CN112813037A - Recombinant mutant adeno-associated virus capable of efficiently infecting primary microglia and related biological material thereof - Google Patents
Recombinant mutant adeno-associated virus capable of efficiently infecting primary microglia and related biological material thereof Download PDFInfo
- Publication number
- CN112813037A CN112813037A CN202110021870.7A CN202110021870A CN112813037A CN 112813037 A CN112813037 A CN 112813037A CN 202110021870 A CN202110021870 A CN 202110021870A CN 112813037 A CN112813037 A CN 112813037A
- Authority
- CN
- China
- Prior art keywords
- associated virus
- aav
- microglia
- sequence
- gly
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14151—Methods of production or purification of viral material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a recombinant mutant adeno-associated virus capable of efficiently infecting primary microglia and a related biological material thereof. The recombinant adeno-associated virus is adeno-associated virus expressing AAV-6X type capsid protein, and AAV-6X type capsid protein is protein with amino acid sequence of sequence 4 in the sequence table. The invention obtains the mutated AAV-6 capsid protein by mutating the wild AAV-6 capsid protein as follows: 7 amino acid residues are inserted between 588-589 th position of the amino acid sequence of the AAV-6 type capsid protein. The efficiency of the recombinant adeno-associated virus rAAV-6X (expressing AAV-6X type capsid protein) to infect microglia in vitro is 8 times of the efficiency of the recombinant adeno-associated virus rAAV-6 (expressing wild AAV-6 type capsid protein) to infect microglia in vitro. The recombinant adeno-associated virus is a rAAV vector for efficiently transducing microglia.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a recombinant mutant adeno-associated virus capable of efficiently infecting primary microglia and a related biological material thereof.
Background
Recent studies have shown that the degree of nerve damage appears to be related to the scale of innate immune activity and the degree of activity. In the central nervous system, microglia (microglia) are the major immune cells, the earliest of which are a class of cells that respond to nerve injury, infection, or changes in pathological state. Besides the function of immune response, microglia plays important roles of supporting, nourishing, protecting and the like in the physiological activities of neurons, and also participates in the shaping and trimming of the connectivity of the neurons in the development process, the formation of synaptic plasticity of the neurons, neurogenesis, learning and the like. There is increasing research evidence that microglia-associated signaling pathways may be central to the risk and pathogenesis of neurodegenerative diseases. Large scale genome-wide association studies (GWAS) have identified more than 20 sites associated with Alzheimer's disease (AD, senile dementia), many of which are expressed or only in microglia. In AD, the neurotoxic effects of amyloid beta (a β) are thought to play a major role in the progression of the AD disease process. Microglial clearance of a β failure may be the key to causing neuroinflammation and neurodegeneration. For example, if the TREM2 gene is knocked out in microglia, the microglia rarely accumulate around a β plaques, and the neuroinflammation and cognitive ability of AD mice worsen. The role of microglia in autism and developmental disorders is becoming a focus of research. Autopsy results of patients with autism show that the number, morphology and neuronal interactions of microglia in the brain of the patients are changed, especially in the dorsolateral prefrontal cortex and other areas that control executive functions. In addition, whole transcriptome analysis found that the expression of many microglia-specific genes in autistic brain tissue was significantly altered, including markers associated with inflammatory states. Although human genetic studies have not found that microglia-expressed genes are directly associated with autism disorders, microglia may cause neural circuit dysfunction in autistic patients by abnormally responding to damage from neurons or other nerve cells.
Adeno-associated virus (AAV) was first discovered in 1960 in the culture medium of experimental adenovirus (AdV) and was subsequently developed as a foreign gene transfer tool. Among many recombinant viruses, recombinant adeno-associated virus (rAAV) has been widely used in gene therapy for various diseases due to its high safety and high stability. In the field of neurobiology, rAAV has been successfully applied to functional studies of foreign genes in astrocytes and neurons. Although the clinical success of rAAV gene therapy is encouraging, rAAV expression systems still face bottleneck problems in many tissues and specific cells, such as low infection rates, poor target specificity, and limited foreign gene size. Until now, there have been many attempts to develop viral vector systems targeting microglia, whether lentiviruses or AAV, which have significant drawbacks in specificity and infection efficiency. AAV-1,2,3,4,5,7,8,9 types have been proved to hardly infect any microglia in vitro and in vivo, and AAV-6 types have an in vitro microglia infection efficiency of less than 10%, which greatly limits the application of AAV in microglia.
Therefore, based on the important biological functions of microglia and the huge application potential of AAV, a highly effective microglial-infecting rAAV is urgently needed to be developed, which in turn advances the fundamental research of microglia in the aspects of functions and regulation mechanism in disease development.
Disclosure of Invention
The invention aims to solve the technical problem of how to improve the efficiency of infecting microglia in vitro by adeno-associated virus.
In order to solve the above technical problems, the present invention provides a recombinant adeno-associated virus.
The recombinant adeno-associated virus provided by the invention is adeno-associated virus expressing AAV-6X type capsid protein, and the AAV-6X type capsid protein is protein with an amino acid sequence of a sequence 4 in a sequence table.
The recombinant adeno-associated virus contains the coding gene of the AAV-6X type capsid protein, and the coding sequence (CDS) of the coding chain of the AAV-6X type capsid protein is nucleotide 2003-4234 of sequence 3 in the sequence table.
In order to solve the above technical problems, the present invention provides a method for constructing a recombinant adeno-associated virus.
The construction method of the recombinant adeno-associated virus provided by the invention comprises the steps of introducing the encoding gene of AAV-6X type capsid protein into a packaging cell to obtain the recombinant adeno-associated virus expressing the AAV-6X type capsid protein; the AAV-6X type capsid protein is a protein with an amino acid sequence of a sequence 4 in a sequence table.
In the above construction method, the packaging cell may be AAV-293 cell line.
In order to solve the above technical problem, the present invention provides any one of the following applications:
p1, the application of the recombinant adeno-associated virus in preparing an exogenous gene transfer tool,
p2, the application of the recombinant adeno-associated virus in introducing the target DNA into microglia,
p3, the application of the recombinant adeno-associated virus in the molecular level modification of microglia,
p4 and application of the recombinant adeno-associated virus in microglia function research.
In order to solve the above technical problems, the present invention provides a protein.
The protein provided by the invention is AAV-6X type capsid protein, and the AAV-6X type capsid protein is protein with an amino acid sequence of a sequence 4 in a sequence table.
The invention also provides a biological material related to AAV-6X type capsid protein, wherein the biological material is any one of C1) to C7):
C1) a nucleic acid molecule encoding an AAV-6 type X capsid protein;
C2) an expression cassette comprising the nucleic acid molecule of C1);
C3) a recombinant vector comprising the nucleic acid molecule of C1), or a recombinant vector comprising the expression cassette of C2);
C4) a recombinant microorganism containing C1) the nucleic acid molecule, or a recombinant microorganism containing C2) the expression cassette, or a recombinant microorganism containing C3) the recombinant vector.
