Preparation and application of recombinant adeno-associated virus capable of efficiently assisting rabies virus transsynapse

Abstract

The invention belongs to the technical field of biology, and discloses preparation and application of a recombinant adeno-associated virus capable of efficiently assisting rabies virus trans-synapse, and a method comprises the following steps: using KpnI and BstEII double enzyme digestion AAV plasmid vector, and inserting the sequence SEQ ID NO.1 into the vector by adopting an enzyme digestion connection mode; the method comprises the following steps: firstly, carrying out double enzyme digestion on a plasmid by adopting NruI and BstEII, and inserting a sequence shown as SEQ ID NO.2 into the plasmid by adopting an enzyme digestion connection mode, so as to obtain cloned pAAV-EF1a-DIO-ooG-hsynP-DIO-TVA-2A-nlsDsRed; co-transfecting cells by the clone and helper plasmids, collecting the cells, cracking and purifying. The recombinant adeno-associated virus prepared by the invention can realize the expression of ooG, TVA and nlsDsRed by a single AAV, can better help the rabies virus to cross synapses, can be used as a neural circuit marking tool, and is convenient to carry out related researches.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to the preparation and application of a recombinant adeno-associated virus that efficiently assists rabies virus transsynapsis. Background Art

[0002] The human brain is one of the most complex systems in nature, and neural networks are the basis for the brain to function. The normal connection of neural networks 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. At present, normal physiological activities and pathogenic mechanisms are unclear, mainly due to the lack of information on brain neural network connections. Therefore, conducting research on brain neural circuits and drawing high-precision brain function connection maps are of great significance for understanding human physiological activities and pathogenic mechanisms. The Chinese government attaches great importance to the study of brain science. The "National Medium- and Long-Term Science and Technology Development Plan" lists "brain science and cognitive science" as one of the eight frontier scientific issues. A group of scientists have devoted themselves to research in this field and have achieved a series of results. In 2012, the Chinese Academy of Sciences launched the strategic leading science and technology project "Brain Function Connection Map Research" and established the "Brain Science Excellence Innovation Center" in 2014. In 2013, the United States and the European Union have begun to implement the human brain map research plan. The brain science research project is another challenging and great project after the Human Genome Project. The research results of this project will benefit mankind just like the results of the Human Genome Project. High-performance neural circuit tracing tools play a very important role in the smooth implementation of this project.

[0003] Rabies virus belongs to the Lyssavirus genus of the Rhabdoviridae family. It can infect nerve cells and transport in neural networks. Its genome is a negative-strand RNA with a length of about 12 kb, encoding five proteins, namely N, P, M, G and L. Among them, the G protein is an outer membrane protein that is packaged into complete virus particles, but it has no effect on the replication of the virus. In 2007, Ian R. Wickersham used 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 construct a visual recombinant rabies virus, and achieved in vivo labeling of neurons in the mouse brain. In 2015, Edward M. Callaway and Liqun Luo clearly pointed out that the output or input signals marked by the current neural circuit tracing tools only present a small part of the real connection. Although the rabies virus-based reverse cross-single-level synapse tool is recognized and widely used, due to the high toxicity and low cross-synaptic efficiency, only part of the upstream network is reversely labeled. Therefore, it is of great significance to establish an efficient trans-synaptic tracing tool. Summary of the invention

[0004] In view of the shortcomings and deficiencies in the prior art, the object of the present invention is to provide a preparation and application of a recombinant adeno-associated virus that efficiently assists rabies virus transsynapsis.

[0005] The first aspect of the present invention provides a method for preparing a recombinant adeno-associated virus that efficiently assists rabies virus transsynapsis, comprising the following steps:

[0006] (1) Construction of clones: The AAV core plasmid vector was double-digested with KpnI and BstEII, and the sequence shown in SEQ ID NO.1 was inserted into the vector by restriction digestion and ligation to obtain a clone named pAAV-EF1a-DIO-ooG; then the pAAV-EF1a-DIO-ooG plasmid was double-digested with NruI and BstEII, and the sequence shown in SEQ ID NO.2 was inserted into the plasmid by restriction digestion and ligation to obtain a clone named pAAV-EF1a-DIO-ooG-hsynP-DIO-TVA-2A-nlsDsRed;

[0007] (2) Construction of adeno-associated virus: The plasmid pAAV-EF1a-DIO-ooG-hsynP-DIO-TVA-2A-nlsDsRed, the auxiliary plasmid pAAV-Helper, and pAAV-RC obtained in step (1) are mixed and transfected into cells, and the cells are collected, lysed, and purified to obtain a recombinant adeno-associated virus that efficiently assists rabies virus transsynapsis.

