CN106701691A - AAV virus capable of efficiently infecting immune cells and preparing method and application thereof - Google Patents
AAV virus capable of efficiently infecting immune cells and preparing method and application thereof Download PDFInfo
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Abstract
The invention provides an AAV virus capable of efficiently infecting immune cells and a preparing method and application thereof. The prepared novel AAV virus with a new structure protein has efficient infection to the immune cells; the virus can serve as a gene carrier, constitute a recombinant adenovirus with a foreign gene, carry the foreign gene to the immune cells, and inject the foreign gene into the immune cells after efficient infection, and accordingly the effect of the foreign gene is exerted; the AAV virus is wide in adaptation to the foreign gene, does not need a complicated preparing process, has ultra-low immunogenicity, is safe to a human body, and provides a wider choice to gene treatment.
Description
Technical field
The present invention relates to a kind of new AAV serotypes, and in particular to it is a kind of can efficient infection immunocyte AAV viruses and preparation method and application.
Background technology
With developing rapidly for molecular biology, gene therapy has turned into one of human genetic disease or acquired disease treatment most efficient method.Compared with traditional therapy, the nature that gene therapy is expressed closer to human body gene, and its key is how safely and effectively to import in human body therapeutic gene, and enable therapeutic gene long-term expression.Compared with adenovirus, slow virus carrier, adeno-associated virus（AAV）By feat of safe, immunogenicity is low, can the mediated gene advantage such as expression steadily in the long term, shown significant potential in many preclinical and clinical research, received significant attention in field of gene.
Adeno-associated virus（AAV）Belong to the Parvoviridae of dependovirus, be initially found as a kind of pollutant component in adenovirus experiment prepared product in nineteen sixty-five.12 naturally occurring AAV serotypes and more than 100 kinds of virus mutation are had now been found that.Wild type AAV is the nonenveloped virus that a class is wrapped in linear ssdna genome, and the base end repetitive sequence of 145bp is contained at its two ends（Inverted terminal repeats, ITR）.ITRs sequences are folded into hairpin structure packaging virus genomic for AAV and initial single stranded DNA is most important to the transformation of double-stranded DNA, and the only known cis-acting elements for needed for infectious viral particle.ITR elements are located at two both sides of ORFs, left side Rep gene codes four non-structural Rep albumen-Rep78, Rep68, Rep52 and Rep40, the duplication and integration of wherein Rep78 and Rep68 regulation and control viruses, Rep52 and Rep40 then plays the active function of solution ATP enzyme and helicase, and they also regulate and control the reproduction process of viral single-stranded genome jointly with Rep78, Rep68；Cap gene codes three structural proteins-VP1, VP2 and the VP3 on right side, are the capsid proteins needed for assembling intact virus, and they play an important role in viral integrase, duplication and assembling.The capsid of AAV is icosahedron, is made up of 60 virocapsomers, and diameter is about 20-25 nm, is the virus of minimum in animal virus.AAV is a kind of defective virus, it is impossible to independent to replicate, it is necessary under conditions of helper virus such as adenovirus, herpes simplex virus are present, synthesis and assembling albumen, the new virion of generation be replicated in host cell.After the host cell of the AAV infection people of wild type, adjustable point is incorporated into the AAVS1 sites of host cell（No. 19 chromosomes, 19q13.4）, this may be related to the collective effect of wild type adeno-associated virus ITR structures and Rep albumen.
The infection clones AAV serotypes 2 that AAV viral vectors general at present is set up for nineteen eighty-two（AAV2）, because AAV2 carriers are almost without pathogenic effects, weak immunogene, host range is wider, can infect the cell and nondividing phase cell of division stage, can the advantage such as mediated gene expression steady in a long-term be widely used in gene therapy.Restructuring AAV2 carriers are tested in research before many Disease Clinicals, such as hemophilia, α1 antitrypsin deficiency hepatopathy, cystic fibrosis, duchenne muscular dystrophy and rheumatoid arthritis.But with increasing for clinical research, the deficiency of AAV2 carriers is also gradually displayed, and such as AAV2 differs greatly to the efficiency of infection organized, and the efficiency of infection in some tissues is very high, and the efficiency of infection in some tissues is very low.In addition, can produce neutrality antibody to the infection of AAV in human body, in adult there is the neutrality antibody for various AAV viruses in about 50-80%, and the neutrality antibody of anti-AAV2 is principal mode.In order to improve the efficiency of infection and host range of AAV viral vectors, people have carried out various trials, such as capsid protein of mutagenesis screening mutation or the chimeric AAV viral vectors methods of structure.The organizing specific Journal of Sex Research of different serotypes AAV viral vectors was widely paid close attention in recent years, using the natural infection characteristic of various serotypes A AV, acquisition has the AAV viral vectors of different transduction potentials to different tissues, most important in the extensive use of gene therapy to AAV.
