CN112210565B - G gene and application thereof in efficient reverse trans-monosynaptic - Google Patents

G gene and application thereof in efficient reverse trans-monosynaptic Download PDF

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CN112210565B
CN112210565B CN202011102419.XA CN202011102419A CN112210565B CN 112210565 B CN112210565 B CN 112210565B CN 202011102419 A CN202011102419 A CN 202011102419A CN 112210565 B CN112210565 B CN 112210565B
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rabies virus
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贾凡
徐富强
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Shenzhen Institute of Advanced Technology of CAS
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Abstract

The invention relates to a novel rabies virus G gene and application thereof in efficient reverse cross-monosynaptic, firstly rearranging a rabies virus G gene sequence, wherein the obtained novel rabies virus G gene nucleotide sequence is shown as SEQ ID NO. 1; then, recombinant adeno-associated virus expressing the novel rabies virus G gene is prepared. The recombinant adeno-associated virus obtained by the preparation method can improve the expression level of the rabies virus G gene and the reverse transmonosynaptic efficiency of the rabies virus, can be used for preparing a high-efficiency rabies virus reverse transsynaptic tracing tool, and has wide application value in mammal neural loop marking.

Description

G gene and application thereof in efficient reverse trans-monosynaptic
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a rabies virus G gene and application thereof in efficient reverse trans-monosynaptic.
Background
The human brain is one of the most complex systems in nature, and neural networks underlie the functioning of the brain. The normal connection of the neural network enables the human body to generate normal physiological activities, such as cognition, learning, memory, fear and the like. Abnormalities in the neural network often lead to the appearance of neurological diseases, such as: alzheimer's disease, parkinson's disease, depression, etc., but there is no effective means for treating these neurological diseases. At present, normal physiological activities and pathogenic mechanisms are not clear, and the main reason is that people lack information connected with a cranial nerve network. Therefore, the research of cranial nerve loop is developed to draw a high-precision brain function connection map, which has important significance for understanding the physiological activities and pathogenic mechanisms of human beings. The government of China highly attaches importance to the research of brain science, and the 'brain science and cognitive science' are one of eight leading-edge scientific problems in 'national middle and long term science and technical development planning', and a group of scientists have invested in the research of the field and have obtained a series of achievements. In 2012, the Chinese academy of sciences started the strategic leading science and technology project brain function linkage map research, and established the brain science excellent innovation center in 2014. In 2013, the united states and the european union have begun to implement a human brain atlas study program. The brain science research program is a challenge great program following the human genome program, and the research result of the program will benefit human beings as well as the human genome program. The excellent neural circuit tracer tool plays an important role in smoothly developing the project.
Rabies viruses belong to the Rhabdoviridae (Rhabdoviridae) genus rabies (Lyssavir), infect nerve cells and are transported in the neural network. Its genome is a negative strand RNA, approximately 12kb in length, and encodes five proteins, N, P, M, G and L, respectively. In which the G protein serves as the outer membrane protein and is packaged as a complete viral particle, but which has no effect on viral replication. The transmission direction of rabies virus between neurons is reverse, i.e. only from the postsynaptic membrane to the presynaptic membrane. In 2007, Ian R.Wickersham takes a rabies virus vaccine strain SAD B19 as an object, a G gene is deleted, an eGFP gene is inserted into the position of the G gene to express a G protein, a visualized recombinant rabies virus is constructed together with N, P, M and L proteins existing in the system, and in-vivo labeling of neurons in a rat brain is realized. In 2015, Edward m.callaway and Liqun Luo clearly indicated that the output or input signals labeled by current neural circuit tracing tools only represent a small fraction of real connections. Although the rabies virus-based reverse transsynaptic tools are recognized and used, the transsynaptic efficiency is low, so that rabies virus is only partially used for reverse labeling of upstream networks.
Sequence optimization is reported on rabies virus G genes, a new gene is obtained and named as oG, the adopted strategy is original virus G protein chimeric by using different strains, and the sequence is optimized by using a codon use frequency method, however, although the strategy can improve the expression level of the G genes to a certain extent and assist the rabies virus reverse transsynaptic, the level of improving the G gene expression is limited, the effect of assisting the rabies virus reverse transsynaptic is not obvious enough, and a recombinant adeno-associated virus capable of obviously improving the rabies virus reverse transsynaptic effect is urgently needed.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a rabies virus G gene and application thereof in high-efficiency reverse cross-monosynaptic, firstly rearranging a rabies virus G gene sequence to obtain a rabies virus G gene, wherein the nucleotide sequence of the rabies virus G gene is shown as SEQ ID NO. 1; then, recombinant adeno-associated virus expressing the rabies virus G gene is prepared.
