A recombinant adeno-associated virus of serotype 13, its preparation method and application

Abstract

The present invention discloses a recombinant adeno-associated virus of serotype 13, its preparation method and application. By constructing a recombinant adeno-associated virus serotype packaging plasmid containing the Rep gene of AAV2 wild virus and the Cap gene of AAV13 wild virus, and preparing based on a triple plasmid transfection system, a high-titer recombinant adeno-associated virus rAAV2 / 13 can be obtained efficiently and rapidly. The recombinant adeno-associated virus of the present invention can be used as a gene transfer vector to transduce a target gene into nerve cells. It has high expression ability and a smaller diffusion range than rAAV2. Based on this, the rAAV2 / 13 vector can carry the target gene for precise labeling of small brain nuclei for circuit labeling and manipulation in neuroscience research, and can also be used as a vector for gene therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a recombinant adeno-associated virus of serotype 13, a preparation method thereof, and applications thereof. Background Art

[0002] The brain mainly includes the left and right cerebral hemispheres, and is the largest and most complex structure in the central nervous system. It is an organ that regulates the functions of the body and is also the material basis for higher nervous activities such as consciousness, spirit, language, learning, memory, and intelligence. Structurally, the brain is divided into parts such as the frontal lobe, temporal lobe, parietal lobe, occipital lobe, and insula. In 1909, the German anatomist Brodmann divided the brain into 52 regions according to the types of cortical cells and the density of fibers, and numbered them. With the progress of technology, the division of different brain regions has become more and more refined. The connections between different brain regions and different types of neurons encode the consciousness and behaviors of organisms. Therefore, mapping the brain connection map and revealing the structure of neuron connections are the basis for decoding the mysteries of brain function.

[0003] Labeling and tracing based on viral vectors has become one of the important methods for analyzing the structural connections of neurons. By using viral vectors to carry fluorescent marker genes to transduce neurons, the complete neuron structure or neural network structure can be labeled. Among them, recombinant adeno-associated virus (rAAV) is a gene transfer vector modified on the basis of non-pathogenic wild-type AAV. Due to its advantages such as high safety, multiple types, wide tropism, and long-term stable expression of mediated foreign genes, it has become one of the most popular gene transfer vectors in the fields of neuroscience and gene therapy (Bedbrook et al, Annual Review of Neuroscience, 2018; Wang et al, Nature Reviews Drug Discovery, 2019). AAV has different serotypes, and the capsid proteins of different serotypes are different. The differences in capsid proteins determine most of their transduction characteristics. Among these different serotypes, serotypes 1, 2, 5, 6, 8, 9, etc. are commonly used as gene transfer vectors, and they have strong transduction ability for nerve cells. However, their infection and spread range is still relatively large, and it is easy to cause infection of non-target brain regions or cells. Therefore, it is necessary to further develop vectors with a smaller infection and spread range suitable for small nucleus labeling.

[0004] At present, a total of 13 serotypes of AAV existing in nature, namely AAV1 to AAV13, have been published. Different serotypes have different targeting properties and applications, and there are also differences in the preparation methods and yields of each serotype of AAV. There are few reports on the application of AAV13. Therefore, it is necessary to develop an efficient preparation method to meet the research on its functions and applications. SUMMARY OF THE INVENTION

[0005] In order to solve the deficiencies in the prior art, the object of the present invention is to provide a recombinant adeno-associated virus of serotype 13, its preparation method and application.

[0006] The specific technical solution of the present invention is as follows:

[0007] The present invention provides a recombinant adeno-associated virus of serotype 13, which is obtained by packaging with a packaging plasmid containing the Rep gene of adeno-associated virus type 2 and the Cap gene of adeno-associated virus type 13.

[0008] Furthermore, the genome of the recombinant adeno-associated virus of serotype 13 contains a foreign gene;

[0009] Preferably, the foreign gene includes a fluorescent marker gene and / or other foreign genes.