So far, from adenovirus or people/primate tissue it is separated go out several AAV serotypes and more than 100 kinds of AAV virus mutation.Due to the alternative of AAV serotypes, not only because its potential transduction efficiency high can reduce carrier load, the neutrality antibody evaded natural infection or produced based on AAV viral therapies is also beneficial to.
Except primate, the genome of AAV can also be separated from other animals and obtained.Because the amino acid of different serotypes AAV capsid proteins constitutes different（The homology of Cap is relatively low）, cause them to be combined with the not isoacceptor of target cells, thus there is significant difference in various histiocytic close preferendum to host.Nonenveloped virus is by the glycosaminoglycan acceptor interaction of capsid protein and cell surface hence into cell.Then viral capsid determines intracellular transport pathway with the secondary interaction of auxiliary acceptor, and the stage is referred to as route of infection（Final transduction efficiency）.The alternative of AAV serotypes has significant impact to final transduction efficiency.In existing AAV serotypes, AAV2 enters host cell with the sulfate-proteoglycan acceptor interaction of cell surface, then AAV2 and auxiliary acceptor human fibroblastic growth factor acceptor 1（FGFR1）, hepatocyte growth factor receptor and the β 1 of 5/ α of beta 2 integrin alpha v β 5 interact also extremely important to the transduction of host cell.AAV6, only six amino acid residues be different from AAV1 and with the homologous capsid sequences of AAV2 about 85%, with reference to heparin sulfate, and can be purified using heparin affinity chromatography.AAV3 has about 87% homology with AAV2 capsids, can equally be combined with heparin sulfate receptors, but affinity is relatively low.AAV4 and AAV5 do not have heparin sulfate to be combined into a little, are combined and conducted with cell by sialic acid receptor, therefore its tissue specificity and AAV2 etc. have very big difference.In recent years, people have been set up transductions of the saliva acid mediated AAV1 or AAV6 to some cell types.AAV8-10 is combined by laminin recepter (LamR) with cell surface, and the cell surface receptor for being used for other AAV serotypes and the combination of AAV virus mutations still has to be determined.
There is very big difference to different tissues transduction efficiency in various serotypes A AV viral vectors.The known transducible most organization types of AAV2, including liver, muscle, lung and central nervous system, with medium transduction efficiency.The same transducible Various Tissues types of AAV9, its transduction efficiency is higher than AAV2.AAV1 and AAV7 has quick infection and high-caliber transduction to skeletal muscle, because AAV6 capsids only have six amino acid residues different from AAV1, therefore also shows that the transduction tended to skeletal muscle.Research finds that various serotypes A AV viral vector infections efficiency are from high to low AAV1 in mouse liver and muscle tissue>AAV5>AAV3>AAV2>AAV4；Sequentially it is AAV5 in rat retina>AAV4>AAV1>AAV2>AAV3.From now on, it is still necessary to widely study to illustrate the tissue tropisms and transduction efficiency of other serotypes.
Some AAV serotypes show the not contransduction pattern to nervous system.Generally, injected from central nervous system, compared with AAV2, AAV1 and AAV5 shows transduction efficiency higher.But AAV2 shows extensive transduction efficiency in whole midbrain, AAV4 then transduces specific cell type, such as endyma and astroglia of subependymal region.In hepatic tissue, AAV8 has proved to be a carrier for transduction efficiency high, is capable of achieving the transduction close to 100% to liver.Additionally, AAV6 is also higher than AAV2 and AAV1 to the transduction efficiency of hepatic tissue.It is worth noting that, AAV8 not only can efficient transduction hepatic tissue, also have transduction efficiency higher to other organs, the AAV8 transduced skeletal fleshes of high dose include diaphragm to whole body, and heart, pancreas, smooth muscle and brain are respectively provided with transducing high.On the other hand, transduction efficiency AAV8 high to skeletal muscle is, it is necessary to VEGF is administered simultaneously, to be passed through the transgene expression that vascular barrier realizes whole body.Generally, due to blood brain and the presence systemic injection AAV of vascular barrier, transduction to brain and skeletal muscle is difficult to realize, however, above-mentioned research shows, the endothelial cell that AAV8 may pass through blood vessel is transduceed these tissues.Therefore, the tissue tropism of AAV serotypes is extremely important（The transduction of non-target tissue can be caused in the transmission of gene）, the successful Application for it in human clinical trial is, it is necessary to strictly control and manipulate their tissue tropisms.On the other hand, the final transduction efficiency of different serotypes is that the dynamic variation of transgene expression is also critically important.Such as AAV2,6 and 8 pairs of transduction hepatic tissues that can be effective；After hepatic portal vein injection AAV2 viral vectors, transgene expression is increased with a relatively slow speed, it usually needs reach within 6-8 weeks the levels of transduction of maximum, in contrast, AAV6 and AAV8 transductions have quick transgene expression, and general administration reaches maximum horizontal after 4 weeks.Because each organization type is different with intracellular transport mechanism to the cellular uptake of AAV serotypes, the structural differences between AAV serotype capsids cause the difference on effect between this serotype.