The invention provides a rabies virus G gene, the nucleotide sequence of the gene is shown as SEQ ID NO. 1:
ATGGTTCCTCAAGCCCTTCTCTTTGTTCCTCTTCTTGTCTTCCCGCTCTGCTTTGGGAAGTTCCCCATCTACACCATTCCTGACAAGCTAGGGCCCTGGAGCCCCATTGACATCCACCACCTCAGCTGCCCCAACAACTTGGTTGTAGAAGATGAAGGCTGCACCAACCTCAGTGGCTTCTCCTACATGGAGCTAAAAGTGGGCTACATCTCGGCCATCAAGATGAATGGCTTCACCTGCACTGGAGTTGTCACTGAAGCAGAGACCTACACCAACTTTGTTGGCTATGTCACCACCACCTTCAAAAGAAAACACTTCCGGCCCACTCCAGATGCCTGCCGCGCGGCCTACAACTGGAAGATGGCGGGGGACCCCCGCTATGAAGAGAGCCTGCACAACCCCTACCCAGACTACCACTGGCTGAGGACTGTGAAGACCACCAAAGAAAGTTTGGTCATCATCAGCCCCAGTGTAGCTGACTTGGACCCCTATGACCGTTCTCTACACAGCCCTGTATTTCCTGGTGGGAACTGCAGTGGTGTGGCTGTCAGCAGCACCTACTGCAGCACCAACCATGACTACACCATCTGGATGCCGGAGAACCCCCGGCTAGGGATGTCCTGTGACATCTTCACCAACAGCCGAGGGAAAAGAGCCAGCAAAGGTTCTGAGACCTGTGGCTTTGTAGATGAGCGTGGCCTCTACAAGAGTTTAAAAGGTGCCTGCAAATTAAAACTCTGTGGTGTTCTTGGTCTTCGGCTCATGGATGGCACCTGGGTGGCCATGCAGACCAGCAATGAGACCAAGTGGTGCCCGCCGGGCCAGCTTGTCAACCTCCATGACTTCCGAAGTGATGAAATAGAACATCTTGTTGTAGAAGAACTTGTCAAGAAAAGAGAAGAATGTTTAGATGCCCTGGAGAGCATCATGACTACCAAGAGTGTCTCCTTCCGTCGCCTCAGCCACCTCAGGAAACTTGTTCCTGGCTTTGGGAAAGCCTACACCATCTTCAACAAGACGCTCATGGAAGCAGATGCCCACTACAAATCTGTCCGCACGTGGAATGAGATCATTCCTTCCAAAGGCTGCCTCCGAGTTGGTGGCCGCTGCCACCCACATGTCAATGGTGTCTTCTTCAATGGCATCATTCTTGGGCCAGATGGAAATGTCCTCATTCCAGAGATGCAGAGCAGCCTGCTGCAGCAGCACATGGAACTTCTTGTCAGCAGTGTCATCCCGCTCATGCACCCGCTGGCAGACCCCAGCACTGTCTTCAAGAATGGAGATGAAGCAGAAGATTTTGTAGAAGTTCATCTTCCTGATGTTCATGAAAGAATTTCTGGTGTGGACTTGGGTCTTCCCAACTGGGGAAAATATGTTCTTCTTTCTGCTGGGGCGCTCACGGCGCTCATGTTAATAATATTCCTCATGACCTGCTGcAGAAGAGTCAACCGCTCGGAGCCCACCCAGCACAACCTTCGTGGCACGGGCCGAGAAGTTTCTGTCACGCCGCAGAGTGGGAAGATCATCTCCTCCTGGGAGAGCCACAAGTCAGGAGGAGAGACGCGCCTG。
the new G gene sequence obtained after rearrangement is different from the original G gene sequence and oG gene with the highest expression efficiency reported in the literature, and is a brand new sequence. The rabies virus G gene can enhance the expression level of rabies virus G protein and efficiently assist the rabies virus to reversely span the monosynaptic junction.