[0010] The present invention also provides a method for preparing the recombinant adeno-associated virus of serotype 13, including: co-transfecting a packaging cell line with a recombinant adeno-associated virus packaging plasmid, an adeno-associated virus core plasmid, and an adenovirus element helper plasmid, and then performing virus harvesting, purification, and concentration to obtain it;

[0011] The recombinant adeno-associated virus packaging plasmid carries the Rep gene of adeno-associated virus type 2 and the Cap gene of adeno-associated virus type 13;

[0012] The adeno-associated virus core plasmid carries the ITR sequence of adeno-associated virus.

[0013] Furthermore, the adenovirus element helper plasmid is pAd-Helper;

[0014] The packaging cell line is HEK-293T cells;

[0015] The plasmid molecule numbers of the recombinant adeno-associated virus packaging plasmid, the adeno-associated virus core plasmid, and the adenovirus element helper plasmid are 1:1:1.

[0016] Furthermore, the nucleotide sequence of the recombinant adeno-associated virus packaging plasmid is as shown in SEQ ID NO: 3.

[0017] Furthermore, the recombinant adeno-associated virus core plasmid inserts a promoter, a foreign gene, a transcriptional regulatory element, and a transcriptional termination sequence in sequence in the middle of the ITR.

[0018] Furthermore, the promoter is a CMV promoter or an EF1α promoter;

[0019] The exogenous gene includes a fluorescent marker gene and / or other exogenous genes;

[0020] The transcriptional regulatory element is WPRE;

[0021] The transcriptional termination sequence is SV40 polyA or hGH polyA.

[0022] The present invention also provides the use of the recombinant adeno-associated virus serotype 13 as a gene transfer vector.

[0023] The present invention also provides the use of the recombinant adeno-associated virus serotype 13 in the preparation of a labeling vector for small nuclei.

[0024] The beneficial effects of the present invention are as follows:

[0025] 1. The present invention constructs a packaging plasmid of recombinant adeno-associated virus rAAV2 / 13, which contains the Rep gene of AAV2 wild virus and the Cap gene of AAV13 wild virus, and produces recombinant adeno-associated virus based on a triple plasmid transfection system, capable of efficiently and rapidly obtaining high-titer recombinant adeno-associated virus rAAV2 / 13, providing technical support for the characterization research and popularization application of recombinant adeno-associated virus rAAV2 / 13.

[0026] 2. The present invention first verifies the labeling characteristics of rAAV2 / 13 in the limited infection of the nervous system. When the rAAV2 / 13 vector carrying the green fluorescent protein gene (EGFP) infects the VTA and S1 brain regions, a very limited small-scale diffusion can be seen. Further, by comparing rAAV2 / 13 with the existing rAAV2 vector commonly used for small-scale labeling, it is proved that the diffusion range of rAAV2 / 13 is smaller than that of rAAV2. These results all indicate the limited labeling characteristics of rAAV2 / 13, which is more suitable for the precise labeling of small nuclei, providing better tools and technical support for neuroscience research, disease model establishment and gene therapy, etc., and having broad application value and market prospects.

[0027] 3. The recombinant adeno-associated virus rAAV2 / 13 of the present invention can be used as a gene transfer vector to transduce the target gene into nerve cells, which has high expression ability and a smaller diffusion range than rAAV2. Based on this, the rAAV2 / 13 vector can carry the target gene for precise labeling of small brain nuclei for circuit labeling and manipulation in neuroscience research, and can also be used as a vector for gene therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1This is the expression vector map of the recombinant adeno-associated virus serotype packaging plasmid pAAV-RC2 / 13.

[0029] Figure 2 This is the silver staining result diagram of rAAV2 / 13.

[0030] Figure 3 This is the infection activity diagram of rAAV2 / 13 on HEK293T cells.

[0031] Figure 4 This is the signal of recombinant adeno-associated virus serotype 13 infecting the primary somatosensory cortex (S1). Among them, the blue fluorescence is the signal of DAPI staining the cell nucleus; the red fluorescence is the signal after staining the neuron marker NeuN (the upper row) and the astrocyte marker GFAP (the bottom row) respectively; the green fluorescence is the cell signal labeled by rAAV2 / 13-CMV-EGFP-WPRE-SV40polyA.