Recent study finds, AAV2, AAV5 and AAV6 can effective transduction human cells of monocytic origin DCs cells（Dendritic cells）, form and functional characteristic to DC cells have no adverse effect, and compared with other serotypes As AV viral vectors, AAV5 is presented the tendentiousness high to people's DC cells, but transduction efficiency is low；And AAV6 has transduction efficiency high to the DC cells that mouse is originated.So far not finding can be with the AAV serotype of efficient infection immunocyte, in order to obtain the AAV virus mutations of transduction efficiency higher, preferably for clinical gene therapy, the AAV viruses of necessary other serotypes of development.
The content of the invention
The purpose of the present invention in order to provide a kind of AAV viruses of new serotype, its can efficient infection immunocyte, for AAV provides new approach as the application of genophore, for gene therapy provides wider selection.
To achieve the above object of the invention, the technical solution adopted by the present invention is：
It is a kind of can efficient infection immunocyte AAV viruses, including capsid protein, non-structural protein and structural proteins, wherein, the amino acid sequence of the structural proteins is SEQ ID NO:Shown in 1.
In above-mentioned technical proposal, amino acid sequence is SEQ ID NO:1 structural proteins can be SEQ ID NO by sequence:2 DNA encoding, it is also possible to by sequence be SEQ ID NO:3 DNA encoding.
Adeno-associated virus（AAV viruses）It is a kind of replication defect type DNA virus, by three kinds of coating proteins（Capsid protein, non-structural protein and structural proteins）Constituted with a single stranded DNA, every DNA is single-stranded including ITR elements and ORFs；AAV viruses of the invention can load foreign gene, and be carried to immunocyte, inject in immunocyte foreign gene after efficient infection, play a role, therefore can be as the application of foreign gene carrier；So as to the invention also discloses based on it is above-mentioned can efficient infection immunocyte AAV virus recombinant adeno-associated virus.By it is above-mentioned can efficient infection immunocyte AAV virus constituted with foreign gene, wherein foreign gene be linear single-stranded structure；The size of foreign gene is 6bp~6000bp.
The invention also discloses the preparation method of above-mentioned recombinant adeno-associated virus, comprise the following steps：
（1）Synthesis SEQ ID NO:2 or SEQ ID NO:3 DNA sequence dna；Then the DNA sequence dna of synthesis is cloned into pAAV-RC carriers；
（2）Then step is taken（1）Clone products convert into E. coli competent, after monoclonal bacterium colony grows, choose monoclonal bacterium colony and add SOC culture mediums, after culture, bacterium solution is transferred in LB culture mediums, add the ampicillin of final concentration of 50ug/mL, carry out shaking bacterium Amplification Culture；Finally extract plasmid and carry out sequence verification and correctly obtain adeno-associated virus plasmid afterwards；
（3）According to 3x10 in culture dish6The density inoculation HEK293T cells of individual/10cm；After 48 hours, in the culture dish of inoculation HEK293T cells, using PEI transfection procedures（2）The adeno-associated virus plasmid of preparation, the pAAV plasmids of foreign gene-carrying and pHelper plasmids；After transfection 48~72 hours, cell is collected, after supernatant is removed in centrifugation, cell pyrolysis liquid is added, by after 3 Frozen-thawed cycleds, being centrifugally separating to obtain adeno-associated virus extract solution；The temperature of the Frozen-thawed cycled is -80~37 degree of degree；Foreign gene is linear single-stranded structure；The size of foreign gene is 6bp~6000bp；
（4）Mass concentration is respectively during 15%, 25%, 40%, 54% Iodixanol solution sequentially adds centrifuge tube；It is subsequently adding step（3）Adeno-associated virus extract solution；Treatment is then centrifuged for, supernatant is removed, recombinant adeno-associated virus are obtained.
In above-mentioned technical proposal, step（1）In, using the DNA sequence dna synthesized described in PacI-AscI double digestions, digestion products are reclaimed, obtain the DNA fragmentation after digestion；Using PacI-AscI double digestion pAAV-RC carriers, digestion products are reclaimed, obtain digestion carrier；DNA fragmentation after digestion and digestion carrier are prepared into T4 coupled reaction systems, 16 DEG C of connections are overnight；So as to DNA sequence dna is cloned into pAAV-RC carriers.
In above-mentioned technical proposal, step（2）In, then clone products are taken to convert into Stbl3 E. coli competents, after monoclonal bacterium colony grows, choose 3-5 monoclonal bacterium colony and add 5mL SOC culture mediums, after 37 degree of 180RPM are cultivated 1 hour, all of liquid is transferred in 800mL LB culture mediums, adds the ampicillin of final concentration of 50ug/mL, 37 degree of Shaking cultures to carry out within 16 hours shaking bacterium Amplification Culture；Finally plasmid being extracted using the big extraction reagent kit of plasmid and carrying out sequence verification correctly obtain adeno-associated virus plasmid afterwards.