[0008] Preferably, the cells are 293T cells.

[0009] The second aspect of the present invention provides a recombinant adeno-associated virus that efficiently assists rabies virus transsynapsis and is prepared by the above-mentioned preparation method.

[0010] The third aspect of the present invention provides the use of the above-mentioned recombinant adeno-associated virus that efficiently assists rabies virus in transsynaptic tracing in the brain neural circuit tracing of mammals.

[0011] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0012] (1) Analyzing the structure of neural circuits is the basis for conducting brain science research, and good tools for neural circuit labeling are of great significance for analyzing the structure of neural circuits. The present invention prepares a recombinant adeno-associated virus that efficiently assists rabies virus transsynapsis, which can achieve single AAV expression of ooG, TVA and nlsDsRed, can better assist rabies virus transsynapsis, and can be used as a neural circuit labeling tool to facilitate related research.

[0013] (2) The present invention provides a method for designing an auxiliary AAV for improving the efficiency of auxiliary transsynaptic viruses, which plays an important role in carrying out related research.

[0014] (3) The present invention provides a new tool that can efficiently assist the transsynaptic expression of rabies virus using a single AAV, facilitating related research.

[0015] (4) The present invention has important practical significance and broad application value for conducting basic research (such as pathogenic mechanism, replication mechanism, etc.) and applied research (such as neural circuit labeling, cell gene therapy, oncolytic virus, new vaccines and diagnostic reagents, etc.) on rabies virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 The schematic diagram of the construction of the recombinant adeno-associated virus clone that efficiently assists rabies virus transsynapsis of the present invention, wherein EF1a and Syn are promoters; the genes to be expressed are ooG, TVA and nlsDsRed respectively; the post-transcriptional regulatory element WPRE3 is a simplified version of WPRE, different from the conventionally used WPRE; the transcription termination elements are SV40pA and BGHpA; 2A is 2A peptide.

[0018] Figure 2 The figure is a schematic diagram of the packaging process of the recombinant adeno-associated virus that efficiently assists rabies virus transsynapsis of the present invention, wherein plasmid A is the core plasmid, that is, the plasmid pAAV-EF1a-DIO-ooG-hsynP-DIO-TVA-2A-nlsDsRed of the present invention, plasmid B and plasmid C are the auxiliary plasmids pAAV-Helper and pAAV-RC for packaging AAV, respectively; the cells used for transfection are 293T cells.

[0019] Figure 3 This is a diagram showing the effect of the recombinant adeno-associated virus of the present invention that efficiently assists rabies virus transsynapsis in improving the efficiency of rabies virus transsynapsis, wherein A is the effect of the new recombinant adeno-associated virus of the present invention in assisting rabies virus transsynapsis, B is the effect of the existing recombinant adeno-associated virus in assisting rabies virus transsynapsis, and C is a comparison result of the transsynaptic efficiency of the recombinant adeno-associated virus of the present invention and the existing recombinant adeno-associated virus. DETAILED DESCRIPTION

[0020] The embodiments of the present invention will be clearly and completely described below in conjunction with the examples, and the described examples are only some examples of the present invention, rather than all examples. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the examples in the present invention, all other examples obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0021] Unless otherwise specified, the instruments and materials used in the following examples are all routinely used in laboratories, and the technical solutions described are all routine techniques in the art.

[0022] Embodiment 1:

[0023] 1. Construction of clones capable of expressing ooG, TVA and nlsDsRed proteins

[0024] The present invention firstly adopts a whole gene synthesis method to respectively synthesize the sequence of DNA fragment 1 containing ooG (Jia F et al., Molecular Brain. 2021), WPRE3 (Shibata AC et al., Nature Communications. 2021) and SV40pA (Shibata AC et al., Nature Communications. 2021) (the sequence is shown in SEQ ID NO. 1) and the sequence of DNA fragment 2 containing Syn promoter, TVA (Osakada F and Callaway EM. Nature Protocols. 2013.), 2A and nlsDsRed (Karig D et al., PNAS. 2018 and Shui S et al., Bioengineering. 2022) (the sequence is shown in SEQ ID NO. 2), and then Figure 1 The sequence shown in SEQ ID NO.1 and the sequence shown in SEQ ID NO.2 were inserted into the AAV core plasmid (Addgene #20298) and the obtained cloned plasmid was named pAAV-EF1a-DIO-ooG-hsynP-DIO-TVA-2A-nlsDsRed.