[0032] Figure 5 This is the signal of recombinant adeno-associated virus serotype 13 infecting the ventral tegmental area (VTA) of the midbrain. Among them, the blue fluorescence is the signal of DAPI staining the cell nucleus; the red fluorescence is the signal after staining the neuron marker NeuN (the upper row) and the astrocyte marker GFAP (the bottom row) respectively; the green fluorescence is the cell signal labeled by rAAV2 / 13-CMV-EGFP-WPRE-SV40 polyA.

[0033] Figure 6 This is the signal of the mixed injection of rAAV2 / 13-EFGFP and rAAV2-mCherry infecting the primary somatosensory cortex (S1). Among them, the blue fluorescence is the signal of DAPI staining the cell nucleus; the red fluorescence is the signal of rAAV2 infecting neurons; the green fluorescence is the signal of rAAV2 / 13 infecting neurons. Detailed implementation mode

[0034] To understand the present invention more clearly, the present invention will be further described with reference to the following examples and accompanying drawings. The examples are only for explanation and do not limit the present invention in any way. In the examples, all the original reagent materials can be obtained commercially. The experimental methods without specific conditions are the conventional methods and conventional conditions well known in the art, or the conditions recommended by the instrument manufacturer.

[0035] Example 1

[0036] I. Construction of the recombinant adeno-associated virus serotype packaging plasmid

[0037] According to the AAV13 genome sequence (GenBank: EU285562), the AAV13 Cap gene sequence was synthesized as a template, and the AAV13 Cap gene fragment was amplified using Takara Primerstar Polymerase (Takara). The sequence of the forward primer Cap13-F is shown in SEQ ID NO.1, and the sequence of the reverse primer Cap13-R is shown in SEQ ID NO.2. The reaction system for PCR was 50 μl: 10 μl of 5×Reaction Buffer, 1 μl of 10 mM dNTPs, 2.5 μl of 10 μM forward primer, 2.5 μl of 10 μM reverse primer, 0.5 μl of template DNA, 0.5 μl of DNA Polymerase, and 33 μl of ddH2O. The amplification conditions were: 98°C for 3 min, 98°C for 20 s, 60°C for 20 s, 72°C for 2 min, 72°C for 10 min, 16°C for 30 min, for 30 cycles; the amplified DNA fragment was recovered using a gel recovery kit (Omega).

[0038] The pAAV-RC2 / 1 vector (purchased from Addgene, catalog number: 112862) and the AAV13 Cap gene fragment were digested with SwaI and AgeI (New England Biolabs) restriction endonucleases respectively, and then the AAV13 Cap gene fragment was inserted into pAAV-RC2 / 1 using T4 ligase. The ligation product was transformed into competent Stbl3, and the clones identified as positive by colony PCR were inoculated into 15 ml of LB liquid medium for culture and plasmid extraction for sequencing. The correctly sequenced clones were named pAAV-RC2 / 13, and the obtained plasmid can encode the AAV13 capsid protein VP1. The map of the constructed recombinant adeno-associated virus serotype packaging plasmid pAAV-RC2 / 13 expression vector is as Figure 1 shown, and its gene sequence is shown in SEQ ID NO.3. All PCR primers, gene synthesis, and sequencing in the present invention were completed by Sangon Biotech (Shanghai) Co., Ltd.