The invention also provides a preparation method of the recombinant adeno-associated virus, which comprises the following steps:
(1) obtaining a rabies virus G gene sequence, wherein the nucleotide sequence of the rabies virus G gene is shown as SEQ ID NO. 1.
(2) Preparing the recombinant adeno-associated virus expressing the rabies virus G gene.
Further, the step (1) comprises a step of obtaining codon pair preference values of the whole gene.
Further, in the step (2), the sequence of SEQ ID NO.2 and SEQ ID NO.1 is inserted into the vector to obtain a clone plasmid, the clone plasmid and the packaging plasmid of the adeno-associated virus are cotransfected with cells, the cells are collected and lysed, and the recombinant adeno-associated virus expressing the rabies virus G gene is obtained. SEQ ID NO.2 is a Syn promoter sequence, and can enhance the expression level of the rabies virus G gene. SEQ ID NO.1 is the rabies virus G gene sequence of the invention, and the rabies virus G gene obtained by the invention is named ooG.
Further, the vector is pdsAAV-CBAp-eGFP, and the cell is HEK293 cell.
The invention also provides a recombinant adeno-associated virus prepared by the preparation method.
The invention provides a method for marking a neural circuit, which comprises the following steps: respectively injecting the recombinant adeno-associated virus and rabies viruses expressing rabies virus N, P, M and L protein into hippocampal brain areas of mammals, anesthetizing the animals after infection, respectively perfusing with physiological saline, then fixing with paraformaldehyde, and then placing brain tissues into a sucrose solution; embedding and freezing brain tissue, and slicing; observation was performed using a fluorescence microscope.
Further, the mammal is any one of mouse, rabbit, monkey, and human. The application objects are not limited to mice, but also can be used for nerve loop markers of monkeys, rabbits and humans.
The invention also provides application of the rabies virus G gene in preparation of a rabies virus reverse trans-synaptic tracing tool.
The invention also provides application of the rabies virus G gene in a neural loop marker.
In summary, compared with the prior art, the invention achieves the following technical effects:
1. the recombinant adeno-associated virus for efficiently assisting the rabies virus reverse transsynaptic is prepared, has higher efficiency of expressing G protein, can better help the rabies virus reverse transsynaptic and is convenient for developing related research.
2. The invention provides a method for improving gene design for assisting the efficiency of a cross-synaptic virus, which has the characteristic of convenience and rapidness for improving the expression efficiency of the virus and has an important function for developing related researches.
3. The invention provides a G protein sequence for efficiently assisting the reverse transsynaptic of rabies viruses, which is convenient for developing related researches.
4. The invention has important practical significance and wide application value for carrying out basic research (such as pathogenesis, replication mechanism and the like) and application research (such as neural loop marker, cell gene therapy, oncolytic virus, novel vaccine, diagnostic reagent and the like) of rabies virus.
5. The analysis of the neural loop structure is the basis for developing brain science research, and a good tool for neural loop marking has important significance for analyzing the neural loop structure. The G protein for efficiently expressing the auxiliary rabies virus reverse transsynaptic can improve the efficiency of the rabies virus reverse transsynaptic and can be used as a neural loop marking tool.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a comparison of codon pair bias values for ooG and oG in a murine whole genome context.
FIG. 2 is a schematic diagram of a construction of a recombinant adeno-associated virus with high efficiency in assisting rabies virus in reverse transsynaptic transmission; wherein the promoter is Syn; the G protein gene of the rabies virus is an optimized brand-new sequence, is named ooG, and is a G protein with better expression efficiency than the original protein and oG protein reported in the literature.
FIG. 3 is a schematic diagram of the application of a recombinant adeno-associated virus with high efficiency in assisting the reverse transsynaptic transmission of rabies virus in neural circuit markers.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, shall fall within the scope of protection of the present invention.
The invention provides a preparation method of recombinant adeno-associated virus, which is characterized by comprising the following steps:
(1) obtaining a bias value of a whole gene codon pair, and carrying out sequence rearrangement on a rabies virus G gene to obtain a rabies virus G gene sequence, wherein the nucleotide sequence of the rabies virus G gene is shown as SEQ ID NO. 1;
(2) preparing the recombinant adeno-associated virus expressing the rabies virus G gene. Respectively inserting the SEQ ID NO.2 and the SEQ ID NO.1 into a vector in sequence to obtain a clone plasmid, co-transfecting the clone plasmid and a packaging plasmid of the adeno-associated virus with cells, collecting the cells, cracking and purifying to obtain the recombinant adeno-associated virus expressing the rabies virus G gene. SEQ ID NO.2 is a Syn promoter sequence and can drive the transcription of the rabies virus G gene. SEQ ID NO.1 is the rabies virus G gene sequence, the rabies virus G gene obtained by the invention is named as ooG, and the rabies virus G gene has the capability of high-efficiency expression.