[0025] Specifically, the AAV core plasmid vector was double-digested with KpnI and BstEII, and the sequence shown in SEQ ID NO.1 was inserted into the vector by enzyme cutting and ligation to obtain a clone named pAAV-EF1a-DIO-ooG; then the pAAV-EF1a-DIO-ooG plasmid was double-digested with NruI and BstEII, and the sequence shown in SEQ ID NO.2 was inserted into the plasmid by enzyme cutting and ligation to obtain a clone named pAAV-EF1a-DIO-ooG-hsynP-DIO-TVA-2A-nlsDsRed.

[0026] Primers for amplifying corresponding sequences: (a) Primers for amplifying SEQ ID NO.1 are SEQ ID NO.3 and SEQ ID NO.4, and the template is SEQ ID NO.1; (b) Primers for amplifying SEQ ID NO.2 are SEQ ID NO.5 and SEQ ID NO.6, and the template is SEQ ID NO.2.

[0027] The primers used in the PCR of the present invention were all synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and the constructed clones were sequenced and verified by Sangon Biotechnology (Shanghai) Co., Ltd.

[0028] 2. Construction of recombinant adeno-associated virus

[0029] (1) Plasmid transfection into 293T cells to package rAAV

[0030] One day in advance, 293T cells (purchased from ATCC) were digested with trypsin (purchased from Thermo Fisher Scientific) and plated on a 15 cm culture dish for adherent culture. The cell confluence was about 80% during transfection. The three plasmids required for packaging rAAV, namely pAAV-EF1a-DIO-ooG-hsynP-DIO-TVA-2A-nlsDsRed core plasmid A, pAAV-Helper plasmid B (Kimura T et al., Scientific Reports. 2019), and pAAV-RC plasmid C (Kimura T et al., Scientific Reports. 2019), were co-transfected into 293T cells, and the culture supernatant and cells were collected 72 hours after transfection.

[0031] (2) Cell lysis and rAAV purification

[0032] The cells collected in step (1) were repeatedly frozen and thawed three times with a lysis solution (50mM Tris-Cl (pH8.0), 2mM MgCl2), and then treated with nuclease (purchased from Sigma-Aldrich) and centrifuged to remove cell debris. The supernatant of the cell lysate was combined with the culture supernatant after nuclease treatment, and then concentrated and centrifuged with PEG4000 to obtain a PBS resuspension containing rAAV. 8mL of 15% iodixanol separation solution, 6mL of 25% iodixanol separation solution, 8mL of 40% iodixanol separation solution, and 5mL of 58% iodixanol separation solution were added to an ultracentrifuge tube in sequence, and then the PBS resuspension containing rAAV was added to the upper layer of the centrifuge tube and sealed, and then centrifuged at 63000rpm for 2h using a Type 70Ti rotor. The liquid in the 40% iodixanol gradient layer was aspirated with a needle, dialyzed against PBS overnight, and then concentrated by centrifugation using an Amicon ultra-4 (100KD cutoff) retention column to a final volume of about 1 mL. After sterile aliquoting, the liquid was frozen at -80°C to obtain the recombinant adeno-associated virus expressing ooG, TVA and nlsDsRed proteins.

[0033] (3) Detection of rAAV titer by fluorescence quantitative PCR

[0034] Specific primers were used for fluorescence quantitative PCR to detect the DNA copy number of rAAV virus particles, i.e., the number of rAAV VG (Viral genomes), which was converted into the content of rAAV per unit volume, i.e., the titer of rAAV, expressed as VG / mL (Viral genomes / mL). 5 μL of the frozen sample in step (2) was added to a 1.5 mL centrifuge tube, and 15 μL of ddH2O was added to 20 μL; 20 μL of 2N NaOH was added, mixed, and placed in a 55°C water bath for 30 min; 20 μL of 2N HCl was added for neutralization. 5 μL of the sample treated with NaOH-HCl was added to a 1.5 mL centrifuge tube, and 45 μL of ddH2O was added to 50 μL, mixed, and used as the sample to be tested for Q-PCR reaction.