In above-mentioned technical proposal, step（3）In, in the culture dish of inoculation HEK293T cells, add step（2）The adeno-associated virus plasmid of preparation, the pAAV plasmids of foreign gene-carrying and pHelper plasmids, the amount according still further to final concentration of 9 μ g/mL add PEI, are transfected；After transfection 48 hours, cell is collected；Add the all-round nucleases of final concentration of 50U/mL（Benzonase）, it is incubated at 37 degree 1 hour, then 3000g centrifugations 30 minutes；Obtain adeno-associated virus extract solution；The pH of the cell pyrolysis liquid is 8.5, including 50mM Tris-HCl and 50mM NaHCO3。
In above-mentioned technical proposal, step（4）In, in centrifuge tube, 15%, 25%, 40%, 54% Iodixanol solution and the volume ratio of adeno-associated virus extract solution are 5: 7: 5: 5: 15；Parameter during centrifugal treating is 28000RPM, 16 degree of 5 hours of centrifugation.
Foreign gene is injected in immunocyte after recombinant adeno-associated virus efficient infection immunocyte of the invention, is played a role；Therefore the application the invention also discloses the recombinant adeno-associated virus in gene therapy medicament is prepared.
Compared with prior art, the new A AV prepared with the new construction albumen viruses of the invention, it has efficient infectious to immunocyte；Recombinant adeno-associated virus can be constituted with foreign gene as genophore, foreign gene is carried to immunocyte, inject in immunocyte foreign gene after efficient infection, so as to play foreign gene effect；To the wide adaptability of foreign gene, and the AAV viruses are not required to the preparation technology of complexity, and extremely low immunogenicity is safe to the human body, for gene therapy provides widely selection.
Brief description of the drawings
Fig. 1 is the effect contrast figure of existing AAV viruses and AAV of the present invention virus packaging GFP gene infection immunity cells；
After Fig. 2 is for existing AAV viruses and AAV of the present invention virus packaging IL15 gene infection immunity cells, the effect contrast figure of destination protein secreting, expressing.
Specific embodiment
Below in conjunction with the accompanying drawings and embodiment the invention will be further described.
Embodiment one
（1）SEQ ID NO are directly synthesized using the method for chemical synthesis:DNA sequence dna shown in 2, referred to as hCap genes, and PacI-AscI double digestions are carried out, obtain the DNA fragmentation after digestion；Using PacI-AscI double digestion pAAV-RC carriers, digestion products are reclaimed, obtain digestion carrier；DNA fragmentation after digestion and digestion carrier are prepared into T4 coupled reaction systems, 16 DEG C of connections are overnight；So as to DNA sequence dna is cloned into pAAV-RC carriers, through sequence verification it is correct after, by the product after connection（Clone products）In conversion to 30uL Stbl3 E. coli competents.After conversion terminates, the SOC culture mediums of 1mL preheatings are added immediately, then in being gone to the centrifuge tube of 15mL, and add 5mLSOC culture mediums, after 37 degree of 180RPM are cultivated 1 hour, all of liquid is transferred in 800mL LB culture mediums, the ampicillin of final concentration of 50ug/mL is added, 37 degree of Shaking cultures 16 hours.Culture terminate after, 3000g is collected by centrifugation bacterial precipitation, the kit isolated plasmid dna carried greatly using plasmid and carry out sequence verification correctly afterwards obtain adeno-associated virus plasmid.Can efficient infection immunocyte AAV virus, it is subzero 80 degree preserve.
Embodiment two
（1）SEQ ID NO are directly synthesized using the method for chemical synthesis:DNA sequence dna shown in 3, referred to as bCap genes, and carry out PacI-AscI double digestions, are cloned into pAAV-RC, through sequence verification it is correct after, the product after connection is converted into 30uL Stbl3 E. coli competents by the method for shocking by electricity.After electricity conversion terminates, the SOC culture mediums of 1mL preheatings are added immediately, then in being gone to the centrifuge tube of 15mL, and add 5mLSOC culture mediums, after 37 degree of 180RPM are cultivated 5 hours, all of liquid is transferred in 500mL LB culture mediums, the ampicillin of final concentration of 50ug/mL is added, 37 degree of Shaking cultures 18 hours.Culture terminate after, 2000g is collected by centrifugation bacterial precipitation, the kit isolated plasmid dna carried greatly using plasmid and carry out sequence verification correctly afterwards obtain adeno-associated virus plasmid.Can efficient infection immunocyte AAV virus.
