The recombinant adeno-associated virus of the present invention can efficiently assist rabies virus in reverse trans-synapse
  • the recombinant adeno-associated virus prepared in Example 2 and the recombinant adeno-associated virus expressing TVA and eGFP reported in the literature were mixed according to a ratio of 1:1; on the other hand, according to 1:1: Mix 1 ratio of recombinant adeno-associated virus reported in literature expressing oG protein (Euiseok J.Kim et al., Cell reports, 2016) and recombinant adeno-associated virus reported in literature expressing TVA and eGFP (Fumitaka Osakada et al., Neuron. 2011) .
  • the recombinant adeno-associated virus expressing TVA and eGFP reported in the literature provides the N, P, M and L proteins required for rabies virus packaging
  • the recombinant adeno-associated virus of the present invention provides the G protein of rabies virus.
  • the trans-synaptic region of Figure 3A shows that the recombinant adeno-associated virus expressing ooG of the present invention is related to the recombinant adeno-associated virus expressing oG gene with the best reverse trans-synaptic effect of assisting rabies virus in the prior art.
  • the presynaptic location showed a marked increase in the number of rabies viruses that had crossed over the virus.
  • the ordinate of Fig. 3B is the trans-synaptic efficiency index.
  • the ooG is significantly higher than the oG, indicating that the adeno-associated virus expressing ooG of the present invention is better than the oG-expressing virus published in the literature.
  • Adeno-associated virus can better help rabies virus to cross synapses and improve the efficiency of rabies virus across synapses.
  • the above results show that a recombinant adeno-associated virus highly expressing ooG of the present invention can assist rabies virus to be transported in neural networks more efficiently, and has the ability to label brain neural circuits.
  • the labeling includes the following steps: taking 0.8 ⁇ l of recombinant adeno-associated virus and injecting it into the ventral hippocampal brain region of Thy1-Cre mice; 21 days after infection, taking 0.15 ⁇ l of rabies virus SAD ⁇ G-mCherry (Fumitaka Osakada et al.) al., Neuron. 2011) was also injected into the ventral hippocampus of Thy1-Cre mice. After 7 days, the animals were anesthetized, perfused with 0.9% (V/V) normal saline, and then with 4% (V/V) paraformaldehyde.
  • the brain tissue was taken out and soaked in 4% (V/V) paraformaldehyde solution, and then the brain tissue was first placed in 20% (V/V) sucrose solution for 1 day, and then placed in 30% (V/V) solution 2 days in sucrose solution; cut the bottom of the brain tissue flat, place it on the base to embed and freeze for 1 hour, then slice; take the brain slice and observe it with a fluorescence microscope, and the position of rabies virus can be tracked according to the fluorescence signal.
  • novel rabies virus G gene of the present invention and the recombinant adeno-associated virus expressing the gene have various applications in the research platform of brain neural circuit, including rearranging any viral gene using the mouse codon pair preference value rearrangement method provided by the present invention , used to increase or decrease the expression level of proteins for cell therapy, gene therapy, oncolytic virus and vaccine development, or to mark mammalian brain neural circuits.
  • the application object is not limited to mice, but can also be used for neural circuit markers in monkeys and other animals; the ooG gene used in the present invention is only used as a paradigm, therefore, it can also be used for rearrangement of other genes.

