Preparation and application of an efficient reverse transsynaptic rabies virus tracing tool expressing green fluorescent protein

Abstract

The present invention belongs to the field of biotechnology, and discloses the preparation and application of an attenuated and efficient reverse transsynaptic rabies virus tracing tool expressing green fluorescent protein. The present invention rearranges and modifies the rabies virus N gene sequence to obtain a novel rabies virus N gene optiN, and then prepares an attenuated and efficient transsynaptic rabies virus expressing green fluorescent protein based on the optiN gene. The optiN gene can reduce the toxicity of rabies virus and improve the efficiency of rabies virus reverse transsynaptic, which can be used as a neural circuit marking tool. The present invention has important practical significance and wide application value for conducting basic research (such as pathogenic mechanism, replication mechanism, etc.) and applied research (such as neural circuit marking, cell gene therapy, oncolytic virus, new vaccine and diagnostic reagent, etc.) on rabies virus.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to the preparation and application of a highly efficient reverse transsynaptic rabies virus tracing tool that attenuates and expresses green fluorescent protein. 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 a new attenuated, efficient, reverse transsynaptic rabies virus tracing tool. Summary of the invention

[0004] In view of the shortcomings and deficiencies in the prior art, the purpose of the present invention is to provide a preparation and application of an efficient reverse transsynaptic rabies virus tracing tool that attenuates and expresses green fluorescent protein. The present invention first rearranges and modifies the rabies virus N gene sequence to obtain a new rabies virus N gene, and then prepares a recombinant rabies virus expressing the new rabies virus N gene.

[0005] The first aspect of the present invention provides a novel rabies virus N gene, which is obtained by rearranging and modifying the rabies virus N gene sequence, and is named as the optiN gene, and its sequence is shown in SEQ ID NO. 2. The optiN gene obtained by rearranging and modifying the rabies virus N gene sequence in the present invention is different from the original sequence of the N gene, and is a completely new sequence. The optiN gene can reduce the toxicity of rabies virus and improve the efficiency of reverse transsynaptic translocation of rabies virus.

[0006] The second aspect of the present invention provides a method for preparing a highly efficient reverse transsynaptic rabies virus tracing tool that attenuates and expresses green fluorescent protein, comprising the following steps:

[0007] (1) Construction of clones: The pSADdeltaG-F3 vector was digested with AscI and SacII, and the sequence shown in SEQ ID NO.1 was inserted into the vector by homologous recombination to obtain a clone named SAD-mNeonGreen; then the SAD-mNeonGreen plasmid was digested with SwaI and ClaI, and the sequence shown in SEQ ID NO.2 was inserted into the plasmid by homologous recombination to obtain a clone named attSAD-mNeonGreen;

[0008] (2) Construction of recombinant rabies virus: The plasmid attSAD-mNeonGreen obtained in step (1) and auxiliary plasmids pN, pP, pG and pL were mixed and transfected into B7GG cells and then infected into EnvA cells. The cell culture supernatant was collected and purified to obtain an efficient reverse transsynaptic rabies virus tracing tool that expresses green fluorescent protein attenuated by attenuation.

[0009] The third aspect of the present invention provides a highly efficient reverse transsynaptic rabies virus tracing tool that is attenuated and expresses green fluorescent protein and is prepared by the above preparation method.

[0010] The fourth aspect of the present invention provides the use of the novel rabies virus N gene or the attenuated green fluorescent protein-expressing efficient reverse transsynaptic rabies virus tracing tool in tracing the brain neural circuits of mammals.

[0011] The fifth aspect of the present invention provides the use of the novel rabies virus N gene or the attenuated green fluorescent protein-expressing efficient reverse transsynaptic rabies virus tracing tool in studying the mechanism of drug inhibition of rabies virus.

[0012] The sixth aspect of the present invention provides the use of the novel rabies virus N gene or the attenuated green fluorescent protein-expressing efficient reverse transsynaptic rabies virus tracing tool in the development of rabies virus vaccines.

[0013] The seventh aspect of the present invention provides the use of the novel rabies virus N gene or the attenuated green fluorescent protein-expressing efficient reverse transsynaptic rabies virus tracing tool in the establishment of an animal model of rabies virus infection.

[0014] The eighth aspect of the present invention provides the use of the novel rabies virus N gene or the attenuated green fluorescent protein-expressing efficient reverse transsynaptic rabies virus tracing tool in the analysis of rabies virus replication and pathogenic mechanism.