Embodiment three
（1）HCap genes are directly synthesized using the method for chemical synthesis, and carry out PacI-AscI double digestions, be cloned into pAAV-RC, through sequence verification it is correct after, for follow-up AAV viruses packaging.Product after connection is converted into 30uL Stbl3 E. coli competents by the method for shocking by electricity.After electricity conversion terminates, the SOC culture mediums of 1mL preheatings are added immediately, then in being gone to the centrifuge tube of 15mL, and add 5mLSOC culture mediums, after 37 degree of 180RPM are cultivated 5 hours, all of liquid is transferred in 500mL LB culture mediums, the ampicillin of final concentration of 50ug/mL is added, 37 degree of Shaking cultures 18 hours.Culture terminate after, 2000g is collected by centrifugation bacterial precipitation, the kit isolated plasmid dna carried greatly using plasmid and carry out sequence verification correctly afterwards obtain adeno-associated virus plasmid.
（2）Prepare 20 15cm culture dishes, 4.5x10^6 HEK293 immunocyte is inoculated with per ware.After 48 hours, 220ug adeno-associated viruses plasmid and 250ug pAAV-IL15 and pHelper plasmids are transfected using PEI.After 48 hours, using cell scrape by cell from culture dish bottom collection to centrifuge tube in, after removing supernatant, use cell pyrolysis liquid（50mM Tris-HCl,pH8.5,50mM NaHCO3）, by 3 Frozen-thawed cycleds（- 80/37 degree of degree）Afterwards, 50U/mL Benzonase are added to be incubated at 37 degree 1 hour, then 2000g centrifugations 20 minutes.
（3）Prepare 15%, 25%, 40%, 50% Iodixanol gradient.In SW28 centrifuge tubes, 5mL 54%, 7mL 40%, 5mL 25%, the Iodixanol gradient of 5mL 15%, 15mLAAV crude extracts are sequentially added.28000RPM, 16 degree of 6 hours of centrifugation, 40% Iodixanol ladder is collected, centrifugal treating removes supernatant, obtains the recombinant adeno-associated virus that foreign gene is IL15 genes, is expressed as bCap- IL15.
By step（1）HCap genes replace with bCap genes, can obtain foreign gene be IL15 genes recombinant adeno-associated virus, be expressed as hCap- IL15.
By step（2）PAAV-IL15 replace with pAAV-EGFP, can obtain foreign gene be GFP genes recombinant adeno-associated virus, be expressed as hCap-GFP.
By step（1）HCap genes replace with bCap genes, step（2）PAAV-IL15 replace with pAAV-EGFP, can obtain foreign gene be GFP genes recombinant adeno-associated virus, be expressed as bCap-GFP.
With GFP genes, IL15 genes as foreign gene, with it is above-mentioned can efficient infection immunocyte AAV virus as carrier, with existing AAV2 as comparison vehicle（The recombinant adeno-associated virus for obtaining are AAV2-GFP, AAV2- IL15）, Prepare restructuring adeno-associated virus, and carry out fluorometric investigation and expression concentration determination.Respectively using the virus infection immunity cells of the AAV containing green fluorescent protein GFP, IL15 of above-mentioned preparation, after 3 days, in the expression of fluorescence microscopy Microscopic observation green fluorescent protein.The expression concentration of antibody in culture medium supernatant is detected using enzyme linked immunological labelling kit.
Fig. 1 is the effect contrast figure that GFP and AAV of the invention virus packaging GFP infection immunity cells are packed using AAV2, it can be seen that using natural AAV2 serotype adeno-associated virus infection immunity cells, efficiency of infection is relatively low, and GFP egfp expressions are relatively weak；And with AAV of the invention virus packaging GFP infection immunity cells, efficiency of infection is higher, the green fluorescent protein brightness of GFP expression is significantly improved.
Respectively IL15 genes are carried using AAV of the invention viral and natural AAV2, difference infection immunity cell, after continuous culture 72 hours, after collecting culture medium supernatant, the secreting, expressing amount of IL15 is detected using the method for ELISA, Fig. 2 is the expression of IL15 in culture medium supernatant, as a result shows that the AAV serum after present invention improvement can effectively infect immunocyte, and high efficient expression IL15.
The new A AV viruses of offer of the invention can to sum up be illustrated has efficient infectivity to immunocyte；Recombinant adeno-associated virus can be constituted with foreign gene as genophore, foreign gene is carried to immunocyte, injected in immunocyte foreign gene after efficient infection, so as to play the therapeutic action of foreign gene；To the wide adaptability of foreign gene, and the AAV viruses are not required to the preparation technology of complexity, safe to the human body, for gene therapy provides widely selection.