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Abstract

提供了一种新型狂犬病毒G基因及其在高效逆向跨单突触中的应用，首先重排狂犬病毒G基因序列，获得的新型狂犬病毒G基因核苷酸序列如SEQ ID NO.1所示；然后制备表达所述新型狂犬病毒G基因的重组腺相关病毒。利用上述制备方法获得的重组腺相关病毒能够提高狂犬病毒G基因的表达水平，能够提高狂犬病毒逆向跨单突触效率，可用于制备高效的狂犬病毒逆向跨突触示踪工具，在哺乳动物神经环路标记中具有广泛的应用价值。

Description

一种新型G基因及其在高效逆向跨单突触中的应用 技术领域
本发明属于生物技术领域，具体涉及一种新型狂犬病毒G基因及其在高效逆向跨单突触中的应用。
背景技术
人脑是自然界中最为复杂的系统之一，而神经网络是大脑行使功能的基础。神经网络的正常连接，使得人体产生正常的生理活动，如认知、学习、记忆和恐惧等。神经网络的异常往往导致神经疾病的出现，如：阿尔茨海默病、帕金森病、抑郁症等，但是还没有有效的手段来治疗这些神经疾病。目前，正常生理活动和致病机制均不清楚，主要的原因在于人们缺乏脑神经网络连接的信息。因此，开展脑神经环路的研究而绘制高精度的脑功能连接图谱，对于了解人的生理活动和致病机制具有重要的意义。我国政府高度重视脑科学的研究，《国家中长期科学和技术发展规划》将“脑科学与认知科学”列为八大前沿科学问题之一，有一批科学家已投身于该领域的研究，并取得了一系列的成果。2012年，中国科学院启动了战略性先导科技专项“脑功能联结图谱研究”，并在2014年成立了“脑科学卓越创新中心”。2013年，美国和欧盟已经开始实施人脑图谱研究计划。脑科学研究计划是继人类基因组计划后又一个具有挑战的伟大计划，该计划的研究成果将会和人类基因组计划的成果一样造福人类。性能优良的神经环路示踪工具对于顺利开展该项目具有十分重要的作用。
狂犬病毒属于弹状病毒科(Rhabdoviridae)狂犬病毒属(Lyssavirus)成员，能感染神经细胞，并在神经网络中运输。其基因组为负链RNA，长度约为12kb，编码五个蛋白，分别为N、P、M、G和L。其中G蛋白作为外膜蛋白，包装成完整的病毒颗粒，但是其对病毒的复制没有影响。狂犬病 毒在神经元之间的传递方向为逆向，即只能由突触后膜传递到突触前膜。2007年，Ian R.Wickersham以狂犬病毒疫苗株SAD B19为对象，缺失G基因，同时将eGFP基因插入到G基因的位置，表达出G蛋白，与体系中存在的N、P、M和L蛋白一起，构建可视化的重组狂犬病毒，并在鼠脑实现了神经元的在体标记。2015年，Edward M.Callaway和Liqun Luo明确指出目前的神经环路示踪工具所标记的输出或者输入的信号仅仅呈现了很小一部分的真实连接。虽然狂犬病毒为基础的逆向跨单级突触工具被认可和使用，但跨突触效率低，使得狂犬病毒仅部分用于逆向标记上游网络。
有报道对狂犬病毒G基因进行了序列优化，获得了新基因命名为oG，采用的策略为使用不同毒株的原始病毒G蛋白嵌合，以及利用密码子使用频率的方法对序列进行优化，然而，上述策略虽然能够在一定程度上提高G基因的表达水平，辅助狂犬病毒逆向跨突触，但是其提高G基因表达的水平有限，辅助狂犬病毒逆向跨突触的效果也并不够明显，亟需一种能够显著提高狂犬病毒逆向跨突触效果的重组腺相关病毒。
发明内容
针对现有技术中的缺陷，本发明提出了一种新型狂犬病毒G基因及其在高效逆向跨单突触中的应用，本发明首先重排狂犬病毒G基因序列，获得新型狂犬病毒G基因，其核苷酸序列如SEQ ID NO.1所示；然后制备表达所述新型狂犬病毒G基因的重组腺相关病毒。
本发明提供一种新型狂犬病毒G基因，所述基因的核苷酸序列如SEQ ID NO.1所示：
Figure PCTCN2020121122-appb-000001
Figure PCTCN2020121122-appb-000002