Sequence optimization of rabies virus G gene is reported, a new gene is obtained and named as oG, the strategy adopted for obtaining the new gene is to use original virus G protein chimeric of different strains, and the sequence is optimized by using a method of codon usage frequency. However, although the above strategy can increase the expression level of the G gene to some extent to assist the rabies virus in reverse transsynaptic junction, the level of increasing the expression of the G gene is limited, and the effect of assisting the rabies virus in reverse transsynaptic junction is not obvious enough. The rabies virus G gene obtained by the invention is named as ooG, the strategy for obtaining a new sequence is different from the strategy for obtaining oG in the literature, the method adopts a method established by Coleman JR to calculate the preference value of the codon pair according to the whole genome sequence information GRCm38 of the mouse, so as to obtain the codon pair preference value of the whole gene of the mouse, and then the primer is obtained by taking oG gene as an object to carry out sequence rearrangement.
The invention also provides an application step of the recombinant adeno-associated virus in neural loop markers: respectively injecting the recombinant adeno-associated virus and rabies viruses expressing rabies virus N, P, M and L protein into hippocampal brain areas of mammals, anesthetizing the animals after infection, respectively perfusing with physiological saline, then fixing with paraformaldehyde, and then placing brain tissues into a sucrose solution; embedding and freezing brain tissue, and slicing; observation was performed using a fluorescence microscope. The mammal is any one of mouse, rabbit, monkey and human. The application objects are not limited to mice, but also can be used for nerve loop markers of monkeys, rabbits and humans.
Example 1: ooG gene sequence in recombinant adeno-associated virus for obtaining high-efficiency auxiliary rabies virus reverse transsynaptic
On the one hand, the codon pair preference value is calculated by the method established by Coleman JR (Coleman JR et al, science.2008) according to the whole genome sequence information GRCm38 of the mouse, and the codon pair preference value refers to: during long-term evolution of viruses and hosts (which have adapted to host characteristics), there is a species-dependent preference for codon pairs of proteins encoded by their genomes, e.g. the combination of codon pairs of amino acid pairs a-E is GCTGAA, GCTGAG, GCCGAA, GCAGAA, GCGGAA and GCGGAG, respectively, which vary widely in frequency of use in different hosts, i.e. a codon pair has a preference in a species. Therefore, the high frequency codon pair is used to increase the protein expression level for a certain species. Thereby obtaining the codon pair preference value of the whole gene of the mouse; on the other hand, around the bottleneck problem of rabies virus widely used for neural loop reverse labeling, low trans-synaptic efficiency, sequence rearrangement was performed with the rabies virus G gene designed by Euiseok J (named oG, Euiseok J. kim et al, Cell reports,2016) as the subject, named ooG, and the obtained sequence information SEQ ID No. 1. In the context of the whole mouse genome, comparing the codon pair preference values of ooG with the codon pair preference values of oG, the results are shown in fig. 1, wherein the codon pair preference value of ooG is significantly increased compared with the codon pair preference value of oG, which indicates that ooG has the advantage of high-efficiency expression in mice compared with oG.
Example 2: preparation of recombinant adeno-associated virus (AAV) with high efficiency in assisting rabies virus reverse transsynaptic
Firstly, preparing clone with high-efficiency expression ooG protein ability
Firstly, respectively synthesizing a Syn promoter (shown as a sequence in SEQ ID NO.2) and ooG (shown as a sequence in SEQ ID NO.1) by adopting a whole-Gene synthesis mode, inserting the Syn promoter and ooG into a double-stranded AAV core plasmid pdsAAV-CBAp-eGFP (Chen Ling et al, Hum Gene ther.2014) according to a mode shown in the figure 2, and obtaining a cloning plasmid named as pscAAV-hsynP-DIO-ooG-bGHpA.