In the above biological material, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc. The nucleic acid molecule can be cDNA molecule or DNA molecule (i.e. AAV-6X type capsid protein gene) of which the coding sequence of the coding strand is 2003-4234 th nucleotide of sequence 3 in the sequence table.
In the above biological material, the expression cassette is a DNA capable of expressing AAV-6X capsid protein in a host cell, and the DNA may include not only a promoter for initiating the gene transcription of AAV-6X capsid protein, but also a terminator for terminating the transcription of AAV-6X capsid protein. Further, the expression cassette may also include an enhancer sequence.
In the above biological material, the recombinant microorganism may be specifically a virus, a yeast, a bacterium, an alga and a fungus.
In order to solve the technical problems, the invention provides the application of the biological material in preparing the recombinant adeno-associated virus.
In order to solve the above technical problem, the present invention provides any one of the following applications:
p5, use of said protein for increasing the efficiency of infection of microglia by adeno-associated virus (use of said protein for conferring increased microglia infectivity by adeno-associated virus).
P6 and the application of the biological material in improving the infection efficiency of the adeno-associated virus to microglia.
In the above application, the protein is used for improving the infection efficiency of the adeno-associated virus to microglia, and the improvement is that compared with AAV-6 type capsid protein, the AAV-6 type capsid protein is protein with an amino acid sequence of sequence 2 in a sequence table. The use of the biomaterial for increasing the efficiency of infection of microglia by adeno-associated virus, as compared to the biomaterial associated with the AAV-6 type capsid protein.
In order to solve the above technical problems, the present invention provides a composition for infecting microglia.
The composition for infecting microglia provided by the invention contains the recombinant adeno-associated virus.
The above-described composition for infecting primary microglia may also contain other agents required for infecting primary microglia, such as a medium for culturing microglia.
Above, the exogenous gene delivery means may be a vector. The exogenous gene transfer means may be a vector for introducing the DNA of interest into microglia. The direct purpose of the application is non-disease diagnostic and/or therapeutic purposes.
The invention obtains the AAV-6 type capsid protein (namely AAV-6X type capsid protein) by mutating the wild type AAV-6 type capsid protein as follows: 7 amino acid residues are inserted between 588-589 th position of the amino acid sequence of the AAV-6 type capsid protein. Experiments prove that the efficiency of the recombinant adeno-associated virus rAAV-6 for expressing the wild AAV-6 type capsid protein to infect microglia in vitro is 8.0% +/-1.9%, and the efficiency of the recombinant adeno-associated virus rAAV-6X (expressing the AAV-6X type capsid protein) to infect microglia in vitro is 67.9% +/-4.6%; the efficiency of the recombinant adeno-associated virus rAAV-6X for infecting microglia in vitro is 8 times of the efficiency of the recombinant adeno-associated virus rAAV-6 for infecting microglia in vitro. The recombinant adeno-associated virus rAAV-6X of the invention is microglial. The recombinant adeno-associated virus rAAV-6X is an rAAV vector for efficiently transducing microglia. Compared with AAV-6 type capsid protein and its gene (wild type), the AAV-6X type capsid protein and its gene of the invention make the infection efficiency of recombinant adeno-associated virus to microglia increase 7 times, and endow adeno-associated virus with enhanced microglia infectivity. The AAV-6X type capsid protein and the gene thereof can be used for constructing rAAV for efficiently transducing microglia.
Drawings
FIG. 1 is a schematic diagram of a plasmid map used for constructing recombinant adeno-associated virus. A is a map of pAAV-6X, and CAP represents AAV-6X type capsid protein gene; b is a map of pAAV-CAG-GFP; c is a map of pAdDeltaF 6; d is the map of pAAV-RC6, and CAP represents AAV-6 type capsid protein gene.
FIG. 2 is a schematic representation of AAV-6 type X capsids and AAV-6 type capsids. CAP6 represents AAV-6 type capsid protein, and CAP6X represents AAV-6X type capsid protein.
FIG. 3 is a graph showing how efficiently rAAV-6X (AAV-6X) and rAAV-6 (AAV-6) infect primary microglia. One-way ANOVA analysis of variance was performed on three replicates of the experiment.
FIG. 4 shows comparison of the results of immunofluorescence staining with antibody Iba1 after primary microglia infection with rAAV-6 and rAAV-6X in vitro.
A is a schematic diagram of the result of the immunofluorescence staining of the Iba1 antibody after primary microglia is infected by rAAV-6 in vitro. B is a schematic diagram of the Iba1 antibody immunofluorescence staining result after primary microglia is infected by rAAV-6X in vitro.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The pAAV-RC6 adeno-associated virus packaging plasmid (hereinafter referred to as pAAV-RC6) in the following examples is a product of ZOMANBIO, and its product number is # ZK 2304. The nucleotide sequence of pAAV-RC6 is shown as sequence 1 in the sequence table. pAAV-RC6 is an expression vector of AAV-6 type capsid protein (hereinafter referred to as wild type capsid protein) gene. The 2003-4213 th nucleotide of the sequence 1 in the sequence table is a coding sequence of a coding chain of AAV-6 type capsid protein gene (AAV-6 gene for short). The AAV-6 type capsid protein gene coding amino acid sequence is the protein of sequence 2 in the sequence table (i.e. AAV-6 type capsid protein).
Example 1 construction of recombinant adeno-associated Virus and analysis of infected Primary microglia
Packaging of recombinant adeno-associated virus (AAV)
1. Preparation of recombinant plasmids (see FIGS. 1A-D):
1.1. preparation of recombinant capsid packaging plasmid pAAV-6X
The sequence to be inserted (underlined sequence in R) is designed on a downstream primer R by taking pAAV-RC6 as an original template, and the exogenous sequence is inserted into the capsid sequence of pAAV-RC6 in a PCR mode. The upstream primer F (nucleotide sequence is 5'-ACAGCAGCTACGCGCACA-3') and the downstream primer R (nucleotide sequence is 5 ' -AGGCTCCCATAACATGCACATCTCCGGTCGCAGGGTCTGT)CAGACGCGTCGGACCAACCGGGCTGCTGCTCTGGAGATTGAC-3') 1. mu.l each (working solution concentration 10. mu.M), 15 cycles later, 1. mu.l of Dpn1 enzyme was added to the PCR product to digest the original template plasmid pAAV-RC 6. Then, the PCR amplification product is transformed into escherichia coli competent cells, and the transformation product is spread on a culture medium and cultured overnight at 37 ℃. The next day, monoclonal strains were picked and subjected to a first generation sequencing validation. The sequencing results indicated that the transformation was successful and the selected monoclonal strain contained the mutated (including the inserted sequence) capsid plasmid, designated pAAV-6X. The nucleotide sequence of pAAV-6X is shown as a sequence 3 in the sequence table. pAAV-6X is an expression vector of a mutant AAV-6 type capsid protein (hereinafter referred to as AAV-6X type capsid protein) gene. The 2003-4234 th nucleotide of the sequence 3 in the sequence table is a coding sequence (CDS) of a coding chain of AAV-6X type capsid protein gene (AAV-6X gene for short). The AAV-6X gene coding amino acid sequence is protein (AAV-6X type capsid protein) of sequence 4 in the sequence table. pAAV-6X is an AAV-6X gene expression vector. pAAV-6X differs from pAAV-RC6 only in the nucleotide sequence of the capsid protein gene of adeno-associated virus, and pAAV-6X in which the capsid protein gene of adeno-associated virus is AAV-6X gene; in pAAV-RC6, the capsid protein gene of adeno-associated virus is AAV-6 gene. The AAV-6X gene is different from AAV-6 gene only in that AAV-6X gene has 21 inserted nucleotides between 1764 and 1765 nucleotides of AAV-6 gene. The AAV-6 type X capsid protein differs from the AAV-6 type X capsid protein only in the amino acid sequence in that the AAV-6 type X capsid protein is an amino acid in the AAV-6 type capsid protein7 amino acid residues are inserted between 588-589 of the sequence (FIG. 2).