[0015] The ninth aspect of the present invention provides the use of the novel rabies virus N gene or the efficient reverse transsynaptic rabies virus tracing tool expressing green fluorescent protein in establishing a drug screening platform for rabies virus.

[0016] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0017] (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 optiN gene obtained by rearranging and modifying the rabies virus N gene sequence in the present invention can reduce the toxicity of rabies virus and improve the efficiency of rabies virus reverse transsynaptic. It can be used as a neural circuit labeling tool to facilitate related research.

[0018] (2) The present invention prepares a highly efficient reverse transsynaptic rabies virus tracing tool based on the optiN gene that expresses green fluorescent protein attenuated by the retinoic acid. The tool has low toxicity and can better help the rabies virus to reversely transsynapse, thus facilitating related research.

[0019] (3) 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

[0020] 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.

[0021] Figure 1 The schematic diagram of the construction of the efficient reverse transsynaptic rabies virus clone expressing green fluorescent protein with attenuation of the present invention is shown, wherein A represents the backbone plasmid pSADdeltaG-F3, B represents the intermediate plasmid SAD-mNeonGreen, C represents the target plasmid attSAD-mNeonGreen of the present invention, MCS represents the multiple cloning site, N, P, M and L represent the four proteins encoded by rabies virus respectively, mNeonGreen represents the green fluorescent protein gene, and optiN represents the rabies virus N gene after sequence rearrangement and modification.

[0022] Figure 2 The figure is a schematic diagram of the packaging process of the attenuated green fluorescent protein-expressing efficient reverse transsynaptic rabies virus of the present invention, wherein plasmid A is the core plasmid, i.e., the target plasmid attSAD-mNeonGreen of the present invention, plasmid B, plasmid C, plasmid D and plasmid E are respectively the auxiliary plasmids pN, pP, pG and pL for packaging rabies virus; the cells used for transfection are B7GG cells, and the cells used for infection are EnvA cells.

[0023] Figure 3 This is a diagram showing the effect of reducing toxicity of the attenuated high-efficiency reverse transsynaptic rabies virus expressing green fluorescent protein of the present invention, wherein A is the toxicity of the existing rabies virus after infecting cells, and B is the toxicity of the recombinant rabies virus constructed by the present invention after infecting cells.

[0024] Figure 4 This is a diagram showing the effect of improving the transsynaptic efficiency of the attenuated high-efficiency reverse transsynaptic rabies virus expressing green fluorescent protein of the present invention, wherein A is the transsynaptic effect of the recombinant rabies virus constructed by the present invention, B is the transsynaptic effect of the existing rabies virus, and C is the comparison result of the transsynaptic efficiency of the recombinant rabies virus constructed by the present invention and the existing rabies virus. DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] Example 1: Obtaining the optiN gene sequence

[0028] The present invention is based on the whole genome sequence information GRCm38 of mice, and the method established by Coleman JR (Coleman JR et al., Science. 2008) is used to calculate the preference value of codon pairs. The so-called codon pair preference value refers to: in the long-term evolution process between the virus and the host (which has adapted to the host characteristics), the codon pairs of the genome encoding proteins have species-dependent preferences, such as the codon pair combinations of the amino acid pair AE are GCTGAA, GCTGAG, GCCGAA, GCCGAG, GCAGAA, GCAGAG, GCGGAA and GCGGAG, respectively. The usage frequencies of these codon pairs in different hosts vary greatly, that is, a certain codon pair has a preference in a certain species. Therefore, taking a certain species as the object, the protein expression level can be improved by using high-frequency codon pairs, thereby obtaining the whole gene codon pair preference value of mice. To address the bottleneck issues of high toxicity and low transsynaptic efficiency of rabies virus, which is widely used for reverse labeling of neural circuits, the rabies virus N gene was used as the object for sequence rearrangement and modification to obtain a new sequence, named optiN gene, whose sequence is shown in SEQ ID NO.2.

[0029] Example 2: Preparation of an attenuated, highly efficient, reverse transsynaptic rabies virus expressing green fluorescent protein

[0030] 1. Building clones

[0031] The pSADdeltaG-F3 (Addgene #32634) vector was digested with AscI and SacII, and the sequence shown in SEQ ID NO.1 was inserted into the vector by homologous recombination to obtain a clone named SAD-mNeonGreen; then the SAD-mNeonGreen plasmid was digested with SwaI and ClaI, and the sequence shown in SEQ ID NO.2 was inserted into the plasmid by homologous recombination to obtain a clone named attSAD-mNeonGreen.