重排后获得的新的G基因的序列与G基因原始序列以及文献中报道的目前表达效率最高的oG基因均不同，是一个全新的序列。所述新型狂犬病毒G基因可以增强狂犬病毒G蛋白的表达水平，高效辅助狂犬病毒逆向跨单突触。
本发明还提供一种重组腺相关病毒的制备方法，包括如下步骤：
(1)获得新型狂犬病毒G基因序列，所述新型狂犬病毒G基因的核苷酸序列如SEQ ID NO.1所示。
(2)制备表达所述新型狂犬病毒G基因的重组腺相关病毒。
进一步的，所述步骤(1)中包括获得全基因密码子对偏好值的步骤。
进一步的，所述步骤(2)中分别将SEQ ID NO.2和SEQ ID NO.1顺次插入到载体，获得克隆质粒，将所述克隆质粒与腺相关病毒的包装质粒共转染细胞，收集细胞裂解并纯化，即获得表达新型狂犬病毒G基因的重组腺相关病毒。SEQ ID NO.2为Syn启动子序列，可以增强本发明中新型狂犬病毒G基因的表达水平。SEQ ID NO.1为本发明的新型狂犬病毒G基因序列，本发明获取的新型狂犬病毒G基因命名为ooG。
进一步的，所述载体为pdsAAV-CBAp-eGFP，所述细胞为HEK293细胞。
本发明还提供一种重组腺相关病毒，由所述的制备方法制备获得。
本发明提供一种神经环路标记的方法，包括如下步骤：取所述的重组腺相关病毒和表达狂犬病毒N、P、M和L蛋白的狂犬病毒分别注射到哺乳动物海马脑区，感染后麻醉动物，分别用生理盐水灌流，然后用多聚甲醛固定，然后将脑组织置于蔗糖溶液中；将脑组织包埋冰冻后切片；使用荧光显微镜观察。
进一步的，所述哺乳动物为鼠、兔、猴、人中的任意一种。所述的应用对象不仅限于鼠，还能用于猴、兔和人的神经环路标记。
本发明还提供所述的新型狂犬病毒G基因在制备狂犬病毒逆向跨突触示踪工具中的应用。
本发明还提供所述的新型狂犬病毒G基因在神经环路标记中的应用。
综上，与现有技术相比，本发明达到了以下技术效果：
1.本发明制备了一种高效辅助狂犬病毒逆向跨突触的重组腺相关病毒，其表达G蛋白的效率较高，能更好帮助狂犬病毒逆向跨突触，便于开展相关的研究。
2.本发明提供了一种提高用于辅助跨突触病毒效率的基因设计的方 法，其对于提高病毒的表达效率具有便捷的特点，对于开展相关的研究具有重要的作用。
3.本发明提供了一种高效辅助狂犬病毒逆向跨突触的G蛋白序列，便于开展相关的研究。
4.本发明对于开展狂犬病毒的基础性研究(如致病机制、复制机制等)和应用性研究(如神经环路标记、细胞基因治疗、溶瘤病毒、新型疫苗和诊断试剂等)具有重要的现实意义和广泛的应用价值。
5.解析神经环路结构是开展脑科学研究的基础，良好的用于神经环路标记的工具对于解析神经环路的结构具有重要意义。高效表达辅助狂犬病毒逆向跨突触的G蛋白，能够提高狂犬病毒逆向跨突触的效率，能作为神经环路标记工具。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为鼠全基因组背景下，ooG的密码子对偏好值与oG的密码子对偏好值的比较。
图2为一种高效辅助狂犬病毒逆向跨突触的重组腺相关病毒的构建示意图；其中的启动子是Syn；狂犬病毒的G蛋白基因是经过优化的全新序列，命名为ooG，是表达效率比原始蛋白以及文献报道的oG蛋白更好的G蛋白。
图3为一种高效辅助狂犬病毒逆向跨突触的重组腺相关病毒在神经环路标记中的应用示意图。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
本发明提供一种重组腺相关病毒的制备方法，其特征在于，包括如下步骤：
(1)获得全基因密码子对偏好值，对狂犬病毒G基因进行序列重排，获得新型狂犬病毒G基因序列，所述新型狂犬病毒G基因的核苷酸序列如SEQ ID NO.1所示；
(2)制备表达所述新型狂犬病毒G基因的重组腺相关病毒。分别将S EQ ID NO.2和SEQ ID NO.1顺次插入到载体，获得克隆质粒，将所述克隆质粒与腺相关病毒的包装质粒共转染细胞，收集细胞裂解并纯化，即获得表达新型狂犬病毒G基因的重组腺相关病毒。SEQ ID NO.2为Syn启动子序列，可以驱动本发明中新型狂犬病毒G基因的转录。SEQ ID NO.1为本发明的新型狂犬病毒G基因序列，本发明获取的新型狂犬病毒G基因命名为ooG，其具有高效表达的能力。