The primers for amplifying the corresponding sequences are as follows:
(a) primers for DNA fragment Syn promoter: 3 and 4, the template is a synthesized fragment SEQ ID NO. 2; (b) primer for DNA fragment rabbit ooG: 5 and 6, and the template is a synthesized fragment SEQ ID NO. 1.
Firstly, the pdsAAV-CBAp-eGFP vector is subjected to double digestion by KpnI and HindIII, and then the SEQ ID NO.2 and the SEQ ID NO.1 are sequentially inserted into the vector by adopting a homologous recombination mode to obtain a clone named as pscAAV-hsynP-DIO-ooG-bGHpA. The primers used by the PCR are all synthesized by the biological engineering (Shanghai) corporation, and the constructed clones are all subjected to sequencing verification by the biological engineering (Shanghai) corporation.
Secondly, preparing adeno-associated virus:
(1) plasmid-transfected HEK293 cells packaging rAAV: one day ahead, HEK293 cells are digested by trypsin and then spread in a 15cm culture dish for adherent culture, and the confluency of the cells is about 80% during transfection. Three plasmids required for packaging rAAV, namely pAAV-Helper plasmid, serotype plasmid pAAV-RC and core expression plasmid pscAAV-hsynP-DIO-ooG-bGHpA, are used for transfecting HEK293 cells by using calcium phosphate or PEI transfection reagent, and culture supernatant and the cells are collected after 72 hours of transfection.
(2) Cell lysis and rAAV purification: cell lysates (50mM Tris-Cl (pH8.0), 2mM MgCl 2 ) And (3) repeatedly freezing and thawing for 3 times, adding nuclease for treatment, and centrifuging to remove cell debris. And (3) merging the cell lysate supernatant and the culture supernatant treated by nuclease, then concentrating and centrifuging by using PEG4000, and carrying out heavy suspension by using a PBS solution to obtain the PBS heavy suspension containing rAAV. Adding 8mL of 15% iodixanol separating medium, 6mL of 25% iodixanol separating medium, 8mL of 40% iodixanol separating medium and 5mL of 58% iodixanol separating medium into an ultracentrifuge tube in sequence, adding the PBS heavy suspension containing the rAAV into the upper layer of the centrifuge tube, sealing, and centrifuging for 2h by using a Type 70Ti rotor at 63000 rpm. Sucking the liquid of the 40% iodixanol gradient layer with a needle, dialyzing with PBS overnight, centrifuging with Amicon ultra-4(100KD cutoff) interception column, concentrating to a final volume of about 1mL, aseptically packaging, and freezing at-80 deg.C.
(3) And (3) detecting the titer of the rAAV by fluorescent quantitative PCR: the DNA copy number of rAAV virions, namely the number VG (represented by Viral genes), is detected by using specific primers to perform fluorescence quantitative PCR, and is converted into the content of rAAV in a unit volume, namely the titer of rAAV, and is represented by VG/mL (Viral genes/mL). Add 5. mu.l of sample to a 1.5ml centrifuge tube, supplement 15. mu.l of ddH 2 O to 20 μ l; adding 20 μ l of 2N NaOH, mixing, and treating in 55 deg.C water bath for 30 min; then, 20. mu.l of 2N HCl was added for neutralization. 5 ul of NaOH-HCl treated sample was added to a 1.5ml centrifuge tube and 45 ul of ddH was added 2 And mixing the obtained product with 50 mu l of the obtained product to be used as a sample to be detected in the Q-PCR reaction.