SEQUENCE LISTING
<110>Ai Kang get biomedical technologies（Suzhou）Co., Ltd
<120>It is a kind of can efficient infection immunocyte AAV viruses and preparation method and application
<160> 3
<170> PatentIn version 3.3
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<211> 735
<212> PRT
<213>Artificial sequence
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Met Ala Ala
Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser
1 5 10 15
Glu Gly Ile
Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro
20 25 30
Lys Pro Ala
Glu Arg His Lys Asp Asp Ser 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
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Val Asn Glu
Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
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Arg Gln Leu
Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala
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Asp Ala Glu
Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly
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Asn Leu Gly
Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
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Leu Gly Leu
Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg
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Pro Val Glu
His Ser Pro Val Phe Pro Asp Ser Ser Ser Gly Phe Gly
145 150 155 160
Lys Ala Gly
Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr
165 170 175
Gly Asp Ala
Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Val Pro Pro
180 185 190
Ala Ala Pro
Ser Gly Leu Gly Ser Ser Thr Met Ala Thr Gly Ser Gly
195 200 205
Ala Pro Met
Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser
210 215 220
Ser Gly Asn
Trp His Cys Asp Ser Thr Trp Met 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 Gln Ser Gly Ala Ser Asn Asp Asn His Tyr
260 265 270
Phe Gly Tyr
Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His
275 280 285
Cys His Phe
Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp
290 295 300
Gly Phe Arg
Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val
305 310 315 320
Lys Glu Val
Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu
325 330 335
Thr Ser Thr
Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr
340 345 350
Val Leu Gly
Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp
355 360 365
Val Phe Met
Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser
370 375 380
Gln Ala Val
Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser
385 390 395 400
Gln Met Leu
Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu
405 410 415
Asp Val Pro
Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg
420 425 430
Leu Met Asn
Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr
435 440 445
Asn Thr Pro
Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln
450 455 460
Ala Gly Ala
Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly
465 470 475 480
Pro Cys Tyr Arg
Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn
485 490 495
Asn Ser Glu
Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly
500 505 510
Arg Asp Ser
Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp
515 520 525
Asp Glu Glu
Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys
530 535 540
Gln Gly Ser
Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr
545 550 555 560
Asp Glu Glu
Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr
565 570 575
Gly Ser Val
Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr
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Ala Asp Val
Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp
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Arg Asp Val
Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr
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Asp Gly His
Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys
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His Pro Pro
Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn
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Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln
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Tyr Ser Thr
Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys
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Glu Asn Ser
Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr
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Asn Lys Ser
Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr
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Ser Glu Pro
Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu
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atggccgccg
atggctacct gcctgactgg ctggaagata ccctgagcga gggcatccgg 60
cagtggtgga
agctgaagcc tggaccccct ccacctaagc ccgccgagag acacaaggac 120
gacagcagag
gactggtgct gcccggctac aagtacctgg gccccttcaa cggcctggac 180
aagggcgagc
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agacagctgg
acagcggcga caacccctac ctgaagtaca accacgccga cgccgagttc 300
caggaacggc
tgaaagagga caccagcttc ggcggcaatc tgggcagagc cgtgtttcag 360
gccaagaaac
gggtgctgga acccctgggc ctggtggaag aacccgtgaa aaccgcccct 420