有报道对狂犬病毒G基因进行了序列优化，获得了新基因并命名为oG，获取新基因采用的策略为使用不同毒株的原始病毒G蛋白嵌合，以及利用密码子使用频率的方法对序列进行优化。然而，上述策略虽然能够在一定程度上提高G基因的表达水平，辅助狂犬病毒逆向跨突触，但是其提高G基因表达的水平有限，辅助狂犬病毒逆向跨突触的效果也并不够明显。本发明获取的新型狂犬病毒G基因命名为ooG，获取新序列的策略与文献中的oG获取的策略不同，本发明的方法根据鼠的全基因组序列信息GRCm38，采用Coleman JR建立的方法计算密码子对的偏好值，获得鼠的全基因密码子对偏 好值，进而以oG基因为对象进行序列重排而获得。
本发明还提供所述的重组腺相关病毒在神经环路标记中的应用步骤：取所述的重组腺相关病毒和表达狂犬病毒N、P、M和L蛋白的狂犬病毒分别注射到哺乳动物海马脑区，感染后麻醉动物，分别用生理盐水灌流，然后用多聚甲醛固定，然后将脑组织置于蔗糖溶液中；将脑组织包埋冰冻后切片；使用荧光显微镜观察。所述哺乳动物为鼠、兔、猴、人中的任意一种。所述的应用对象不仅限于鼠，还能用于猴、兔和人的神经环路标记。
实施例1：获得高效辅助狂犬病毒逆向跨突触的重组腺相关病毒中ooG基因序列
一方面，根据鼠的全基因组序列信息GRCm38，采用Coleman JR建立的方法(Coleman JR et al.,Science.2008)计算密码子对的偏好值，所谓的密码子对偏好值是指：病毒与宿主在长期的进化过程中(其已适应宿主特性)，其基因组编码蛋白的密码子对存在物种依赖的偏好性，例如氨基酸对A-E的密码子对组合分别为GCTGAA，GCTGAG，GCCGAA，GCCGAG，GCAGAA，GCAGAG，GCGGAA和GCGGAG，这些密码子对在不同宿主中的使用频率差异很大，即某一密码子对在某一物种中具有偏好。因此，以某个物种为对象，使用高频密码子对即可提高蛋白表达水平。从而获得鼠的全基因密码子对偏好值；另一方面，围绕广泛用于神经环路逆向标记的狂犬病毒的瓶颈问题—低跨突触效率，以Euiseok J设计的新型狂犬病毒G基因(命名为oG，Euiseok J.Kim et al.,Cell reports,2016)为对象进行序列重排，命名为ooG，获得的序列信息SEQ ID NO.1。在鼠全基因组背景下，比较ooG的密码子对偏好值和oG的密码子对偏好值，结果如图1所示，ooG的密码子对偏好值较oG的密码子对偏好值显著增加，表明ooG在鼠体内比oG具有高效表达的优势。
实施例2：制备高效辅助狂犬病毒逆向跨突触的重组腺相关病毒
一、制备具有高效表达ooG蛋白能力的克隆
首先采用全基因合成的方式分别合成Syn启动子(序列见SEQ ID NO.2)和ooG(序列见SEQ ID NO.1)，按照图2的方式将Syn启动子和ooG插入至双链AAV核心质粒pdsAAV-CBAp-eGFP(Chen Ling et al.,Hum Gene Ther.2014)，获得克隆质粒命名为pscAAV-hsynP-DIO-ooG-bGHpA。
扩增相应序列的引物分别如下：
(a)DNA片段Syn启动子的引物：SEQ ID NO:3和SEQ ID NO:4，模板为合成的片段SEQ ID NO.2；(b)DNA片段兔ooG的引物：SEQ I D NO:5和SEQ ID NO:6，模板为合成的片段SEQ ID NO.1。
首先使用KpnI和HindIII双酶切pdsAAV-CBAp-eGFP载体，然后采用同源重组的方式将SEQ ID NO.2和SEQ ID NO.1顺次插入到载体，获得克隆命名为pscAAV-hsynP-DIO-ooG-bGHpA。本发明的PCR所用引物均由生工生物工程(上海)股份有限公司合成，且构建的克隆均在生工生物工程(上海)股份有限公司进行测序验证。