Preparation of pAAV-CAG-GFP, pAdDeltaF6
pAAV-CAG-GFP is an adeno-associated virus (AAV) vector transfer plasmid, contains a fluorescent reporter gene GFP thereon, and is used for subsequently detecting the infection efficiency of the recombinant adeno-associated virus containing the 1.1 recombinant capsid plasmid; pAdDeltaF6 is an adeno-associated virus helper packaging plasmid used for packaging recombinant adeno-associated virus.
The pAAV-CAG-GFP (Addgene, #37825)) plasmid contains two ITR sequences of AAV-2 and a CAG promoter sequence, a GFP gene coding sequence, and an SV40 polyA sequence; the pAdDeltaF6(Addgene, #112867) plasmid contained adenovirus-derived E4, E2a, and VA gene coding sequences.
2. Recovery of AAV-293 cells (Procell, # CL-0019)
2.1, setting the temperature to be 37-42 ℃ in a water bath.
2.2, taking out the frozen AAV-293 cells from the liquid nitrogen tank, quickly throwing the cells into a water bath pot and quickly shaking the cells to ensure that the cell solution is completely dissolved within 1-2min as far as possible.
2.3 transfer the cell solution to a 15mL centrifuge tube and add 1mL fresh complete medium to it, mix well and centrifuge at 1000rpm for 5 min.
2.4, removing the supernatant, adding 5ml of fresh complete culture medium, uniformly mixing and precipitating, and transferring into a 10cm culture dish.
2.5, placing the culture dish at 37 deg.C and 5% CO2And 95% relative humidity.
And 2.6, observing the cell survival rate the next day. Cell growth was observed daily thereafter.
3. Generation of AAV-293 cells
3.1. When the cell grows to reach 80-90% of confluence rate, the cell needs to be subjected to passage operation to expand the cell number and maintain a good growth state of the cell.
3.2. Discarding the original culture medium, washing the cells with 0.01M PBS for 2 times, removing 0.01M PBS, adding 1-2ml of 0.25% trypsin, placing in an incubator at 37 deg.C for digestion for 2min, adding complete culture medium containing fetal calf serum after the cells are shed, and stopping digestion. After that, the cells were completely blown down and centrifuged at 2000rpm for 2 min.
3.3, adding a complete culture medium for resuspension after cell centrifugation is finished, and dividing the cells into 15cm culture dishes for culture according to specific conditions.
4. Recombinant adeno-associated virus rAAV-6X packaging and concentration
4.1. Plasmid amplification
Coli containing the constructed AAV vector transfer plasmid (pAAV-CAG-GFP), recombinant capsid packaging plasmid (pAAV-6X) and helper plasmid (pAdDeltaF6) is subjected to plasmid extraction by using a Tiangen endotoxin-free plasmid macroextraction kit (specific operation reference instruction), the plasmid concentration is required to be more than 1 mug/mul, and A260/280 is used for packaging viruses between 1.7 and 1.8.
4.2. Large scale amplification of AAV-293 cells
The culture medium in a 15cm culture dish for culturing AAV-293 cells is completely aspirated, the cells are washed 2 times with 0.01M PBS, 1-2mL of 0.25% pancreatin solution is added after the PBS is removed, the flask bottom is uniformly covered, the flask is placed in an incubator at 37 ℃ for 2min, the cells are taken out, the cells are separated from the bottom by shaking, 3mL of DMEM medium (containing 10% FBS) preheated in water bath at 37 ℃ is added, a 10mL pipette is used for blowing and beating the cells, and the cells are placed in a 15mL centrifuge tube. After that, the cells were centrifuged at 2000rpm for 2min and, as the case may be, the cells were dispensed into 15cm dishes. The cell density was observed on the day of transfection and was 70-80% full.
4.3. Viral packaging by plasmid transfection
AAV virus packaging was achieved by plasmid transfection of AAV-293 cells using Polyethyleneimine (PEI) (1 mg/mL). Plasmid usage was as per 15cm dish: transfection was carried out with 6. mu.g of vector transfer plasmid (pAAV-CAG-GFP), 10. mu.g of capsid packaging plasmid (pAAV-6X), and 12. mu.g of helper plasmid (pAdDeltaF 6).
4.4. Recombinant adeno-associated virus rAAV-6X for detoxification
Virus recovery was initiated 72h after packaging, and viral particles were present in both the packaging cells and the culture supernatant. Both the cells and the culture supernatant can be collected to obtain the best yield.
1) Preparing a dry ice ethanol bath and a 37 ℃ water bath
2) The toxigenic cells were collected into a 15mL centrifuge tube along with the culture medium. When collecting cells, the culture tray is inclined at a certain angle to scrape the cells into the culture medium;
3) centrifuging at 1000rpm for 3min, separating cells and supernatant, and resuspending the cells with 3ml PBS;
4) the cell suspension was repeatedly transferred in a dry ice ethanol bath and a water bath at 37 ℃ and freeze-thawed 3 times. After each melting, the mixture was shaken slightly. Note that: each freeze and thaw took approximately ten minutes.
5)10,000g, centrifuging to remove cell debris, filtering the supernatant by using a 0.22 mu m filter membrane, transferring the supernatant into a new centrifuge tube, and collecting the recombinant adeno-associated virus rAAV-6X virus particles.
4.5. Purification of recombinant adeno-associated virus rAAV-6X
1) 15%, 25%, 40%, 60% iodixanol solution was added to the ultracentrifuge tube in sequence, with 25%, 60% layers containing phenol red solution. After the gradient solution is prepared, the supernatant cracked in the step 4.4 is slowly added into a density gradient centrifugation medium, and after balancing, an ultracentrifuge 36,0000g is used for centrifugation for 2 hours at 16 ℃.
2) And (3) puncturing the junction of 40-60% layers by using a needle, and recovering the solution of 40% layers. And then concentrating by using a Millpore 100kd ultrafiltration tube, centrifuging at 3500rpm for 30min, repeatedly blowing and beating the intercepted virus particles by using 300-.
4.6 rAAV physical Titer assay
1) DNAse I was used to digest DNA that was not packaged into the viral capsid. DNAse I + DNAse digestion buffer was prepared, the dispensed virus was added to vortex for 1-2 seconds and mixed, and then pipetted into 3 tubes separately. Vortexed briefly and spun down briefly and incubated in a 37 ℃ water bath for 1 h. DNAse was then inactivated, 5 μ l EDTA was added per tube, briefly vortexed, and incubated in a dry bath at 70 ℃ for 10 minutes.