[0039] II. Preparation of recombinant adeno-associated virus

[0040] Recombinant adeno-associated virus serotype packaging plasmid pAAV-RC2 / 13 expression vector was used to package recombinant adeno-associated virus. The plasmid pAAV-CMV-EGFP-WPRE-hGH polyA carrying the core elements, the AAV capsid plasmid of serotype AAV-RC2 / 13, and the adenovirus element helper plasmid pAd-Helper were co-transfected into HEK-293T cells at a plasmid molecular ratio of 1:1:1. After 72 hours of transfection, the supernatant and cell pellet were collected and processed separately: The HEK-293T cell pellet containing AAV virus particles was resuspended in lysis buffer (9 mL for 15 dishes of cell volume), repeatedly frozen and thawed in liquid nitrogen and 37 °C water bath, then Benzonase nuclease (Sigma, E1014-25KU) was added and digested at 37 °C for 1 hour. Then, NaCl with a final concentration of 150 mM was added and shaken at 37 °C for 30 min; After the cell supernatant was treated with nuclease, PEG8000 (Solarbio, 214B0310) and 0.5 mol / L NaCl solution were added, mixed well, and left at 4 °C for 16 h to precipitate proteins. The next day, the treated supernatant was centrifuged at 4 °C at 10000 g to discard the supernatant, then the cell sample and the supernatant precipitate sample were combined, centrifuged at 3000 g for 10 min, and the cell debris precipitate was discarded to obtain the virus concentrate. Iodixanol solutions with concentrations of 15%, 25%, 40%, and 58% were prepared in a Beckman ultracentrifugation tube in sequence, then 10 mL of the virus concentrate was added, and finally the centrifuge tube was filled with PBS and sealed. After balancing, it was placed in a Ti70 rotor and centrifuged at 64000 rpm for 2 hours at 18 °C in a Beckman ultracentrifuge. After centrifugation, a syringe was used to aspirate the solution at the separation layer of 40% and 58% iodixanol to recover it. The iodixanol layer solution containing rAAV aspirated was placed in a dialysis bag and dialyzed in PBS buffer at 4 °C for 16 h; The dialyzed virus solution was filtered through a 0.22 μm filter membrane to sterilize it, then added to an ultrafiltration tube (Minipore, UFC910024), centrifuged to desalt and concentrated to a volume of about 200 μL, and then Pluronic F68 (Thermo Fisher Scientific, 24040032) with a final concentration of 0.001% was added, aliquoted, and stored in a -80 °C refrigerator. Finally, the titer of recombinant adeno-associated virus was detected by SYBR Green qPCR method, and the titer of rAAV2 / 13-CMV-EGFP-WPRE-hGH polyA virus was finally obtained as 1.0×10 13 VG / mL. The assembly effect of capsid proteins was identified by silver staining, and the silver staining results are as Figure 2 shown. The above results indicate that the adeno-associated virus serotype packaging plasmid pAAV-RC2 / 13 can prepare rAAV2 / 13 with high titer.

[0041] III. Activity test of recombinant adeno-associated virus

[0042] To verify the infectivity of the prepared recombinant adeno-associated virus rAAV2 / 13, the recombinant adeno-associated virus was used at an MOI of 2.5×10 5 to infect adherent HEK293T cells. By observing the expression level of the carried reporter gene EGFP, the infectivity of the recombinant adeno-associated virus rAAV2 / 13 can be identified. As Figure 3 shown, high-abundance expression of green fluorescent protein can be observed 48 hours after virus infection of the cells. The results indicate that the recombinant adeno-associated virus rAAV2 / 13 has high infectivity for cells cultured in vitro.

[0043] Example 2

[0044] (1) The recombinant adeno-associated virus rAAV2 / 13-CMV-EGFP-WPRE-SV40polyA was prepared using the method described in Example 1. The prepared rAAV2 / 13-CMV-EGFP-WPRE-SV40 polyA (200 nL / animal) virus was injected into the primary somatosensory cortex (S1) and ventral tegmental area (VTA) of 8- to 10-week-old C57BL / 6 mice (purchased from Hunan Slack Jingda Laboratory Animal Co., Ltd.) by stereotaxic injection into the brain. Three weeks later, the mice were perfused and the brains were removed. The mouse brain tissues were fixed in a DEPC-treated PFA solution for 4 hours and then dehydrated in a DEPC-treated 30% sucrose-PBS solution for 48 hours. The dehydrated brain tissues were fully embedded with tissue embedding agent and cut into 40-μm-thick brain slices using a cryostat. The brain slices containing S1 and VTA were immunostained with GFAP antibody and NeuN antibody, and then mounted and imaged using a slide scanner microscope. The in vivo detection results show that ( Figure 4 and Figure 5 ) after rAAV2 / 13-CMV-EGFP-WPRE-SV40 polyA infects the central nervous system, it has a very limited labeling range, and the vast majority of the transduced green fluorescent signals co-localize with neurons.