[0035] Example 2

[0036] On the one hand, the adeno-associated virus expressing ooG (Jia Fet al., Molecular Brain. 2021) and the recombinant adeno-associated virus expressing TVA and nlsmCherry (purchased from Wuhan Shumi Brain Science Technology Co., Ltd.) reported in the literature were mixed in a volume ratio of 1:1; on the other hand, the recombinant adeno-associated virus expressing ooG, TVA and nlsDsRed proteins prepared in Example 1 was taken. 0.08 μL of the recombinant adeno-associated virus of the above two aspects was injected into the ventral hippocampus of mice (purchased from Guangdong Weitong Lihua Experimental Animal Technology Co., Ltd.) respectively. 21 days after infection, 0.15 μL of rabies virus expressing green fluorescent protein (purchased from Wuhan Shumi Brain Science Technology Co., Ltd.) was injected into the corresponding ventral hippocampus area where the two groups of recombinant adeno-associated viruses had been injected, and the animals were anesthetized 7 days later. The brain tissues were perfused with 0.9% (V / V) saline and then fixed with 4% (V / V) paraformaldehyde. The brain tissues were taken out and immersed in 4% (V / V) paraformaldehyde solution. The brain tissues were then placed in 20% (V / V) sucrose solution for 1 day and then in 30% (V / V) sucrose solution for 2 days. The bottom of the brain tissue was cut flat, placed on a base, embedded and frozen for 1 hour, and then sliced. The brain slices were taken and observed using a fluorescence microscope.

[0037] The results are as follows Figure 3 As shown, Figure 3 A shows that after the recombinant adeno-associated virus of the present invention is injected into the mouse brain, it can help the rabies virus expressing green fluorescence to cross the synapse, and the green fluorescent protein signal can be seen. The number of fluorescent cells is large, indicating that the virus has completed the cross-synapse; Figure 3 B shows that after the recombinant adeno-associated virus in the current field is injected into the mouse brain, it can help the rabies virus expressing green fluorescence to cross the synapse. The green fluorescent protein signal is visible, but the number of fluorescent cells is relatively small. Figure 3A is significantly less than that of the recombinant adeno-associated virus of the present invention, indicating that the virus has completed transsynaptic. However, the efficiency is significantly lower than that of the recombinant adeno-associated virus of the present invention. Figure 3 The vertical axis C is the transsynaptic efficiency index. It was found that the single AAV adeno-associated virus of the present invention is significantly higher than the double AAV adeno-associated virus used in the current field, indicating that the recombinant adeno-associated virus of the present invention can better help the rabies virus to cross the synapse and improve the efficiency of rabies virus cross-synapse compared with the adeno-associated virus in the prior art.

[0038] The above results indicate that the novel recombinant adeno-associated virus of the present invention can assist rabies virus in being transported in neural networks more efficiently and has the ability to mark brain neural circuits.

[0039] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A method for preparing a recombinant adeno-associated virus that efficiently assists rabies virus transsynapsis, characterized in that: The following steps are involved: (1) Construction of clones: The AAV core plasmid vector was double-digested with KpnI and BstEII, and the sequence shown in SEQ ID NO.1 was inserted into the vector by restriction digestion and ligation to obtain a clone named pAAV-EF1a-DIO-ooG; then the pAAV-EF1a-DIO-ooG plasmid was double-digested with NruI and BstEII, and the sequence shown in SEQ ID NO.2 was inserted into the plasmid by restriction digestion and ligation to obtain a clone named pAAV-EF1a-DIO-ooG-hsynP-DIO-TVA-2A-nlsDsRed; (2) Construction of adeno-associated virus: The plasmid pAAV-EF1a-DIO-ooG-hsynP-DIO-TVA-2A-nlsDsRed, the auxiliary plasmid pAAV-Helper, and pAAV-RC obtained in step (1) are mixed and transfected into cells, and the cells are collected, lysed, and purified to obtain a recombinant adeno-associated virus that efficiently assists rabies virus transsynapsis.

2. The preparation method according to claim 1, characterized in that: The cells are 293T cells.

3. A recombinant adeno-associated virus that efficiently assists rabies virus transsynapsis, prepared by the preparation method according to any one of claims 1-2.

4. Use of the recombinant adeno-associated virus that efficiently assists rabies virus transsynapsis as described in claim 3 in tracing brain neural circuits in mammals.

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