Example 3: the recombinant adeno-associated virus can efficiently assist the reverse transsynaptic junction of the rabies virus
In one aspect, according to 1: 1 ratio mixing of the recombinant adeno-associated virus prepared in example 2 with a literature report of recombinant adeno-associated virus expressing TVA and eGFP (Fumitaka Osakada et al, neuron.2011); on the other hand, according to 1: 1 ratio mixing of recombinant adeno-associated virus (Euiseok j. kim et al., Cell reports,2016) expressing oG protein and recombinant adeno-associated virus (sumitaka Osakada et al., neuron.2011) expressing TVA and eGFP. Among them, the literature reports that recombinant adeno-associated viruses expressing TVA and eGFP provide N, P, M and L proteins required for rabies virus packaging, and the recombinant adeno-associated viruses of the present invention provide G proteins of rabies viruses. Respectively taking 0.8 mu L of the mixed recombinant adeno-associated virus, positioning and injecting the mixed recombinant adeno-associated virus into the ventral hippocampus brain region of a Thy1-Cre mouse, respectively injecting 0.15 mu L of rabies virus SAD delta G-mCherry (Fumitoka Osakada et al, neuron.2011) expressing red fluorescent protein into the ventral hippocampus brain region into which two corresponding recombinant adeno-associated viruses are injected 21 days after infection, anesthetizing the animal 7 days later, respectively perfusing the animal with 0.9% (V/V) physiological saline, then fixing the animal with 4% (V/V) paraformaldehyde, taking out the brain tissue, soaking the brain tissue in 4% (V/V) paraformaldehyde solution, then placing the brain tissue in 20% (V/V) sucrose solution for 1 day, and then placing the brain tissue in 30% (V/V) sucrose solution for 2 days; cutting the bottom of the brain tissue flat, placing on a base, embedding and freezing for 1h, and then slicing; the brain slices were taken and observed using a fluorescence microscope. The result is shown in fig. 3, after the recombinant virus is injected into the rat brain, a red fluorescent protein signal can be seen, which is shown in the injection region and the transsynaptic region of fig. 3A, and the gray scale in the figure indicates that the recombinant virus can mediate the expression of the foreign protein in the rat brain nerve cell; FIG. 3A shows that the presynaptic position of ooG-expressing recombinant adeno-associated virus shows a significant increase in the number of crossed rabies viruses compared to oG-expressing recombinant adeno-associated virus, which is the best known means for assisting rabies virus in reverse transsynaptic transmission. FIG. 3B is a graph with ordinate representing a trans-synaptic efficiency index, and through calculating the trans-synaptic efficiencies of oG in the literature and ooG in the present invention, ooG is found to be significantly higher than oG, which indicates that the adeno-associated virus expressing ooG in the present invention can better help the rabies virus to transsynaptic and improve the efficiency of the rabies virus to transsynaptic compared with the oG-expressing adeno-associated virus published in the literature. The result shows that the recombinant adeno-associated virus capable of efficiently expressing ooG can assist rabies viruses to be more efficiently transported in a neural network, and has the capability of marking cranial nerve loops.
Example 4: labeling of the cranial nerve loop of a mammal
Specifically, the marking comprises the following steps: injecting 0.8 mu l of recombinant adeno-associated virus into the ventral hippocampus brain region of a Thy1-Cre mouse, injecting 0.15 mu l of rabies virus SAD delta G-mChery (Fumitoka Osakada et al, neuron.2011) expressing red fluorescent protein into the ventral hippocampus brain region of the Thy1-Cre mouse 21 days after infection, anesthetizing the animal 7 days later, perfusing with 0.9% (V/V) physiological saline respectively, fixing with 4% (V/V) paraformaldehyde, taking out the brain tissue, soaking the brain tissue in 4% (V/V) paraformaldehyde solution, placing the brain tissue in 20% (V/V) sucrose solution for 1 day, and then placing the brain tissue in 30% (V/V) sucrose solution for 2 days; cutting the bottom of the brain tissue flat, placing on a base, embedding and freezing for 1h, and then slicing; the position of the rabies virus can be tracked according to the fluorescent signal by observing the brain slice with a fluorescent microscope.