2) Protease K is used to digest the viral capsid and release the viral genome. Proteinase K + proteinase K buffer was prepared, vortexed briefly and spun, and incubated at 50 ℃ for 2 h. Thereafter, proteinase K is inactivated at a high temperature of 95 ℃.
3) The qPCR mix was prepared and 1. mu.l 10 was added separately10,109,108,107,106,105Mu.l of plasmid standard (pAAV-CAG-GFP) with 6 copy number gradients, and 1. mu.l of the prepared viral genomic DNA sample was added to mix. Negative controls were set up with 3 replicates per group. The physical titer of the virus was then calculated by establishing a standard curve. The physical titer of the obtained recombinant adeno-associated virus rAAV-6X is determined to be 5.45 multiplied by 1012VG/mL。
5. Recombinant adeno-associated virus rAAV-6 packaging and concentration
The method for preparing the recombinant adeno-associated virus rAAV-6 only differs from the step 4 in that pAAV-6X is replaced by pAAV-RC6, and other operations are completely the same. The physical titer of the recombinant adeno-associated virus rAAV-6 is 6.02 multiplied by 1012VG/mL。
Secondly, comparing the efficiency of infecting primary microglia by recombinant adeno-associated virus rAAV-6X and rAAV-6
The process of infecting primary microglia by two recombinant adeno-associated viruses, rAAV-6X and rAAV-6, is as follows, and the experiment comprises three repetitions.
1. First, microglia cells were isolated from neonatal mice for primary culture.
1.1 tissue sampling
The ice bath was started in 1 XPBS buffer and 2ml aliquots were added to 15ml centrifuge tubes and labeled. Operating in a biological safety cabinet, taking out the brain tissue of the mouse, and removing the hippocampus and the blood membrane coated outside the tissue; the cerebral cortex of 4 homoparental mice was pooled into a 15ml centrifuge tube.
1.2 Single cell suspension preparation
(1) Centrifuging at normal temperature at 1000rpm for 1min, precipitating the tissue, and removing the supernatant;
(2) adding 1ml pancreatin, digesting at 37 deg.C for 8min, and shaking the centrifuge tube for 2-3 times;
(3) adding serum for neutralization, stopping digestion, and centrifuging at normal temperature for 1min at 1000 rpm;
(4) adding 2ml of fresh glial cell culture medium DMEM + 10% FBS + 1% P/S (adding FBS (GIBCO) and P/S (Hyclone) to DMEM (GIBCO) to obtain a liquid culture medium, wherein in DMEM + 10% FBS + 1% P/S, the content of FBS is 10% and the content of P/S is 1%), blowing and beating the tissue by using a 1ml pipette gun to enable the tissue fragments without macroscopic view to be centrifuged at normal temperature, and rotating at 2000rpm for 3 min;
(5) discarding the supernatant, adding 2ml of fresh glial cell culture medium again, beating uniformly, centrifuging at normal temperature, and rotating at 2000rpm for 3 min;
(6) finally, 2ml of fresh glial cell culture medium was added and blown into a single cell suspension using a 1ml pipette.
1.3 Mixed cell culture Process
(1) Transferring the single cell suspension in the 15ml centrifuge tube to a T75 culture bottle with a corresponding mark, and adding 13ml culture medium;
(2) mixing, standing in incubator with 5% CO2Culturing at 37 deg.C for 3 days;
(3) after 3 days, the culture medium was changed, and 17ml of fresh medium was added to continue the culture.
1.4 separation of microglia from astrocytes
(1) Observing under an optical microscope, and finding that the astrocytes are positioned on the lower layer of the bottom of the culture dish at the bottom of the culture dish, and the microglia glial cells are attached to the astrocytes;
(2) after the astrocytes were confluent, the T75 flask was placed on a shaker at 200rpm,
3h。
1.5 cultivation of microglia
(1) Treating the culture bottle after the separation treatment, collecting and centrifuging the supernatant to obtain microglia;
(2) counting with a blood counting chamber, spreading the microglia in a 12 or 24-pore plate coated in advance (37 ℃, 2h) by PDL, wherein each pore of the 12-pore plate is 40 ten thousand cells, and each pore of the 24-pore plate is 10 ten thousand cells;
(3) the liquid is changed the next day.
2. Infection was performed by adding adeno-associated virus to primary cultured microglia.
1) According to MOI value 1: 500000 two recombinant adeno-associated viruses were added to a medium containing primary cultured microglia cells, respectively, and infected for 120 h.
2) After 120h fixation with 4% PFA was performed for 15min, after which the medium was discarded and washed 3 times with PBS.
3) Adding blocking solution containing 2% BSA, and blocking for more than 60 min.
4) Most of the serum blocking solution was aspirated off, and a blocking solution diluted primary anti-Iba1 polyclonal antibody (Wako, 019-19741) was added (1: 1000) at 4 ℃, for 16-18 h or overnight.
5) PBS was washed 3 times for 5 minutes each. Diluted Alexa Flour 594nm labeled secondary goat anti-rabbit IgG (1: 1000) and DAPI (1: 1000) were added and incubated at room temperature for 2 h.
6) PBS was washed 3 times, then patches were photographed, live imaging was performed to observe the pattern of EGFP expression and the morphology of infected cells, primary cultured cells were stained with the microglia-specific protein calcium ion binding adaptor molecule 1(Iba1) antibody, as shown under fluorescent microscope: the Iba1 antibody specific staining results can be coincided with the green fluorescent protein expressed by virus transduction, the total number of microglia (b) is determined by counting the number of microglia cells coincided with the green fluorescent protein expressed by virus transduction through the Iba1 antibody specific staining (a), and the total number of microglia cells (b) is determined by counting the Iba1 antibody specific staining and cell nucleus staining, and then the formula is further defined as follows: infection efficiency (%). a/b the efficiency of rAAV infection of primary microglia was calculated, assuming 3 replicates.