二、腺相关病毒的制备：
(1)质粒转染HEK293细胞包装rAAV：提前一天将HEK293细胞用胰蛋白酶消化后铺到15cm的培养皿中贴壁培养，转染时细胞汇合度80％左右。将包装rAAV所需的三种质粒，即pAAV-Helper质粒，血清型质粒pAAV-RC，核心表达质粒pscAAV-hsynP-DIO-ooG-bGHpA用磷酸钙或PEI转染试剂转染HEK293细胞，转染72小时后，收集培养上清和细胞。
(2)细胞的裂解与rAAV的纯化：细胞用裂解液(50mM Tris-Cl(PH8.0)，2mM MgCl 2)反复冻融3次，加入核酸酶处理，离心去除细胞碎片。将细胞裂解液上清与核酸酶处理后的培养上清液合并，然后用PEG4000进行浓缩离心沉淀，用PBS溶液重悬即获得含有rAAV的PBS重悬液。在超速离心管中依次加入15％碘克沙醇分离液8mL，25％碘克沙醇分离液6mL，40％碘克沙醇分离液8mL，58％碘克沙醇分离液5mL，然后将含 有rAAV的PBS重悬液加入离心管的上层密封，再用Type 70 Ti转子，63000rpm离心2h。用针头吸取40％碘克沙醇梯度层的液体，PBS透析过夜，然后用Amicon ultra-4(100KD cutoff)截留柱离心浓缩至终体积1mL左右，无菌分装后-80℃冻存。
(3)荧光定量PCR检测rAAV的滴度：利用特异性的引物进行荧光定量PCR检测rAAV病毒粒子的DNA拷贝数，即rAAV的数量VG(Viral genomes表示)，换算成单位体积中rAAV的含量，即rAAV的滴度，用VG/mL(Viral genomes/mL)表示。取5μl样品加入1.5ml的离心管，补加15μl ddH 2O至20μl；再加入20μl的2N NaOH，混匀放置到55℃的水浴锅中处理30min；再加入20μl的2N HCl中和处理。取5μl的NaOH-HCl处理后的样品加入1.5ml的离心管补加45μl ddH 2O至50μl，混匀，作为Q-PCR反应的待测样品。
实施例3：本发明的重组腺相关病毒能够高效辅助狂犬病毒逆向跨突触
一方面，按照1：1比例混合实施例2制备的重组腺相关病毒和文献报道的表达TVA和eGFP的重组腺相关病毒(Fumitaka Osakada et al.,Neuron.2011)；另一方面，按照1：1比例混合文献报道表达oG蛋白的重组腺相关病毒(Euiseok J.Kim et al.,Cell reports,2016)和文献报道的表达TVA和eGFP重组腺相关病毒(Fumitaka Osakada et al.,Neuron.2011)。其中，文献报道的表达TVA和eGFP的重组腺相关病毒提供狂犬病毒包装所需的N、P、M和L蛋白，本发明的重组腺相关病毒提供狂犬病毒的G蛋白。分别取上述混合的重组腺相关病毒0.8μl定位注射到Thy1-Cre鼠腹侧海马脑区，感染后21天后，取0.15μL表达红色荧光蛋白的狂犬病毒SADΔG-mCherry(Fumitaka Osakada et al.,Neuron.2011)分别注射到对应的两个重组腺相关病毒已经注射的腹侧海马脑区，7天后麻醉动物，分别用0.9％(V/V)生理盐水灌流，然后用4％(V/V)多聚甲醛固定，取出脑组织浸泡于4％(V/V)多聚甲醛液中，然后将脑组织先置于20％(V/V) 蔗糖溶液中1天，然后置于30％(V/V)蔗糖溶液中2天；将脑组织底部切平，置于底座上包埋冰冻1h后切片；取脑片后使用荧光显微镜观察。结果如图3所示，重组病毒注射到鼠脑后，可见红色荧光蛋白信号，见图3A的注射区域和跨突触区域，图中以灰度表示，表明重组病毒能够介导外源蛋白在鼠脑神经细胞中表达；图3A的跨突触区域显示，本发明的表达ooG的重组腺相关病毒与目前现有技术中辅助狂犬病毒逆向跨突触效果最好的表达oG基因的重组腺相关病毒相比，突触前位置显示已跨过的狂犬病毒的数量明显增多。图3B纵坐标为跨突触效率指数，通过计算文献的oG和本发明的ooG的跨突触效率，发现ooG显著高于oG，说明本发明表达ooG的腺相关病毒比文献发表的表达oG的腺相关病毒能够更好的帮助狂犬病毒跨突触，提高狂犬病毒跨突触的效率。上述结果表明本发明的一种高效表达ooG的重组腺相关病毒能够辅助狂犬病毒能够更高效的在神经网络中运输，具有标记脑神经环路的能力。