[0032] 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.

[0033] 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.

[0034] 2. Construction of recombinant rabies virus

[0035] (1) Preparation of recombinant rabies virus packaged by G

[0036] One day in advance, B7GG cells (Osakada F and Callaway EM. Nature Protocols. 2013.) 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 five plasmids required for packaging rabies virus, namely core plasmid A (attSAD-mNeonGreen or SAD-mNeonGreen, where SAD-mNeonGreen was used as a control experiment), pN plasmid B, pP plasmid C, pG plasmid D, and pL plasmid E (sources of pN, pP, pG, and pL refer to "Osakada F and Callaway EM. Nature Protocols. 2013"), were transfected into B7GG cells using transfection reagent lipo2000 (purchased from Thermo Fisher Scientific). After transfection, the culture supernatant was collected to obtain the recombinant rabies virus packaged by G protein. The packaging process is as follows: Figure 2 shown.

[0037] Next, the virus titer was determined by taking 15 μL of the collected supernatant and adding it to a 1.5 mL centrifuge tube, adding 135 μL of culture medium to 150 μL, and then diluting it by 10-fold dilution. 100 μL of the original solution and the diluted virus were taken to infect 293T cells (purchased from ATCC). Since the recombinant rabies virus can express green fluorescent protein, the virus titer was calculated by recording the number of cells expressing green fluorescent protein observed under a microscope.

[0038] (2) Preparation of recombinant rabies virus packaged by EnvA

[0039] One day in advance, the EnvA cells (Osakada F and Callaway EM. Nature Protocols. 2013.) were digested with trypsin and plated on a 15 cm culture dish for adherent culture. The cell confluence was about 90% during infection. The recombinant rabies virus packaged by G prepared in step (1) was used to infect the cells expressing EnvA. After infection, the culture supernatant was collected to obtain the recombinant rabies virus packaged by EnvA. The packaging process is as follows: Figure 2 shown.

[0040] Next, the virus titer was determined by taking 15 μL of the collected supernatant and adding it to a 1.5 mL centrifuge tube, adding 135 μL of culture medium to 150 μL, and then diluting it by 10-fold dilution. 100 μL of the original solution and the diluted virus were taken to infect TVA800 cells (Osakada F and Callaway EM. Nature Protocols. 2013.). Since the recombinant rabies virus can express green fluorescent protein, the virus titer was calculated by recording the number of cells expressing green fluorescent protein observed under a microscope.

[0041] Example 3: Toxicity test of attenuated high-efficiency reverse transsynaptic rabies virus expressing green fluorescent protein

[0042] Take 0.15 μL of the recombinant rabies virus expressing green fluorescent protein packaged by G (i.e., attSAD-mNeonGreen and SAD-mNeonGreen) in Example 2 of the present invention and inject them into the ventral hippocampus of mice (purchased from Guangdong Weitong Lihua Experimental Animal Technology Co., Ltd.). After 21 days, anesthetize the animals, perfuse with 0.9% (V / V) saline, and then fix with 4% (V / V) paraformaldehyde. Take out the brain tissue and immerse it in 4% (V / V) paraformaldehyde solution. Then, place the brain tissue in 20% (V / V) sucrose solution for 1 day, and then place it in 30% (V / V) sucrose solution for 2 days. Cut the bottom of the brain tissue flat, place it on a base, embed and freeze it for 1 hour, and then slice it. Take the brain slices and observe them under a fluorescence microscope. The observation results are as follows: Figure 3 As shown, after the recombinant virus was injected into the mouse brain, green fluorescent protein signals were visible. Figure 3 The injection areas of A and 3B show that the recombinant virus can mediate the expression of foreign proteins in mouse brain neurons; Figure 3 A has a smaller number of fluorescent cells, indicating that the rabies virus in the current field causes neuronal cell death due to its high toxicity; Figure 3 The number of fluorescent signals of B is obviously large, indicating that the toxicity of the recombinant rabies virus of the present invention is significantly reduced compared with the current existing technology.

[0043] The results of this example show that the attenuated green fluorescent protein-expressing high-efficiency reverse transsynaptic rabies virus tracing tool prepared by the present invention can infect nerve cells more efficiently and more toxicly, and has the ability to mark brain neural circuits.