TABLE 1 efficiency of rAAV infection of microglia in vitro (%)
| rAAV | Repetition of 1 | |
Repetition of 3 |
| rAAV-6 | 7.2 | 6.7 | 10.2 |
| rAAV-6X | 63.1 | 72.3 | 68.3 |
The results are shown in table 1, fig. 3 and 4: the efficiency of the recombinant adeno-associated virus rAAV-6 to infect the microglia in vitro is 8.0% +/-1.9%, and the efficiency of the recombinant adeno-associated virus rAAV-6X to infect the microglia in vitro is 67.9% +/-4.6%; the efficiency of the recombinant adeno-associated virus rAAV-6X for infecting microglia in vitro is 8 times of the efficiency of the recombinant adeno-associated virus rAAV-6 for infecting microglia in vitro. The recombinant adeno-associated virus rAAV-6X can efficiently infect primary microglia. Compared with AAV-6 type capsid protein and its gene, AAV-6 type capsid protein and its gene can improve the infection efficiency of recombinant adeno-associated virus to microglia by 7 times, and endow adeno-associated virus with enhanced microglia infectivity.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> institute of animal research of Chinese academy of sciences
<120> a recombinant mutant adeno-associated virus capable of efficiently infecting primary microglia and related biological material thereof
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 7330
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atcgttaacg ccccgcgccg gccgctctag aactagtgga tcccccggaa gatcagaagt 60
tcctattccg aagttcctat tctctagaaa gtataggaac ttctgatctg cgcagccgcc 120
atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacga gcatctgccc 180
ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt gccgccagat 240
tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga gaagctgcag 300
cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct tttctttgtg 360
caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac caccggggtg 420
aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat tcagagaatt 480
taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac cagaaatggc 540
gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt gctccccaaa 600
acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag cgcctgtttg 660
aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc gcagacgcag 720
gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag atcaaaaact 780
tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac ctcggagaag 840
cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc caactcgcgg 900
tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac taaaaccgcc 960
cccgactacc tggtgggcca gcagcccgtg gaggacattt ccagcaatcg gatttataaa 1020
attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct gggatgggcc 1080
acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac taccgggaag 1140
accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt aaactggacc 1200
aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg ggaggagggg 1260
aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag caaggtgcgc 1320
gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat cgtcacctcc 1380
aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca ccagcagccg 1440
ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga ctttgggaag 1500
gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt ggttgaggtg 1560
gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc cagtgacgca 1620
gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac gtcagacgcg 1680
gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca cgtgggcatg 1740
aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc aaatatctgc 1800
ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc tcaacccgtt 1860
tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat gggaaaggtg 1920
ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg catctttgaa 1980
caataaatga tttaaatcag gtatggctgc cgatggttat cttccagatt ggctcgagga 2040
caacctctct gagggcattc gcgagtggtg ggacttgaaa cctggagccc cgaaacccaa 2100
agccaaccag caaaagcagg acgacggccg gggtctggtg cttcctggct acaagtacct 2160
cggacccttc aacggactcg acaaggggga gcccgtcaac gcggcggatg cagcggccct 2220
cgagcacgac aaggcctacg accagcagct caaagcgggt gacaatccgt acctgcggta 2280
taaccacgcc gacgccgagt ttcaggagcg tctgcaagaa gatacgtctt ttgggggcaa 2340
cctcgggcga gcagtcttcc aggccaagaa gagggttctc gaacctcttg gtctggttga 2400
ggaaggtgct aagacggctc ctggaaagaa acgtccggta gagcagtcgc cacaagagcc 2460
agactcctcc tcgggcattg gcaagacagg ccagcagccc gctaaaaaga gactcaattt 2520
tggtcagact ggcgactcag agtcagtccc cgacccacaa cctctcggag aacctccagc 2580
aacccccgct gctgtgggac ctactacaat ggcttcaggc ggtggcgcac caatggcaga 2640
caataacgaa ggcgccgacg gagtgggtaa tgcctcagga aattggcatt gcgattccac 2700
atggctgggc gacagagtca tcaccaccag cacccgaaca tgggccttgc ccacctataa 2760
caaccacctc tacaagcaaa tctccagtgc ttcaacgggg gccagcaacg acaaccacta 2820
cttcggctac agcaccccct gggggtattt tgatttcaac agattccact gccatttctc 2880
accacgtgac tggcagcgac tcatcaacaa caattgggga ttccggccca agagactcaa 2940
cttcaagctc ttcaacatcc aagtcaagga ggtcacgacg aatgatggcg tcacgaccat 3000
cgctaataac cttaccagca cggttcaagt cttctcggac tcggagtacc agttgccgta 3060
cgtcctcggc tctgcgcacc agggctgcct ccctccgttc ccggcggacg tgttcatgat 3120
tccgcagtac ggctacctaa cgctcaacaa tggcagccag gcagtgggac ggtcatcctt 3180
ttactgcctg gaatatttcc catcgcagat gctgagaacg ggcaataact ttaccttcag 3240
ctacaccttc gaggacgtgc ctttccacag cagctacgcg cacagccaga gcctggaccg 3300
gctgatgaat cctctcatcg accagtacct gtattacctg aacagaactc agaatcagtc 3360
cggaagtgcc caaaacaagg acttgctgtt tagccggggg tctccagctg gcatgtctgt 3420
tcagcccaaa aactggctac ctggaccctg ttaccggcag cagcgcgttt ctaaaacaaa 3480
aacagacaac aacaacagca actttacctg gactggtgct tcaaaatata accttaatgg 3540
gcgtgaatct ataatcaacc ctggcactgc tatggcctca cacaaagacg acaaagacaa 3600
gttctttccc atgagcggtg tcatgatttt tggaaaggag agcgccggag cttcaaacac 3660
tgcattggac aatgtcatga tcacagacga agaggaaatc aaagccacta accccgtggc 3720
caccgaaaga tttgggactg tggcagtcaa tctccagagc agcagcacag accctgcgac 3780
cggagatgtg catgttatgg gagccttacc tggaatggtg tggcaagaca gagacgtata 3840
cctgcagggt cctatttggg ccaaaattcc tcacacggat ggacactttc acccgtctcc 3900
tctcatgggc ggctttggac ttaagcaccc gcctcctcag atcctcatca aaaacacgcc 3960
tgttcctgcg aatcctccgg cagagttttc ggctacaaag tttgcttcat tcatcaccca 4020
gtattccaca ggacaagtga gcgtggagat tgaatgggag ctgcagaaag aaaacagcaa 4080
acgctggaat cccgaagtgc agtatacatc taactatgca aaatctgcca acgttgattt 4140
cactgtggac aacaatggac tttatactga gcctcgcccc attggcaccc gttacctcac 4200
ccgtcccctg taattgtgtg ttaatcaata aaccggttaa ttcgtgtcag ttgaactttg 4260
gtctcatgtc gttattatct tatctggtca ccagatcccc gtagataagt agcatggcgg 4320
gttaatcatt aactacagcc cgggcgttta aacagcgggc ggaggggtgg agtcgtgacg 4380
tgaattacgt catagggtta gggaggtcct gtattagagg tcacgtgagt gttttgcgac 4440
attttgcgac accatgtggt ctcgctgggg gggggggccc gagtgagcac gcagggtctc 4500
cattttgaag cgggaggttt gaacgagcgc tggcgcgctc actggccgtc gttttacaac 4560
gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt 4620
tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca 4680
gcctgaatgg cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt 4740
ttgttaaatc agctcatttt tttaaccaat aggccgaaat cggcaaaatc ccttataaat 4800
caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat 4860
taagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact 4920
acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg 4980
gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag 5040
aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac 5100
gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg 5160
cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 5220
tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa 5280
gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 5340
tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 5400
tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 5460
ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 5520
atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 5580
cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 5640
attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 5700
gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 5760
ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 5820
gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 5880
agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 5940
gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 6000
gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 6060
ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 6120
tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 6180
tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 6240
catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 6300
gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 6360
aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 6420
gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta 6480
gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 6540
gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 6600
atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 6660
cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 6720
cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 6780
agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 6840
tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 6900
gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 6960
catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 7020
agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 7080
ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag 7140
ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag 7200
ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg 7260
tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa 7320
gcgcgccgat 7330
<210> 2
<211> 736
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser
1 5 10 15
Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro
20 25 30
Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro
35 40 45
Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60
Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
65 70 75 80
Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala
85 90 95
Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly
100 105 110
Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125
Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg
130 135 140
Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly
145 150 155 160
Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr
165 170 175
Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro
180 185 190
Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly
195 200 205
Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala
210 215 220
Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile
225 230 235 240
Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu
245 250 255
Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His
260 265 270
Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe
275 280 285
His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn
290 295 300
Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln
305 310 315 320
Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn
325 330 335
Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro
340 345 350
Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala
355 360 365
Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly
370 375 380
Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro
385 390 395 400
Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe
405 410 415
Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp
420 425 430
Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg
435 440 445
Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser
450 455 460
Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro
465 470 475 480
Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn
485 490 495
Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn
500 505 510
Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys
515 520 525
Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly
530 535 540
Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile
545 550 555 560
Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg
565 570 575
Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala
580 585 590
Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln
595 600 605
Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His
610 615 620
Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu
625 630 635 640
Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala
645 650 655
Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr
660 665 670
Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln
675 680 685
Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn
690 695 700
Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu
705 710 715 720
Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu
725 730 735
<210> 3
<211> 7351
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atcgttaacg ccccgcgccg gccgctctag aactagtgga tcccccggaa gatcagaagt 60
tcctattccg aagttcctat tctctagaaa gtataggaac ttctgatctg cgcagccgcc 120
atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacga gcatctgccc 180
ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt gccgccagat 240
tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga gaagctgcag 300
cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct tttctttgtg 360
caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac caccggggtg 420
aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat tcagagaatt 480
taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac cagaaatggc 540
gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt gctccccaaa 600
acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag cgcctgtttg 660
aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc gcagacgcag 720
gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag atcaaaaact 780
tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac ctcggagaag 840
cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc caactcgcgg 900
tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac taaaaccgcc 960
cccgactacc tggtgggcca gcagcccgtg gaggacattt ccagcaatcg gatttataaa 1020
attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct gggatgggcc 1080
acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac taccgggaag 1140
accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt aaactggacc 1200
aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg ggaggagggg 1260
aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag caaggtgcgc 1320
gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat cgtcacctcc 1380
aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca ccagcagccg 1440
ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga ctttgggaag 1500
gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt ggttgaggtg 1560
gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc cagtgacgca 1620
gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac gtcagacgcg 1680
gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca cgtgggcatg 1740
aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc aaatatctgc 1800
ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc tcaacccgtt 1860
tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat gggaaaggtg 1920
ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg catctttgaa 1980
caataaatga tttaaatcag gtatggctgc cgatggttat cttccagatt ggctcgagga 2040
caacctctct gagggcattc gcgagtggtg ggacttgaaa cctggagccc cgaaacccaa 2100
agccaaccag caaaagcagg acgacggccg gggtctggtg cttcctggct acaagtacct 2160
cggacccttc aacggactcg acaaggggga gcccgtcaac gcggcggatg cagcggccct 2220
cgagcacgac aaggcctacg accagcagct caaagcgggt gacaatccgt acctgcggta 2280
taaccacgcc gacgccgagt ttcaggagcg tctgcaagaa gatacgtctt ttgggggcaa 2340
cctcgggcga gcagtcttcc aggccaagaa gagggttctc gaacctcttg gtctggttga 2400
ggaaggtgct aagacggctc ctggaaagaa acgtccggta gagcagtcgc cacaagagcc 2460
agactcctcc tcgggcattg gcaagacagg ccagcagccc gctaaaaaga gactcaattt 2520
tggtcagact ggcgactcag agtcagtccc cgacccacaa cctctcggag aacctccagc 2580
aacccccgct gctgtgggac ctactacaat ggcttcaggc ggtggcgcac caatggcaga 2640
caataacgaa ggcgccgacg gagtgggtaa tgcctcagga aattggcatt gcgattccac 2700
atggctgggc gacagagtca tcaccaccag cacccgaaca tgggccttgc ccacctataa 2760
caaccacctc tacaagcaaa tctccagtgc ttcaacgggg gccagcaacg acaaccacta 2820
cttcggctac agcaccccct gggggtattt tgatttcaac agattccact gccatttctc 2880
accacgtgac tggcagcgac tcatcaacaa caattgggga ttccggccca agagactcaa 2940
cttcaagctc ttcaacatcc aagtcaagga ggtcacgacg aatgatggcg tcacgaccat 3000
cgctaataac cttaccagca cggttcaagt cttctcggac tcggagtacc agttgccgta 3060
cgtcctcggc tctgcgcacc agggctgcct ccctccgttc ccggcggacg tgttcatgat 3120
tccgcagtac ggctacctaa cgctcaacaa tggcagccag gcagtgggac ggtcatcctt 3180
ttactgcctg gaatatttcc catcgcagat gctgagaacg ggcaataact ttaccttcag 3240
ctacaccttc gaggacgtgc ctttccacag cagctacgcg cacagccaga gcctggaccg 3300
gctgatgaat cctctcatcg accagtacct gtattacctg aacagaactc agaatcagtc 3360
cggaagtgcc caaaacaagg acttgctgtt tagccggggg tctccagctg gcatgtctgt 3420
tcagcccaaa aactggctac ctggaccctg ttaccggcag cagcgcgttt ctaaaacaaa 3480
aacagacaac aacaacagca actttacctg gactggtgct tcaaaatata accttaatgg 3540
gcgtgaatct ataatcaacc ctggcactgc tatggcctca cacaaagacg acaaagacaa 3600
gttctttccc atgagcggtg tcatgatttt tggaaaggag agcgccggag cttcaaacac 3660
tgcattggac aatgtcatga tcacagacga agaggaaatc aaagccacta accccgtggc 3720
caccgaaaga tttgggactg tggcagtcaa tctccagagc agcagcccgg ttggtccgac 3780
gcgtctgaca gaccctgcga ccggagatgt gcatgttatg ggagccttac ctggaatggt 3840
gtggcaagac agagacgtat acctgcaggg tcctatttgg gccaaaattc ctcacacgga 3900
tggacacttt cacccgtctc ctctcatggg cggctttgga cttaagcacc cgcctcctca 3960
gatcctcatc aaaaacacgc ctgttcctgc gaatcctccg gcagagtttt cggctacaaa 4020
gtttgcttca ttcatcaccc agtattccac aggacaagtg agcgtggaga ttgaatggga 4080
gctgcagaaa gaaaacagca aacgctggaa tcccgaagtg cagtatacat ctaactatgc 4140
aaaatctgcc aacgttgatt tcactgtgga caacaatgga ctttatactg agcctcgccc 4200
cattggcacc cgttacctca cccgtcccct gtaattgtgt gttaatcaat aaaccggtta 4260
attcgtgtca gttgaacttt ggtctcatgt cgttattatc ttatctggtc accagatccc 4320
cgtagataag tagcatggcg ggttaatcat taactacagc ccgggcgttt aaacagcggg 4380
cggaggggtg gagtcgtgac gtgaattacg tcatagggtt agggaggtcc tgtattagag 4440
gtcacgtgag tgttttgcga cattttgcga caccatgtgg tctcgctggg ggggggggcc 4500
cgagtgagca cgcagggtct ccattttgaa gcgggaggtt tgaacgagcg ctggcgcgct 4560
cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 4620
gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 4680
gcccttccca acagttgcgc agcctgaatg gcgaatggaa attgtaagcg ttaatatttt 4740
gttaaaattc gcgttaaatt tttgttaaat cagctcattt ttttaaccaa taggccgaaa 4800
tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag 4860
tttggaacaa gagtccacta ttaagaacgt ggactccaac gtcaaagggc gaaaaaccgt 4920
ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag 4980
gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg 5040
aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc 5100
gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc 5160
gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 5220
atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 5280
tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 5340
cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 5400
agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 5460
taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 5520
tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 5580
catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 5640
ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 5700
ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 5760
catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 5820
aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt 5880
aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 5940
taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 6000
atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 6060
gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 6120
tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 6180
ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 6240
gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 6300
agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 6360
aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 6420
agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 6480
tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 6540
atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 6600
taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 6660
gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 6720
gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 6780
aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 6840
tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 6900
gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 6960
cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa 7020
ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag 7080
cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg 7140
ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga 7200
gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat 7260
gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag 7320
ctatgaccat gattacgcca agcgcgccga t 7351
<210> 4
<211> 743
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser
1 5 10 15
Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro
20 25 30
Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro
35 40 45
Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60
Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
65 70 75 80
Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala
85 90 95
Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly
100 105 110
Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125
Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg
130 135 140
Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly
145 150 155 160
Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr
165 170 175
Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro
180 185 190
Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly
195 200 205
Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala
210 215 220
Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile
225 230 235 240
Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu
245 250 255
Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His
260 265 270
Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe
275 280 285
His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn
290 295 300
Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln
305 310 315 320
Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn
325 330 335
Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro
340 345 350
Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala
355 360 365
Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly
370 375 380
Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro
385 390 395 400
Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe
405 410 415
Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp
420 425 430
Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg
435 440 445
Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser
450 455 460
Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro
465 470 475 480
Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn
485 490 495
Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn
500 505 510
Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys
515 520 525
Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly
530 535 540
Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile
545 550 555 560
Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg
565 570 575
Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Pro Val Gly Pro
580 585 590
Thr Arg Leu Thr Asp Pro Ala Thr Gly Asp Val His Val Met Gly Ala
595 600 605
Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro
610 615 620
Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro Ser Pro
625 630 635 640
Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile
645 650 655
Lys Asn Thr Pro Val Pro Ala Asn Pro Pro Ala Glu Phe Ser Ala Thr
660 665 670
Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val
675 680 685
Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro
690 695 700
Glu Val Gln Tyr Thr Ser Asn Tyr Ala Lys Ser Ala Asn Val Asp Phe
705 710 715 720
Thr Val Asp Asn Asn Gly Leu Tyr Thr Glu Pro Arg Pro Ile Gly Thr
725 730 735
Arg Tyr Leu Thr Arg Pro Leu
740
Claims (10)
1. A recombinant adeno-associated virus, characterized in that: the recombinant adeno-associated virus is adeno-associated virus expressing AAV-6X type capsid protein, and the AAV-6X type capsid protein is protein with an amino acid sequence of sequence 4 in a sequence table.
2. The recombinant adeno-associated virus according to claim 1, wherein: the recombinant adeno-associated virus contains the coding gene of the AAV-6X type capsid protein, and the coding sequence of the coding chain of the AAV-6X type capsid protein is nucleotide 2003-4234 of sequence 3 in the sequence table.
3. The method for constructing a recombinant adeno-associated virus according to claim 1 or 2, wherein the method comprises introducing a gene encoding an AAV-6X-type capsid protein into a packaging cell to obtain a recombinant adeno-associated virus expressing the AAV-6X-type capsid protein; the AAV-6X type capsid protein is a protein with an amino acid sequence of a sequence 4 in a sequence table.
4. The application is characterized in that: the application is any one of the following:
the use of P1 and the recombinant adeno-associated virus according to claim 1 or 2 for preparing a foreign gene delivery means,
use of P2, the recombinant adeno-associated virus according to claim 1 or 2 for introducing a DNA of interest into microglia,
use of P3, the recombinant adeno-associated virus according to claim 1 or 2 for molecular level engineering of microglia,
use of P4, the recombinant adeno-associated virus according to claim 1 or 2 for studying microglial function.
5. A protein characterized by: the protein is AAV-6X type capsid protein, and the AAV-6X type capsid protein is protein with an amino acid sequence of sequence 4 in a sequence table.
6. The biomaterial related to the protein of claim 5, wherein the biomaterial is any one of the following C1) to C7):
C1) a nucleic acid molecule encoding the protein of claim 5;
C2) an expression cassette comprising the nucleic acid molecule of C1);
C3) a recombinant vector comprising the nucleic acid molecule of C1), or a recombinant vector comprising the expression cassette of C2);
C4) a recombinant microorganism containing C1) the nucleic acid molecule, or a recombinant microorganism containing C2) the expression cassette, or a recombinant microorganism containing C3) the recombinant vector.
7. The biomaterial of claim 5, wherein: the nucleic acid molecule is a cDNA molecule or DNA molecule of which the coding sequence is the 2003-4234 th nucleotide of the sequence 3 in the sequence table.
8. The application is characterized in that: the application is the application of the biological material of claim 6 or 7 in preparing recombinant adeno-associated virus.
9. The application is characterized in that: the application is any one of the following:
the use of P5 or the protein of claim 5 for increasing the efficiency of infection of microglia by adeno-associated virus.
Use of the biomaterial of P6, claim 6 or 7 for increasing the efficiency of infection of microglia by adeno-associated virus.