The rabies virus G gene and the recombinant adeno-associated virus expressing the gene have multiple applications in researching a cranial nerve loop platform, and the application comprises the step of rearranging any virus gene by using the mouse codon pair preference rearrangement method provided by the invention to improve the expression level of protein or reduce the expression level of the protein, so that the rabies virus G gene and the recombinant adeno-associated virus expressing the gene are used for cell therapy, gene therapy, oncolytic virus and vaccine development or marking the cranial nerve loop of mammals. The application object is not limited to a mouse, and can also be used for nerve loop marking of animals such as monkeys and the like; the ooG gene used in the present invention is only one example, and thus, can be used for rearrangement of other genes.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
SEQUENCE LISTING
<110> Shenzhen advanced technology research institute of Chinese academy of sciences
<120> novel G gene and application thereof in efficient reverse trans-monosynaptic
<130> 20201015
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1572
<212> DNA
<213> Rabies virus
<400> 1
atggttcctc aagcccttct ctttgttcct cttcttgtct tcccgctctg ctttgggaag 60
ttccccatct acaccattcc tgacaagcta gggccctgga gccccattga catccaccac 120
ctcagctgcc ccaacaactt ggttgtagaa gatgaaggct gcaccaacct cagtggcttc 180
tcctacatgg agctaaaagt gggctacatc tcggccatca agatgaatgg cttcacctgc 240
actggagttg tcactgaagc agagacctac accaactttg ttggctatgt caccaccacc 300
ttcaaaagaa aacacttccg gcccactcca gatgcctgcc gcgcggccta caactggaag 360
atggcggggg acccccgcta tgaagagagc ctgcacaacc cctacccaga ctaccactgg 420
ctgaggactg tgaagaccac caaagaaagt ttggtcatca tcagccccag tgtagctgac 480
ttggacccct atgaccgttc tctacacagc cctgtatttc ctggtgggaa ctgcagtggt 540
gtggctgtca gcagcaccta ctgcagcacc aaccatgact acaccatctg gatgccggag 600
aacccccggc tagggatgtc ctgtgacatc ttcaccaaca gccgagggaa aagagccagc 660
aaaggttctg agacctgtgg ctttgtagat gagcgtggcc tctacaagag tttaaaaggt 720
gcctgcaaat taaaactctg tggtgttctt ggtcttcggc tcatggatgg cacctgggtg 780
gccatgcaga ccagcaatga gaccaagtgg tgcccgccgg gccagcttgt caacctccat 840
gacttccgaa gtgatgaaat agaacatctt gttgtagaag aacttgtcaa gaaaagagaa 900
gaatgtttag atgccctgga gagcatcatg actaccaaga gtgtctcctt ccgtcgcctc 960
agccacctca ggaaacttgt tcctggcttt gggaaagcct acaccatctt caacaagacg 1020
ctcatggaag cagatgccca ctacaaatct gtccgcacgt ggaatgagat cattccttcc 1080
aaaggctgcc tccgagttgg tggccgctgc cacccacatg tcaatggtgt cttcttcaat 1140
ggcatcattc ttgggccaga tggaaatgtc ctcattccag agatgcagag cagcctgctg 1200
cagcagcaca tggaacttct tgtcagcagt gtcatcccgc tcatgcaccc gctggcagac 1260
cccagcactg tcttcaagaa tggagatgaa gcagaagatt ttgtagaagt tcatcttcct 1320
gatgttcatg aaagaatttc tggtgtggac ttgggtcttc ccaactgggg aaaatatgtt 1380
cttctttctg ctggggcgct cacggcgctc atgttaataa tattcctcat gacctgctgc 1440
agaagagtca accgctcgga gcccacccag cacaaccttc gtggcacggg ccgagaagtt 1500
tctgtcacgc cgcagagtgg gaagatcatc tcctcctggg agagccacaa gtcaggagga 1560
gagacgcgcc tg 1572
<210> 2
<211> 452
<212> DNA
<213> human
<400> 2
agtgcaagtg ggttttagga ccaggatgag gctgggtggg ggtgcctacc tgacgaccga 60
ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga 120
gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg 180
gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc 240
gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg 300
cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg 360
cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg 420
aggagtcgtg tcgtgcctga gagcgcagtc ga 452

Claims (8)

1. A rabies virus G gene is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.
2. A method for preparing recombinant adeno-associated virus, which is characterized by comprising the following steps:
(1) obtaining a rabies virus G gene sequence, wherein the nucleotide sequence of the rabies virus G gene is shown as SEQ ID NO. 1;
(2) preparing the recombinant adeno-associated virus expressing the rabies virus G gene.
3. The method according to claim 2, wherein the step (1) comprises a step of obtaining codon pair bias values for the whole gene.
4. The method according to claim 2, wherein in step (2), the recombinant adeno-associated virus expressing rabies virus G gene is obtained by inserting SEQ ID No.2 and SEQ ID No.1 into a vector in sequence to obtain a cloned plasmid, co-transfecting the cloned plasmid with a packaging plasmid for adeno-associated virus into cells, collecting the cells, lysing and purifying the cells.
5. The method of claim 4, wherein the vector is pdsAA V-CBAp-eGFP and the cells are HEK293 cells.
6. A recombinant adeno-associated virus produced by the production method according to any one of claims 2 to 5.
7. Use of the rabies virus G gene according to claim 1 in the preparation of a murine rabies virus reverse transsynaptic tracer tool.
8. The use of the rabies G gene according to claim 1 in murine neural circuit markers.
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