ggcaaaaagc
ggcccgtgga acacagcccc gtgttccctg atagcagcag cggctttggc 480
aaggccggac
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agcgtgccag
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tctacaatgg
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gtgggcaaca
gctccggcaa ttggcactgc gacagcacct ggatgggcga cagagtgatc 720
accaccagca
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agcagccagt
ccggcgccag caacgacaac cactacttcg gctacagcac cccctggggc 840
tacttcgact
tcaaccggtt ccactgccac ttcagcccca gagactggca gcggctgatc 900
aacaacaact
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aaagaagtga
cccagaacga cggcaccacc acaatcgcca acaacctgac cagcaccgtg 1020
caggtgttca
ccgacagcga gtaccagctg ccctacgtgc tgggatctgc ccaccaggga 1080
tgcctgcctc
cctttcccgc cgacgtgttc atggtgcccc agtacggcta cctgaccctg 1140
aacaatggca
gccaggccgt gggcagaagc agcttctact gcctggaata cttccccagc 1200
cagatgctgc
ggaccggcaa caacttcacc ttcagctaca ccttcgagga cgtgcccttc 1260
cacagcagct
acgcccactc tcagagcctg gacagactga tgaaccccct gatcgaccag 1320
tacctgtact
acctgagccg gaccaacacc cccagcggca ccacaacaca gagccggctg 1380
cagttttctc
aggctggcgc ctccgacatc cgggaccaga gcagaaattg gctgcctggc 1440
ccctgctacc
ggcagcagag agtgtctaag accagcgccg ataacaacaa tagcgagtac 1500
tcctggaccg
gcgccaccaa gtaccacctg aacggcagag actccctcgt gaaccctgga 1560
cctgccatgg
ccagccacaa ggatgacgag gaaaagttct tcccacagtc cggggtgctg 1620
atcttcggca
agcagggcag cgaaaagacc aacgtggaca tcgagaaagt gatgatcacc 1680
gacgaggaag
agatccggac caccaaccca gtggccaccg agcagtatgg cagcgtgtcc 1740
accaacctgc
agcggggcaa tagacaggcc gccaccgccg atgtgaatac tcagggcgtg 1800
ctgccaggca
tggtgtggca ggatagggac gtgtacctgc agggccccat ctgggccaag 1860
atccctcaca
ccgatggcca cttccacccc agccctctga tgggcggatt cggcctgaag 1920
caccccccac
cccagatcct gatcaagaac acccccgtgc ccgccaaccc cagcacaaca 1980
ttttccgccg
ccaagttcgc cagcttcatc acccagtaca gcacaggcca ggtgtccgtg 2040
gaaatcgagt
gggagctgca gaaagaaaac agcaagcggt ggaaccccga gatccagtac 2100
acctccaact
acaacaagag cgtgaacgtg gacttcaccg tggacaccaa cggcgtgtac 2160
agcgagccca
gacccatcgg caccagatac ctgacacgga acctgtaa 2208
<210> 3
<211> 2208
<212> DNA
<213>Artificial sequence
<400> 3
atggccgctg
atggctacct gcctgactgg ctggaagata ccctgtccga gggcatccgg 60
cagtggtgga
agctgaagcc tggaccccct ccacctaagc ccgccgagag acacaaggac 120
gactccagag
gactggtgct gcccggctac aagtacctgg gccctttcaa cggcctggac 180
aagggcgagc
ctgtgaacga ggctgatgcc gccgctctgg aacacgacaa ggcctacgac 240
cggcagctgg
actctggcga caacccctac ctgaagtaca accacgccga cgccgagttc 300
caggaacggc
tgaaagagga cacctccttc ggcggcaatc tgggcagagc cgtgtttcag 360
gccaagaaac
gggtgctgga acccctgggc ctggtggaag aacccgtgaa aaccgcccct 420
ggcaaaaagc
ggcccgtgga acactccccc gtgttccctg attcctccag cggctttggc 480
aaggccggac
agcagcccgc cagaaagaga ctgaacttcg gccagaccgg cgacgccgac 540
tctgtgcctg
atcctcagcc tctgggagtg cctcctgctg ctccttctgg cctgggctcc 600
tctaccatgg
ctaccggatc tggcgcccct atggccgaca acaatgaagg cgcagacggc 660
gtgggcaact
cctccggcaa ttggcactgc gactccacct ggatgggcga cagagtgatc 720
accacctcca
cccggacatg ggccctgccc acctacaaca accacctgta caagcagatc 780
agctcccagt
ccggcgcctc caacgacaac cactacttcg gctactccac cccctggggc 840
tacttcgact
tcaaccggtt ccactgccac ttcagccctc gggactggca gcggctgatc 900
aacaacaact
ggggcttccg gcccaagcgg ctgaacttca agctgttcaa tattcaagtg 960
aaagaagtga
cccagaacga cggcaccacc acaatcgcca acaacctgac ctccaccgtg 1020
caggtgttca
ccgactccga gtatcagctg ccctacgtgc tgggctctgc tcaccaggga 1080
tgtctgcctc
ccttccccgc cgacgtgttc atggtgcctc agtacggcta cctgaccctg 1140
aacaacggct
ctcaggccgt gggccggtcc agcttctact gcctggaata cttccccagc 1200
cagatgctgc
ggaccggcaa caacttcacc ttcagctaca ccttcgagga cgtgcccttc 1260
cactcctcct
acgcccactc ccagtctctg gacagactga tgaaccccct gatcgaccag 1320
tacctgtact
acctgtcccg gaccaacacc ccctccggca ccacaacaca gtcccggctg 1380
cagttctctc
aggctggcgc ctctgacatc cgggaccagt ccagaaattg gctgcctggc 1440
ccttgctacc
ggcagcagag agtgtccaag acctccgccg ataacaacaa cagcgagtac 1500
tcttggaccg
gcgctaccaa gtaccacctg aacggccggg actctctcgt gaatcctggc 1560
cctgccatgg
cctcccacaa ggatgacgag gaaaagttct tcccacagtc tggcgtgctg 1620
atcttcggca
agcagggctc cgaaaagacc aacgtggaca tcgagaaagt gatgatcacc 1680
gacgaggaag
agatccggac caccaaccct gtggccaccg agcagtatgg ctccgtgtcc 1740
accaacctgc
agcggggcaa tagacaggcc gccaccgccg atgtgaatac ccagggcgtg 1800
ctgccaggca
tggtgtggca ggatagggac gtgtacctgc agggccccat ctgggccaag 1860
atccctcaca
ccgatggcca cttccacccc agccctctga tgggcggatt cggcctgaag 1920
caccccccac
cccagatcct gatcaagaac acccccgtgc ccgccaaccc cagcaccacc 1980
ttttctgccg
ccaagttcgc ctccttcatc acccagtact ccacaggcca ggtgtccgtg 2040
gaaatcgagt
gggagctgca gaaagaaaac tccaagcggt ggaaccccga gatccagtac 2100
accagcaact
acaacaagtc cgtgaacgtg gacttcaccg tggacaccaa cggcgtgtac 2160
tccgagccca
gacccatcgg caccagatac ctgaccagaa acctgtaa 2208
Claims (10)
1. it is a kind of can efficient infection immunocyte AAV viruses, including capsid protein, non-structural protein and structural proteins, it is characterised in that：The amino acid sequence of the structural proteins is SEQ ID NO:Shown in 1.