[0044] Example 4: Transsynaptic testing of an attenuated highly efficient reverse transsynaptic rabies virus expressing green fluorescent protein

[0045] 0.1 μL of recombinant adeno-associated virus expressing G and TVA (purchased from Wuhan Shumi Brain Science Technology Co., Ltd.) was injected into the ventral hippocampus of the mouse. 21 days after infection, 0.15 μL of the recombinant rabies virus expressing green fluorescent protein packaged by EnvA in Example 2 of the present invention (i.e., attSAD-mNeonGreen and SAD-mNeonGreen) was injected into the area where the recombinant adeno-associated virus had been injected. After 14 days, the animals were anesthetized, perfused with 0.9% (V / V) saline, and then fixed with 4% (V / V) paraformaldehyde. The brain tissue was taken out and immersed in 4% (V / V) paraformaldehyde solution, and then placed in 20% (V / V) sucrose solution for 1 day, and then placed 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; brain slices were taken and observed using a fluorescence microscope. The results are as follows Figure 4 As shown, after the recombinant virus was injected into the mouse brain, green fluorescent protein signals were visible. Figure 4 The transsynaptic regions of A and 4B show that the recombinant virus can mediate the expression of foreign proteins in mouse brain neurons and help rabies virus to cross synapses; Figure 4 The transsynaptic region in A shows many green fluorescent signals; Figure 4 The transsynaptic region of B shows less green fluorescent signal. Therefore, compared with the current prior art, the recombinant rabies virus of the present invention has significantly more transsynaptic rabies viruses. Figure 4 The ordinate of C is the transsynaptic efficiency index, and it is found that the transsynaptic efficiency of the recombinant rabies virus of the present invention is significantly higher than that of the rabies virus in the current field.

[0046] The results of this example show that the efficient reverse transsynaptic rabies virus tracing tool expressing green fluorescent protein prepared by the present invention can be more efficiently transported in neural networks and has the ability to mark brain neural circuits.

[0047] 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 novel rabies virus N gene, characterized in that: The novel rabies virus N gene is obtained by rearranging and modifying the rabies virus N gene sequence. The sequence of the novel rabies virus N gene is shown in SEQ ID NO.

2.

2. A method for preparing a highly efficient reverse transsynaptic rabies virus tracing tool expressing green fluorescent protein attenuated by virulence, characterized in that: The following steps are involved: (1) Construction of clones: The pSADdeltaG-F3 vector was digested with AscI and SacII, and the sequence shown in SEQ ID NO.1 was inserted into the vector by homologous recombination to obtain a clone named SAD-mNeonGreen; then the SAD-mNeonGreen plasmid was digested with SwaI and ClaI, and the sequence shown in SEQ ID NO.2 was inserted into the plasmid by homologous recombination to obtain a clone named attSAD-mNeonGreen; (2) Construction of recombinant rabies virus: The plasmid attSAD-mNeonGreen obtained in step (1) and auxiliary plasmids pN, pP, pG and pL were mixed and transfected into B7GG cells and then infected into EnvA cells. The cell culture supernatant was collected and purified to obtain an efficient reverse transsynaptic rabies virus tracing tool that expresses green fluorescent protein attenuated by attenuation.

3. The highly efficient reverse transsynaptic rabies virus tracing tool expressing green fluorescent protein and attenuated by the preparation method of claim 2.

4. Use of the novel rabies virus N gene of claim 1 or the attenuated green fluorescent protein-expressing efficient reverse transsynaptic rabies virus tracing tool of claim 3 in tracing brain neural circuits of mammals.

5. Use of the novel rabies virus N gene of claim 1 or the attenuated green fluorescent protein-expressing efficient reverse transsynaptic rabies virus tracing tool of claim 3 in studying the mechanism of drug inhibition of rabies virus.

6. Use of the novel rabies virus N gene of claim 1 or the attenuated green fluorescent protein-expressing efficient reverse transsynaptic rabies virus tracing tool of claim 3 in the development of rabies virus vaccines.

7. Use of the novel rabies virus N gene of claim 1 or the attenuated green fluorescent protein-expressing efficient reverse transsynaptic rabies virus tracing tool of claim 3 in the establishment of an animal model of rabies virus infection.

8. Use of the novel rabies virus N gene of claim 1 or the attenuated green fluorescent protein-expressing efficient reverse transsynaptic rabies virus tracing tool of claim 3 in the analysis of rabies virus replication and pathogenic mechanism.

9. Use of the novel rabies virus N gene of claim 1 or the attenuated green fluorescent protein-expressing efficient reverse transsynaptic rabies virus tracing tool of claim 3 in establishing a drug screening platform for rabies virus.

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