实施例4：对哺乳动物的脑神经环路进行标记
具体的，标记包括如下步骤：取0.8μl重组腺相关病毒定位注射到Thy1-Cre鼠腹侧海马脑区，感染后21天后，取0.15μl表达红色荧光蛋白的狂犬病毒SADΔG-mCherry(Fumitaka Osakada et al.,Neuron.2011)也注射到Thy1-Cre鼠腹侧海马脑区，7天后麻醉动物，分别用0.9％(V/V)生理盐水灌流，然后用4％(V/V)多聚甲醛固定，取出脑组织浸泡于4％(V/V)多聚甲醛液中，然后将脑组织先置于20％(V/V)蔗糖溶液中1天，然后置于30％(V/V)蔗糖溶液中2天；将脑组织底部切平，置于底座上包埋冰冻1h后切片；取脑片后使用荧光显微镜观察，可以根据荧光信号追踪到狂犬病毒的位置。
本发明的新型狂犬病毒G基因及表达该基因的重组腺相关病毒在研究脑神经环路平台中具有多种应用，包括利用本发明提供的鼠密码子对偏好值重排方法重新编排任何病毒基因，用于提高蛋白的表达水平或降低蛋白 的表达水平，从而用于细胞治疗、基因治疗、溶瘤病毒和疫苗开发、或是对哺乳动物的脑神经环路进行标记。所述的应用对象不仅限于鼠，还能用于猴等动物的神经环路标记；本发明所用的ooG基因只是作为一个范式，因此，还可用于其他基因的重排。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
Figure PCTCN2020121122-appb-000003
Figure PCTCN2020121122-appb-000004
Figure PCTCN2020121122-appb-000005

Claims (10)

  1. 一种新型狂犬病毒G基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO.1所示。
  2. 一种重组腺相关病毒的制备方法，其特征在于，包括如下步骤：
    (1)获得新型狂犬病毒G基因序列，所述新型狂犬病毒G基因的核苷酸序列如SEQ ID NO.1所示；
    (2)制备表达所述新型狂犬病毒G基因的重组腺相关病毒。
  3. 根据权利要求2所述的制备方法，其特征在于，所述步骤(1)中包括获得全基因密码子对偏好值的步骤。
  4. 根据权利要求2所述的制备方法，其特征在于，所述步骤(2)中分别将SEQ ID NO.2和SEQ ID NO.1顺次插入到载体，获得克隆质粒，将所述克隆质粒与腺相关病毒的包装质粒共转染细胞，收集细胞裂解并纯化，即获得表达新型狂犬病毒G基因的重组腺相关病毒。
  5. 根据权利要求4所述的制备方法，其特征在于，所述载体为pdsAA V-CBAp-eGFP，所述细胞为HEK293细胞。
  6. 一种重组腺相关病毒，其特征在于，由权利要求2-5任一项所述的制备方法制备获得。
  7. 一种神经环路标记的方法，其特征在于，包括如下步骤：取权利要求6所述的重组腺相关病毒和表达狂犬病毒N、P、M和L蛋白的狂犬病毒分别注射到哺乳动物海马脑区，感染后麻醉动物，分别用生理盐水灌流，然后用多聚甲醛固定，然后将脑组织置于蔗糖溶液中；将脑组织包埋冰冻后切片；使用荧光显微镜观察。
  8. 根据权利要求7所述的应用，其特征在于，所述哺乳动物为鼠、兔、猴、人中的任意一种。
  9. 权利要求1所述的新型狂犬病毒G基因在制备狂犬病毒逆向跨突触 示踪工具中的应用。
  10. 权利要求1所述的新型狂犬病毒G基因在神经环路标记中的应用。
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