2. according to claim 1 can efficient infection immunocyte AAV virus, it is characterised in that：The DNA sequence dna for encoding the structural proteins amino acid sequence is SEQ ID NO:2 or SEQ ID NO:Shown in 3.
3. described in claim 1 can efficient infection immunocyte AAV virus as foreign gene carrier application；The foreign gene is linear single-stranded structure；The size of the foreign gene is 6bp~6000bp.
4. a kind of preparation method of recombinant adeno-associated virus, it is characterised in that comprise the following steps：
（1）Synthesis SEQ ID NO:2 or SEQ ID NO:3 DNA sequence dna；Then the DNA sequence dna of synthesis is cloned into pAAV-RC carriers；
（2）Then step is taken（1）Clone products convert into E. coli competent, after monoclonal bacterium colony grows, choose monoclonal bacterium colony and add SOC culture mediums, after culture, bacterium solution is transferred in LB culture mediums, add the ampicillin of final concentration of 50ug/mL, carry out shaking bacterium Amplification Culture；Finally extract plasmid and carry out sequence verification and correctly obtain adeno-associated virus plasmid afterwards；
（3）According to 3x10 in culture dish6The density inoculation HEK293T cells of individual/10cm；After 48 hours, in the culture dish of inoculation HEK293T cells, using PEI transfection procedures（2）The adeno-associated virus plasmid of preparation, the pAAV plasmids of foreign gene-carrying and pHelper plasmids；After transfection 48~72 hours, cell is collected, after supernatant is removed in centrifugation, cell pyrolysis liquid is added, by after 3 Frozen-thawed cycleds, being centrifugally separating to obtain adeno-associated virus extract solution；The temperature of the Frozen-thawed cycled is -80~37 degree of degree；The foreign gene is linear single-stranded structure；The size of the foreign gene is 6bp~6000bp；
（4）Mass concentration is respectively during 15%, 25%, 40%, 54% Iodixanol solution sequentially adds centrifuge tube；It is subsequently adding step（3）Adeno-associated virus extract solution；Centrifugal treating, removes supernatant, obtains recombinant adeno-associated virus.
5. the preparation method of recombinant adeno-associated virus according to claim 4, it is characterised in that：Step（1）In, using the DNA sequence dna synthesized described in PacI-AscI double digestions, digestion products are reclaimed, obtain the DNA fragmentation after digestion；Using PacI-AscI double digestion pAAV-RC carriers, digestion products are reclaimed, obtain digestion carrier；DNA fragmentation after digestion and digestion carrier are prepared into T4 coupled reaction systems, 16 DEG C of connections are overnight；So as to DNA sequence dna is cloned into pAAV-RC carriers.
6. the preparation method of recombinant adeno-associated virus according to claim 4, it is characterised in that：Step（2）In, clone products are taken to convert into Stbl3 E. coli competents, after monoclonal bacterium colony grows, choose 3-5 monoclonal bacterium colony and add 5mL SOC culture mediums, after 37 degree of 180RPM are cultivated 1 hour, all of liquid is transferred in 800mL LB culture mediums, adds the ampicillin of final concentration of 50ug/mL, 37 degree of Shaking cultures to carry out within 16 hours shaking bacterium Amplification Culture；Finally plasmid being extracted using the big extraction reagent kit of plasmid and carrying out sequence verification correctly obtain adeno-associated virus plasmid afterwards.
7. the preparation method of recombinant adeno-associated virus according to claim 4, it is characterised in that：Step（3）In, in the culture dish of inoculation HEK293T cells, add step（2）The adeno-associated virus plasmid of preparation, the pAAV plasmids of foreign gene-carrying and pHelper plasmids, the amount according still further to final concentration of 9 μ g/mL add PEI, are transfected；After transfection 48 hours, cell is collected；Cell pyrolysis liquid is added, by after 3 Frozen-thawed cycleds, adding the all-round nucleases of final concentration of 50U/mL, is incubated at 37 degree 1 hour, then 3000g centrifugations 30 minutes；Obtain adeno-associated virus extract solution；The pH of the cell pyrolysis liquid is 8.5, is to include 50mM tri-（Methylol）Aminomethane and 50mM
The hydrochloride buffer of sodium acid carbonate.
8. the preparation method of recombinant adeno-associated virus according to claim 4, it is characterised in that：Step（4）In, in centrifuge tube, 15%, 25%, 40%, 54% Iodixanol solution and the volume ratio of adeno-associated virus extract solution are 5: 7: 5: 5: 15；Parameter during centrifugal treating is 28000RPM, 16 degree of 5 hours of centrifugation.
9. the recombinant adeno-associated virus that prepared by the preparation method of any one recombinant adeno-associated virus according to claim 4~8.
10. application of the recombinant adeno-associated virus described in claim 9 in gene therapy medicament is prepared.
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