[0045] (2) The recombinant adeno-associated virus rAAV2 / 13-EF1α-EGFP-WPRE-SV40polyA was prepared by the method described in Example 1, which differed from Example 1 in the plasmid carrying the core elements. The rAAV2 / 13-EF1α-EGFP-WPRE-SV40 polyA carrying the green fluorescent protein gene was mixed with the rAAV2-EF1α-mCherry-WPRE-SV40 polyA carrying the red fluorescent protein gene at a ratio of 1:1 (200 nL / animal), and was injected into the primary somatosensory cortex (S1) of 8-10-week-old C57BL / 6 mice (purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.) by stereotaxic injection. Three weeks later, the mice were perfused and their brains were removed. The mouse brain tissues were fixed with PFA solution treated with DEPC for 4 hours and then dehydrated with 30% sucrose-PBS solution treated with DEPC for 48 hours. The dehydrated brain tissues were fully embedded with tissue embedding agent and cut into brain slices with a thickness of 40 μm using a cryostat. As can be seen from the in vivo detection results ( Figure 6 ), after rAAV2 / 13-EF1α-EGFP-WPRE-SV40 polyA infects the central nervous system, it has a smaller labeling range compared to rAAV2-EF1α-mCherry-WPRE-SV40polyA.

[0046] Obviously, the above examples are only for clear illustration and not for limitation of the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. Use of a recombinant adeno-associated virus of serotype 13 in the preparation of a labeling vector for small nuclei, characterized in that, The recombinant adeno-associated virus of serotype 13 is obtained by packaging with a packaging plasmid containing the Rep gene of adeno-associated virus type 2 and the Cap gene of adeno-associated virus type 13.

2. The application according to claim 1, wherein The genome of the recombinant adeno-associated virus of serotype 13 contains a foreign gene.

3. The application according to claim 2, wherein The foreign gene includes a fluorescent marker gene and / or other foreign genes.

4. The application according to claim 1, characterized in that The preparation method of the recombinant adeno-associated virus of serotype 13 includes: co-transfecting a packaging cell line with a recombinant adeno-associated virus packaging plasmid, an adeno-associated virus core plasmid, and an adenovirus element helper plasmid, and then performing virus harvesting, purification, and concentration to obtain it. The recombinant adeno-associated virus packaging plasmid carries the Rep gene of adeno-associated virus type 2 and the Cap gene of adeno-associated virus type 13. The adeno-associated virus core plasmid carries the ITR sequence of adeno-associated virus.

5. The application according to claim 4, wherein The adenovirus element helper plasmid is pAd-Helper. The packaging cell line is HEK-293T cells. The plasmid molecule numbers of the recombinant adeno-associated virus packaging plasmid, the adeno-associated virus core plasmid, and the adenovirus element helper plasmid are 1:1:

1.

6. The application according to claim 4, wherein The nucleotide sequence of the recombinant adeno-associated virus packaging plasmid is as shown in SEQ ID NO:

3.

7. The application according to claim 4, characterized in that In the recombinant adeno-associated virus core plasmid, a promoter, a foreign gene, a transcriptional regulatory element, and a transcriptional termination sequence are inserted in sequence in the middle of the ITR.

8. The application according to claim 7, wherein The promoter is a CMV promoter or an EF1α promoter. The foreign gene includes a fluorescent marker gene and / or other foreign genes. The transcriptional regulatory element is WPRE. The transcriptional termination sequence is SV40 polyA or hGH polyA.

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