10. A composition for infecting microglia, comprising: the composition comprises the recombinant adeno-associated virus according to claim 1 or 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110021870.7A CN112813037B (en) | 2021-01-08 | 2021-01-08 | Recombinant mutant adeno-associated virus capable of efficiently infecting primary microglia and related biological material thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110021870.7A CN112813037B (en) | 2021-01-08 | 2021-01-08 | Recombinant mutant adeno-associated virus capable of efficiently infecting primary microglia and related biological material thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112813037A true CN112813037A (en) | 2021-05-18 |
| CN112813037B CN112813037B (en) | 2022-05-10 |
Family
ID=75869049
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110021870.7A Active CN112813037B (en) | 2021-01-08 | 2021-01-08 | Recombinant mutant adeno-associated virus capable of efficiently infecting primary microglia and related biological material thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112813037B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114057840A (en) * | 2021-10-14 | 2022-02-18 | 和元生物技术（上海）股份有限公司 | Recombinant adeno-associated viral particles comprising variant AAV9 capsid proteins |
| CN114195859A (en) * | 2021-12-10 | 2022-03-18 | 和元生物技术（上海）股份有限公司 | Adeno-associated virus mutant suitable for specifically infecting U251 cells |
| CN114213505A (en) * | 2021-12-10 | 2022-03-22 | 和元生物技术（上海）股份有限公司 | Adeno-associated virus mutant suitable for specifically infecting U87-MG cells |
| WO2023198050A1 (en) * | 2022-04-12 | 2023-10-19 | 合肥星眸生物科技有限公司 | Fusion type adeno-associated virus and use thereof |
| WO2023215947A1 (en) * | 2022-05-13 | 2023-11-16 | Children's Medical Research Institute | Adeno-associated virus capsids |
| WO2024077815A1 (en) * | 2022-10-09 | 2024-04-18 | 广州派真生物技术有限公司 | Adeno-associated virus mutant and use thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6156303A (en) * | 1997-06-11 | 2000-12-05 | University Of Washington | Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom |
| CN104592364A (en) * | 2013-10-30 | 2015-05-06 | 北京大学 | Site-directed mutagenesis and site-directed modification adeno-associated viruses, preparation method and application thereof |
| CN110437317A (en) * | 2019-01-30 | 2019-11-12 | 上海科技大学 | Adeno-associated virus and application thereof with variant capsids albumen |
| CN110950934A (en) * | 2019-12-31 | 2020-04-03 | 复旦大学 | Adeno-associated virus capsid protein, vector, construction method and application thereof |
| CN111808175A (en) * | 2020-09-01 | 2020-10-23 | 和元生物技术（上海）股份有限公司 | Recombinant adeno-associated virus particles and application thereof |
-
2021
- 2021-01-08 CN CN202110021870.7A patent/CN112813037B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6156303A (en) * | 1997-06-11 | 2000-12-05 | University Of Washington | Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom |
| CN104592364A (en) * | 2013-10-30 | 2015-05-06 | 北京大学 | Site-directed mutagenesis and site-directed modification adeno-associated viruses, preparation method and application thereof |
| CN110437317A (en) * | 2019-01-30 | 2019-11-12 | 上海科技大学 | Adeno-associated virus and application thereof with variant capsids albumen |
| CN110950934A (en) * | 2019-12-31 | 2020-04-03 | 复旦大学 | Adeno-associated virus capsid protein, vector, construction method and application thereof |
| CN111808175A (en) * | 2020-09-01 | 2020-10-23 | 和元生物技术（上海）股份有限公司 | Recombinant adeno-associated virus particles and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| APRIL R. GILES等: "Mapping an Adeno-associated Virus 9-Specific Neutralizing Epitope To Develop Next-Generation Gene Delivery Vectors", 《JOURNAL OF VIROLOGY》 * |
| JHEEL PANDYA等: "Rationally designed capsid and transgene cassette of AAV6 vectors for dendritic cell-based cancer immunotherapy", 《IMMUNOLOGY AND CELL BIOLOGY》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114057840A (en) * | 2021-10-14 | 2022-02-18 | 和元生物技术（上海）股份有限公司 | Recombinant adeno-associated viral particles comprising variant AAV9 capsid proteins |
| CN114195859A (en) * | 2021-12-10 | 2022-03-18 | 和元生物技术（上海）股份有限公司 | Adeno-associated virus mutant suitable for specifically infecting U251 cells |
| CN114213505A (en) * | 2021-12-10 | 2022-03-22 | 和元生物技术（上海）股份有限公司 | Adeno-associated virus mutant suitable for specifically infecting U87-MG cells |
| WO2023103661A1 (en) * | 2021-12-10 | 2023-06-15 | 和元生物技术（上海）股份有限公司 | Adeno-associated virus mutant suitable for specific infection of u251 cells |
| WO2023198050A1 (en) * | 2022-04-12 | 2023-10-19 | 合肥星眸生物科技有限公司 | Fusion type adeno-associated virus and use thereof |
| WO2023215947A1 (en) * | 2022-05-13 | 2023-11-16 | Children's Medical Research Institute | Adeno-associated virus capsids |
| WO2024077815A1 (en) * | 2022-10-09 | 2024-04-18 | 广州派真生物技术有限公司 | Adeno-associated virus mutant and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112813037B (en) | 2022-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112813037B (en) | Recombinant mutant adeno-associated virus capable of efficiently infecting primary microglia and related biological material thereof | |
| KR102191739B1 (en) | Modified foot-and-mouth disease virus 3C protease, composition and method thereof | |
| CN110951741B (en) | Bacillus subtilis polygene editing and expression regulation system based on CRISPR Cpf1 | |
| CN109943581B (en) | Plasmid and phage-assisted continuous directed evolution system and directed evolution method | |
| CN104498493B (en) | The method of CRISPR/Cas9 specific knockdown hepatitis type B viruses and the gRNA for selectively targeted HBV DNA | |
| KR20170076650A (en) | Mutant microorganisms resistant to lactose killing | |
| CN109055544B (en) | Molecular marker of atherosclerosis and application thereof | |
| CN109182503B (en) | Molecular marker of atherosclerosis and application thereof | |
| JP6228982B2 (en) | Development of NADP (H) sensor and alcohol dehydrogenase | |
| CN107574175A (en) | A kind of expression vector and its construction method based on recombined adhenovirus | |
| CN110951763B (en) | Potato Yvirus induced gene silencing system and application thereof | |
| CN111269294A (en) | Mutant site of cellooligosaccharide transporter LacY, mutant transporter LacY, and preparation method and application thereof | |
| CN103014045B (en) | Recombinant HBV (Hepatitis B Virus) vector for expressing specific HBVshRNA (Hepatitis B Virus Short Hairpin Ribonucleic Acid) as well as construction method and application of vector | |
| CN114634923B (en) | Adenosine deaminase, base editor fusion protein, base editor system and use | |
| CN107988250B (en) | Construction method of universal chlamydomonas foreign gene expression vector | |
| KR102320656B1 (en) | A biological method for preparing acetins | |
| DK2571998T3 (en) | METHOD AND KIT FOR IDENTIFICATION OF CHEMICAL COMPOUNDS MADE TO INHIBIT HUMAN PAPILLOMA VIRUS REPLICATION | |
| CN110964725A (en) | sgRNA for specifically identifying pig KIT gene and coding DNA, KIT and application thereof | |
| CN110295181B (en) | Method for identifying antibiotic species by using bioluminescence report system | |
| CN113604500B (en) | Construction and application of sugarcane streak mosaic virus full-length cDNA invasive clone | |
| CN109136228B (en) | Application of long-chain non-coding RNA-NKILA in bone tissue injury repair | |
| CN113061605B (en) | Construction method and application of fucose transferase 8 (FUT 8) function-deficient cell strain | |
| CN110241129B (en) | Luminous report system and application thereof in antibiotic discovery and screening | |
| CN110286118B (en) | Method for judging antibiotic action mechanism by using bioluminescence report system | |
| CN110241098A (en) | The truncated-type high specific variant of the CRISPR nuclease SpCas9 of streptococcus pyogenes and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |