CN101586096A

CN101586096A - Recombinant virus with coexpression of anthropogenic glutamate decarboxylase and cell factor

Info

Publication number: CN101586096A
Application number: CNA200810028196XA
Authority: CN
Inventors: 王尚武; 朱雅南; 徐评议
Original assignee: Individual
Current assignee: Individual
Priority date: 2008-05-21
Filing date: 2008-05-21
Publication date: 2009-11-25

Abstract

The invention relates to a recombinant virus containing a gene expression box expressing an anthropogenic GAD gene and a cell factor, in particular to a method for constructing a recombinant adeno-associated virus with the coexpression of an anthropogenic GAD 65 gene and a GDNF gene, an application and a meaning thereof. Anthropogenic GAD 65 gene and the GDNF gene protein products generated by the expression of the recombinant virus have favorable synergistic effect on the treatment of diseases relative to Parkinson's disease and the central nervous system and have important values for treatment of relative diseases.

Description

The recombinant virus of a kind of coexpression of anthropogenic glutamate decarboxylase and cytokine

Technical field

The present invention relates to the structure and preparation method thereof of recombinant virus, the especially recombinant adeno-associated virus of a kind of coexpression L-Glutamic decarboxylase and cytokine, the technical field at its place is relevant with life science and biological medicine technology.

Background technology

The most of neuronic neurotransmitter that studies have shown that mammals brain striatum and striatum projected area is γ-An Jidingsuan (GABA, down together), GABA is the main inhibitory nerve mediator of mammals cerebral tissue, and L-Glutamic decarboxylase (GAD, down with) be synthetic GABA rate-limiting enzyme, GAD have GAD 65 and 67 two kinds of isozyme of GAD ( Lindefors N.et al: Prog.Neuropsycho.pharmacol. Biol Psychiatry.1993; 17 (6): 887-903).There is the researchist to use the adeno-associated virus (AAV that expresses the GAD65 gene, down together) the animal model Parkinson's disease is carried out therapeutic studies, the result shows that the Parkinson symptom has clear improvement, electrophysiologic study also proves increases GAD65 in the neuronic expression of nucleus of inferior colliculus, can make the nucleus of inferior colliculus neurone from the excitability output state change into the inhibition output state ( Lee B, et al: Gene Ther.2005 Aug; 12 (15): 1215-22; Emborg ME, et al: J Cereb Blood Flow Metab.2007 Mar; 27 (3): 501-9.), these researchs lay a solid foundation for GAD treatment Ren Pajinsenshi disease.

Recently report, through approval, the U.S. 12 late period the parkinsonian use the AAV that expresses the GAD65 gene and Parkinson's disease has been carried out the clinical I phase study, the result shows that the Parkinson symptom is significantly improved, well-tolerated, safety, effective ( Kaplitt MG, et al:Lancet.2007 Jun23; 369 (9579): 2056-8).Domestic researchist uses the herpes virus of human body no pathogenicity is expressed GAD research as expression vector at stellate cell, but as yet not animal or human's body to Parkinson's disease treat research ( Liu W, et al: Intervirology.2005Sep-Oct; 48 (5): 329-35; Liu WEt al: Mol Ther.2007).

In addition, studies have shown that GABA synthetic enzyme GAD is relevant with the generation of convulsions, epilepsy and cerebellar ataxia disease.Automatic epilepsy takes place in the mouse of disappearance GAD65, the mouse epileptic seizures susceptibility of the GAD65 disappearance that produces of backcrossing significantly increase ( Kash SF, et al: Proc Natl Acad Sci USA.1997 Dec 9; 94 (25): 14060-5)); And the cell that animal epileptic model brain region of interest transplant to be expressed GABA synthetic enzyme GAD can reduce or block epilepsy generation ( Gernert M, et al: Exp Neurol.2002 Jul; 176 (1): 183-92;

W, et al: J Neurosci Res.1998 Jan 15; 51 (2): 196-209; Vulliemoz S, et al: J Neurol Neurosurg Psychiatry).

Undoubtedly, above-mentioned clinical research confirmation GAD to be used for the treatment of Parkinson's disease as the synthetic rate-limiting enzyme of the main inhibitory nerve mediator GABA of mammals cranial nerve cell be safe and effective.But, obviously, this treatment is a kind of symptomatic smelting treatment of Parkinson's disease that improves, and can not change pathology process and consequent other clinical symptom of Parkinson.

Manyly studies have shown that parkinsonian generation mainly is an a variety of causes, drawing together inherited genetic factors, environmental factors, interior metabolism product free radical and brain blood brain factor etc. causes due to striatum and the relevant projected area nucleus dopamine neuron generation apoptosis thereof, cause Dopamine HCL synthetic, production and secretion reduce and the initiation Parkinson's disease.Therefore, slow down or prevent that the dopamine neuron apoptosis from being that parkinsonian link of crucial importance is treated in smelting.

Known at present, neurocyte nutritional factor GDNF is to slowing down or preventing that the dopamine neuron apoptosis from having better effect.GDNF is that the cerebral nerve spongiocyte produces, has very important, the biological function neurocyte factor widely, multiple neurocyte to maincenter and periphery has neurotrophic effect comparatively widely, be find at present to dopamine neuron and the most significant class neurotrophic factor of motor neuron effect, the effect that promotes neure growth and differentiation is more widely arranged, especially to the strongest (the Lin LT et al.Science 1993.260 (5111): 1130-2 of dopamine neuron effect; Henderson et al.Science 1994,266 (5183): 1062-4).GDNF plays the protein kinase A kt/PKB of crux effect in cell survival by the phosphorylation activation, promote the propagation and the differentiation of neurocyte, the apoptosis that suppresses neurocyte, protection Dopamine HCL neurocyte, can protect Dopamine HCL neurocyte antagonism injurious factor, as superoxide, free radical, the infringement that ischemic, anoxic cause; Strengthen the ability of Dopamine HCL neurocyte picked-up Dopamine HCL, increase storage, the release of Dopamine HCL; Promote neural system regeneration that important effect is arranged.

The function of neurotrophic factor GDNF has been carried out extensive studies both at home and abroad, proved GDNF, obviously relaxed the concentrated and dna break of chromatin that 6-hydroxydopamine (6-OHDA) is brought out by the mediation of PI3K/Akt path; Start a series of anti-apoptotic signal approach and neuroprotective unit, GDNF is considered to neuroprotective medicine likely (Do Thi NA, Saillour P, Ferrero L, DedieuJF, Mallet J, Paunio T.Gene Ther.2004 Jan 15; Patel NK et al, AnnNeurol.2005 Feb; 57 (2): 298-302).

Studies show that in the parkinsonian treatment of human body, directly GDNF is input to parkinsonian's brain lenticular nucleus, can significantly improve patient's symptom, after 1 year and do not have a serious clinical side effects; Positron scanning shows that patient's lenticular nucleus Dopamine HCL is stored, picked-up increases by 28% after 18 months, show GDNF to dopamine neuron have direct effect ( Heywood P.et al: Nat Med.2003 May; 9 (5): 589-95.Epub 2003 Mar 31; Slevin JT, et al: J Neurosurg.2005 Feb; 102 (2): 401. Slevin JT, et al: Neurosurg Focus.2006 May 15; 20 (5): E1).

Another member Neurturin (NTN) of GDNF family and midbrain stellate cell derived neurotrophic factor MANF be proved to be to dopamine neuron have special neurotization function ( Petrova PEt al: J Mol Neurosci.2003 Apr; 20 (2): 173-88; Liu WGEt al: Mol Neurodegener.2007 Oct 1; 2 (1): 19).And the conservative Dopamine HCL neurotrophic factor CDNG (conserved dopamine neurotrophic factor, CDNF) that finds recently has the recovery similar with GDNF and prevent the effect that dopamine neuron degenerates ( Lindholm P, et al: Nature.2007 Jul5; 448 (7149): 73-7).

Meaningfully, nearest studies show that the epilepsy model animal adopt the gene smelting of GDNF to treat effectively to suppress or reduce the epilepsy generation ( Kanter-Schlifke I, et al: Mol Ther.2007Jun; 15 (6): 1106-13).

Equally, another kind of human brain cell source neurotrophic factor (BDNF, down together) is by brain cell generation, a kind of neurocyte nutritional factor that the important biomolecule function is arranged of excretory.BDNF has neurotrophic effect comparatively widely to the multiple neurocyte of maincenter and periphery, can promote some neural group's existence and differentiation, a piece of wood serving as a brake to halt a carriage power of regulation and control spinous process axle, and relevant with study, memory and the spinal cord Theory of Pain Mechanism of hippocampus.

BDNF also plays an important role to the existence and the growth of dopamine neuron.It has the death that stops apoptotic cell, the growth of the neuronic existence of increase tyrosine hydrolysis enzyme positive and this neurofibril ( Ostergaard K, et al: Exp Neurol.1996 Dec; 142 (2): 340-50; Tang FI, et al: Exp Brain Res.1998 Apr; 119 (3): 287-96; Singh S, et al: Indian J Exp Biol.2006 Sep; 44 (9): 699-704; Hu Xudong etc. The neuroanatomy magazine, 1998 02).Therefore, BDNF is as a kind of potent neuroprotective factor, be considered to treat some central nervous system disease good candidate's factor ( Pezet S, et al: Expert Opin Ther Targets.2004 Oct; 8 (5): 391-9).

Relevant research proves that also transcription factor Nurr1 can make embryonic stem cell be divided into the dopamine neuron that can secrete Dopamine HCL, and the direct regulation and control by BDNF is expressed, influence the growth of midbrain Dopamine HCL nerve and function ( Volpicelli F, 2007 Jul; 102 (2): 441-53; Hara K, J Neurosci Res.2007May 1; 85 (6): 1240-51; Park CH, FASEB J.2006 Dec; 20 (14): 2553-5; Sakurada K, Development.1999 Sep; 126 (18): 4017-26).And transcription factor Pitx3 can regulate the expression of tyrosine hydrolysis enzyme (TH, together following) because of it, and is most important to the growth and the function of midbrain dopamine neuron; The black substance of Pitx3 disappearance mouse have early stage dopamine neuron dysplasia, selectivity lose dopamine neuron ( Simon HH, et al: Ann N Y Acad Sci.2003 Jun; 991:36-47; Smidt MP, et al: Cell Tissue Res.2004 Oct; 318 (1): 35-43; Hwang DY, et al: Brain Res Mol Brain Res.2003 Jun 10; 114 (2): 123-31; Smidt MP, et al: Development.2004 Mar; 131 (5): 1145-55; Maxwell SL, et al: Dev Biol.2005 Jun 15; 282 (2): 467-79; Messmer K, et al: Int J Dev Neurosci.2007Feb; 25 (1): 29-37).Recently the Pitx3 that studies show that has function (the Changgeng Peng that raises neurotrophic factor BDNF and GDNF expression, et al:FEBS Letters, 2007,581 (7): 1357-1361), think that Pitx3 may be the parkinsonian good target gene of treatment.

Recently, method that there are some researches prove the applying transgene neural stem cell, will with Dopamine HCL synthetic genes involved, import in the Parkinson's disease animal model murine brain as tyrosine hydrolysis enzyme (TH) gene or GTP cyclohydrolase gene, can significantly increase the synthetic of Dopamine HCL, and improve symptoms of Parkinson's Disease ( Ryu MY, Cell Transplant.2005; 14 (4): 193-202).

In addition, discover that oxide compound stress is an important amboceptor of Parkinson's disease Dopamine HCL nerve cell death, copper/zinc peroxide dismutase (SOD1, down together) as a kind of crux enzyme, plays crucial effect in polyphenoils stress.Studies have shown that compare with the normal people, Parkinson's disease people's substantia nigra SOD1 obviously reduces; Cell and experimentation on animals show that adenovirus mediated SOD1 has very strong neuroprotective; can effectively prevent methyl-4-phenylpyridiniumion (MPP; together following) and 1-methyl-4-phenyl-1; 2; 3; 6-tetrahydropyridine (MPTP, down together) inductive nervus retrogression process ( Peng JEt al: J Biol Chem.2005 Aug 12; 280 (32): 29194-8; Kunikowska G, et al: Brain brain Res.2003 Apr 11; 968 (2): 206-18; Tatton WG, et al: Ann Neurol.1998 Sep; 44 (3 Suppl 1): S134-41).

More than studies show that above-mentioned cytokine; have anti-apoptotic, neurocyte protection and restore funcitons and synthesize relevant gene as GDNF and family member, CDNG, transcription factor Nurr1 and Pitx3, MANF, BDNF and SOD1 etc. with Dopamine HCL; as tyrosine hydrolysis enzyme (TH) or GTP cyclohydrolase gene etc.; the multiple disease of center neural system there is treatment effect, and synergy is mutually arranged better with the GAD factor.Therefore, based on above research, make up the recombinant virus of a kind of coexpression GAD and above-mentioned cytokine, both can significantly improve relative disease patient's clinical symptom, can stop the further apoptosis of neurocyte again, promoting nervous cell regenerating, is possible with the target that reaches more effective smelting treatment central nervous system relative disease.

Summary of the invention

The objective of the invention is to make up the recombinant virus of a kind of coexpression human GAD and relevant cell factor and utilize its expression product to treat as the central nervous system relative disease described in this specification sheets.

Content of the present invention is the recombinant virus that makes up coexpression GAD and cytokine, and the recombinant adeno-associated virus of preferred coexpression GAD65 and gdnf gene combination (AAV, down together) is an example; The combination of this recombinant virus expressed genes not only includes the coexpression of GAD and GDNF, also comprised GAD gene and other have neuroprotective, short nervous cell regenerating, anti-oxidant, prevent the coexpression that the cytokine gene apoptosis function or synthetic relevant with Dopamine HCL makes up; Applied virus carrier system also is not limited only to AAV virus system, has also comprised and has used the constructed coexpression GAD of other virus carrier systems and the recombinant virus of above-mentioned cytokine.

The present invention is expression vector with AAV, is expression vector with AAV helper-free system more particularly, makes up the reorganization AAV virus that contains coexpression human GAD65 and GDNF expression cassette structure, and its principal character is:

1), the expression system of coexpression of anthropogenic GAD and gdnf gene is a helper-free AAV system;

2), the GAD gene behaviour source GAD65 gene of Biao Daing;

3), the people source GDNF of Biao Daing is the adult form gene, be that its N-end is than 58 amino acid of wild-type people source GDNF disappearance from first to the 58, and the terminal leader that the Human Inter Leukin-2 is arranged that merges of its N-, it is constant that its C-terminal sequence is kept amino acid;

4), the people source GAD65 of coexpression is to be connected by internal ribosome binding site (IRES) fragment with gdnf gene;

5), the expression cassette structure of coexpression of anthropogenic GAD65 and gdnf gene is made of CMV promotor, beta-globin exon, people source GAD65 gene, internal ribosome binding site (IRES) fragment, people source gdnf gene and human growth hormone poly VITAMIN B4 sequence.

Description of drawings

The PCR product of accompanying drawing 1 people source L-Glutamic decarboxylase (GAD) GAD65 gene:

Extract total Yeast Nucleic Acid of cerebral tissue and synthetic cDNA, using artificial synthetic Auele Specific Primer is that template is carried out the PCR reaction with cerebral tissue cDNA, and its reaction product is separated in 1.5% agarose electrophoresis; Clone behind the purifying.First swimming lane is a dna molecular amount marker, and second swimming lane is the big fragment PCR products of GAD65 gene, and the 3rd swimming lane is a GAD65 gene small segment PCR product.Arrow is the PCR reaction product.

The clone of accompanying drawing 2 people source L-Glutamic decarboxylase GAD65 genes:

After RT-PCR reaction, with the PCR fragment cloning of purifying in the T-Easy carrier; After order-checking confirms, small segment PCR product is spliced on big fragment PCR products by correct direction, constitute the amino acid encode fragment that contains total length people source L-Glutamic decarboxylase GAD65 gene, with restriction endonuclease EcoR1 digestion, 1.5% agarose gel electrophoresis separates.First swimming lane is that dna molecular amount marker, second swimming lane are that restriction enzyme mapping, the 3rd swimming lane of the big fragment PCR products T-Easy carrier of GAD65 gene is that restriction enzyme mapping, the 4th swimming lane of GAD65 gene small segment PCR product T-Easy carrier is the segmental restriction enzyme mapping of total length people source L-Glutamic decarboxylase GAD65 aminopeptidase gene acid encoding (the arrow indication is a GAD65 full-length gene fragment).

Accompanying drawing 3 people source neurocyte nutritional factor gdnf gene cDNA fragment (RT-PCR) products: first swimming lane is deoxynucleotide (DNA, a down together) molecular weight standard; The second and the 3rd swimming lane is reverse transcriptase-polymerase chain reaction (RT-PCR) product of this gene.The arrow indication is the position of this gdnf gene cDNA fragment on agarose electrophoresis.

The fusion of accompanying drawing 4 people source gdnf genes and Human Inter Leukin-2 (IL-2) leader: adopt PCR method, with the gdnf gene is template, the nucleotide primer of using 5 '-not terminal amino acid of coding Human Inter Leukin-2 (IL-2) leader polypeptide and GDNF carries out the PCR reaction, finishes the fusion of human-derived neurotrophic factor GDNF and Human Inter Leukin-2 (IL-2) leader polypeptide.First road is that dna molecular amount standard, the second and the 3rd road are PCR product (arrow indication).

Accompanying drawing 5 merges the expression of gdnf gene in intestinal bacteria: behind the fusion gdnf gene procaryotic cell expression plasmid transformation escherichia coli of structure, through abduction delivering, analyze with 12%SDS-PAGE glue.The first road protein molecular weight standard thing; Second road non-inductive e. coli protein product; The 3rd road inductive e. coli protein product.The fusion gdnf protein product of arrow indication for expressing.

Accompanying drawing 6 is expressed the Western Blotting reaction of gdnf protein product: the first road protein molecular weight standard thing; Second road non-inductive e. coli protein product; The 3rd road inductive e. coli protein product.The arrow indication is a gdnf protein matter.

Accompanying drawing 7 coexpression of anthropogenic GAD65 and gdnf gene expression cassette structure iron: from left to right, be respectively that pCMV promotor, beta-globin intron, people GAD65, IRES, people GDNF and human growth hormone poly VITAMIN B4 are formed.

Accompanying drawing 8 contains the recombinant adeno-associated virus genome structure figure of coexpression of anthropogenic GAD65 and people source gdnf gene expression cassette: from left to right, be respectively the expression cassette and the terminal repetition son (ITR) that reverses of counter-rotating terminal repetition son (ITR), coexpression of anthropogenic GAD65 and gdnf gene.

The structure schema of accompanying drawing 9 coexpression of anthropogenic GAD65 and people source GDNF recombinant adeno-associated virus: coexpression human GAD that the present invention makes up and the recombinant adeno-associated virus of GDNF adopt AAV helper-free expression system to make up.The first step at first is clone GAD65 and a gdnf gene from human brain tissue, and does necessary modification; Second step was inserted the pIRES-neo carrier with GAD65 and gdnf gene, made GAD65 connect gdnf gene via IRES; The 3rd step was to be cloned on the carrier of the pAAV-MSC that contains ITR with the dna fragmentation of the GAD65/IRES/GDNF of suitable restriction endonuclease, was built into the pAAV-MSC vector plasmid that contains coexpression GAD65 and GDNF expression cassette; The 4th step then was to use reorganization pAAV carrier, pAAV-RC and the pHelper plasmid co-transfection AAV-293 cell of coexpression GAD65 and GDNF expression cassette to obtain the AAV virion of coexpression human GAD65 and GDNF.

Concrete enforcement

Make up the specific implementation process of the recombinant virus of coexpression of anthropogenic GAD and people source GDNF expression structure for the ease of narration, the present invention adopts adeno-associated virus (AAV, expression system down together), more particularly adopt helper-free AAV expression system, make up the AAV recombinant virus of coexpression GAD and GDNF expression structure.

The AAV recombinant virus specific implementation process that makes up coexpression GAD and GDNF expression structure is as follows:

One, the clone of people source L-Glutamic decarboxylase GAD65 gene:

1). consider that the GAD65 gene is bigger, decision is divided into two fragments with this gene clones, and then utilizes suitable restriction enzyme site, finishes the splicing of GAD65 full-length gene.

At first extract the total Yeast Nucleic Acid of cerebral tissue (total RNA), and utilize Poly (T) primer synthetic cerebral tissue cDNA under the ThermoScript II effect; Then with human brain tissue cDNA as template, using artificial synthetic Auele Specific Primer is that template is carried out PCR reaction with cerebral tissue cDNA, produces two PCR fragments that vary in size respectively.

Design of primers is as follows:

A: the N-end that produces GAD65 is to the segmental primer in the terminal restriction endonuclease Stu1 site of C-,

5’-ccggatccg atggcatctccgggctctggcttttgg-3’

5’-tgtaaggccttgtctccagtgtcatag-3’

B. the restriction endonuclease Stu1 site that produces GAD65 is to the synthetic segmental primer of terminator of C-end amino acid

5’-gacaaggccttacagtgcggacgc-3’

5’-gagcggccgctctagattataaatcttgtccaaggcgttctatt-3’

Pcr amplification product is respectively through tailing reaction, add VITAMIN B4 (T) after, be connected to the T-easy carrier, behind the transform bacteria, amplification, extracting, purifying are determined through dna sequencing;

2). after dna sequencing was correct, application restriction endonuclease Stu1 and Sca1 digested respectively and contain the segmental T-easy plasmid of above-mentioned PCR; Contain the fragment of above-mentioned PCR with electrophoretic separation, purifying, use the ligase reaction, the fragment assembly of these two PCR; Then, the transformed competence colibacillus bacterium is increased, and finishes the clone of total length people source GAD65 gene;

Two, people's gdnf gene clone and N thereof do not hold the fusion with Human Inter Leukin-2's leader:

The step of the complementary DNA Nucleotide (cDNA) of synthetic human brain tissue is the same; Then with human brain tissue cDNA as template, 2 forward primers of using artificial synthetic, promptly 1. contain the amino acid whose nucleotide primer of Human Inter Leukin-2 guide of partly encoding, 2. contain primer and 1 reverse primer of the nucleotide sequence of partly the encode amino acid whose nucleotide sequence of Human Inter Leukin-2 guide and people's gdnf gene-terminal amino acid, be that people source gdnf gene C-terminal nucleotide sequence is carried out pcr amplification, adopt two to go on foot PCR methods human cloning source gdnf gene, and finish its N and do not hold the reconstruction of merging with Human Inter Leukin-2's leader.

The forward primer design is as follows:

1、5’-tcgagctcaggaggaaacgccatggagaaagaaacctggtgggaaacctggtggaccg-3’

2、5’-cctggtggaccgaatggtctcagccgaaaaaaaaacgtaaagtgtcaccagataaacaaatgg-3’

The reverse primer design is as follows:

5’-tcaagcttcagatacatccacaccttt-3’

Pcr amplification product reacts through tailing, add VITAMIN B4 (T) after, be connected to the T-easy carrier, behind the transform bacteria, amplification, extracting, purifying are determined through dna sequencing.

Three, the structure that contains the pAAV-MCS carrier of coexpression GAD65 and GDNF expression cassette:

1), uses the multiple clone site of pAAV-MCS carrier in the AAV helper-free expression system, with the GAD65 gene with restriction endonuclease BamH1 and Xbal1 nucleotide fragments, purified and carry out ligation with pAAV-MCS carrier that BamH1 and Xbal1 enzyme are cut digestion, purifying after, the transformed competence colibacillus bacterium, increase, and be coated on and contain on the suitable antibiotic culture dish 37 ℃ of incubator overnight growth;

2), a plurality of clone's bacterium colonies of screening are in containing suitable antibiotic nutrient solution, 37 ℃ of concussion overnight incubation, in extraction in second day, purifying pAAV-MCS plasmid DNA, and cut digestion with BamH1 and Xbal1 enzyme, with the plasmid DNA and the clone strain of the pAAV-MCS carrier of the GAD65 gene fragment of determining to contain BamH1 and Xbal1;

3), determine to contain after the pAAV-MCS of GAD65 gene fragment, contain the pAAV-MCS plasmid DNA of GAD65 gene with restriction endonuclease Hind111 and Bgl11 complete digestion, 1% agarose separates, purifying is standby;

4), for obtaining the IRES fragment, with restriction endonuclease Hind111 and Smal1 complete digestion pIRESneo3 carrier DNA (CloneTech.Lab.Inc), 1% agarose separates, purifying IRES fragment is standby;

5), contain the segmental T-easy carrier DNA of gdnf gene with restriction endonuclease Smal1 and Bgl11 complete digestion, 1% agarose separates, purifying gdnf gene fragment is standby;

6), the IRES fragment of above-mentioned restriction endonuclease Hind111 and Smal1 digestion and the gdnf gene fragment of restriction endonuclease Smal1 and Bgl11 digestion are reacted through ligase, be spliced on the pAAV-MCS vector plasmid DNA that contains the GAD65 gene of above-mentioned restriction endonuclease Hind111 and Bgl11 complete digestion, the transformed competence colibacillus bacterium, cultivate amplification for 37 ℃, and be coated on and contain on the suitable antibiotic culture dish 37 ℃ of incubator overnight growth;

7), a plurality of clone's bacterium colonies of screening are in containing suitable antibiotic nutrient solution, 37 ℃ of concussion overnight incubation, in extraction in second day, purifying pAAV-MCS plasmid DNA, and with above-mentioned restriction endonuclease digestion pAAV-MCS plasmid DNA, determine to contain the pAAV-MCS plasmid of GAD65 gene, IRES fragment and gdnf gene;

Four, the preparation of the adeno-associated virus of coexpression of anthropogenic GAD65 and gdnf gene:

The method that the AAV Helper Free system specialization handbook that the preparation of adeno-associated virus provides by Stratagene company is described is carried out.Specific operation process is as follows:

1), in transfection preceding 48 hours, about 300 ten thousand the AAV-293 cells of inoculation in 100 millimeters culture dish that add 10ml DMEM grown cultures liquid, cell density should be 70 ~ 80% after 2 days;

2), pAAV-MCS, pAAV-RCH and the pHelper plasmid DNA of thawing and containing above-mentioned expression cassette, adjusting every kind of plasmid DNA with the TE damping fluid of pH7.5 is 1mg/ml;

3), in 15 milliliters of culture tubes, from above-mentioned 3 kinds of plasmid solution, respectively get 10 microlitre dna solutions, add 1 milliliter of 0.4M CaCl again ₂, mixing gently;

4), in another 15 milliliters of culture tubes, add 1 milliliter of 2x HBS solution, again will on reach 3) in 1.03 milliliters DNA/CaCl ₂Splash in 1 milliliter of 2x HBS solution, the reversing culture tube is mixing repeatedly;

5), immediately with DNA/CaCl ₂/ HBS suspension splashes in the AAV-293 Tissue Culture Dish, and it is evenly distributed in the nutrient solution;

6), the AAV-293 Tissue Culture Dish was put into 37 ℃ of incubators 6 hours, the nutrient solution in the subsequent removal AAV-293 Tissue Culture Dish adds 10 milliliters of fresh DMEM grown cultures liquid, cultivates 72 hours in 37 ℃ of incubators;

7), observe after 3 days, as observe the nutrient solution colour-change or see having cellular form to become the garden, disengaging plate face or swim in the nutrient solution, then showing has reorganization AAV virion to produce;

8), this moment collecting cell nutrient solution and collecting cell, freeze/melt method, lysing cell by 4 times, the AAV virus of recombinating in its born of the same parents is discharged, at last at room temperature with 10, centrifugal 10 minutes of 000Xg, supernatant liquor is transferred in another new pipe, is stored in-80 ° of refrigerators and preserves.This is recombinant adeno-associated virus (AAV) mother liquor of expressing human source GAD65 and gdnf gene, and next step can carry out the detection of virus amplification, virus titer detection and GAD65 and gdnf gene expression product.

Sequence table

Individual?Applicant

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Country: China

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PhoneNumber：020-32290208

FaxNumber：020-38491559

EmailAddress：shangwu_w@yahoo.com

＜110〉LastName: king

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Application?Project

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＜120〉Title: the recombinant virus of a kind of coexpression of anthropogenic glutamate decarboxylase and cytokine

<130>AppFileReference：

<140>CurrentAppNumber：1

<141>CurrentFilingDate：2008-05-19

Sequence

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<210>1

<211>1792

<212>DNA

＜213〉people source GAD65 artificial sequence

<400>1

gctgaattcc?cggatccgat?ggcatctccg?ggctctggct?tttggtcttt?cgggtcggaa 60

gatggctctg?gggattccga?gaatcccggc?acagcgcgag?cctggtgcca?agtggctcag 120

aagttcacgg?gcggcatcgg?aaacaaactg?tgcgccctgc?tctacggaga?cgccgagaag 180

ccggcggaga?gcggcgggag?ccaacccccg?cgggccgccg?cccggaaggc?cgcctgcgcc 240

tgcgaccaga?agccctgcag?ctgctccaaa?gtggatgtca?actacgcgtt?tctccatgca 300

acagacctgc?tgccggcgtg?tgatggagaa?aggcccactt?tggcgtttct?gcaagatgtt 360

atgaacattt?tacttcagta?tgtggtgaaa?agtttcgata?gatcaaccaa?agtgattgat 420

ttccattatc?ctaatgagct?tctccaagaa?tataattggg?aattggcaga?ccaaccacaa 480

aatttggagg?aaattttgat?gcattgccaa?acaactctaa?aatatgcaat?taaaacaggg 540

catcctagat?acttcaatca?actttctact?ggtttggata?tggttggatt?agcagcagac 600

tggctgacat?caacagcaaa?tactaacatg?ttcacctatg?aaattgctcc?agtatttgtg 660

cttttggaat?atgtcacact?aaagaaaatg?agagaaatca?ttggctggcc?agggggctct 720

ggcgatggga?tattttctcc?cggtggcgcc?atatctaaca?tgtatgccat?gatgatcgca 780

cgctttaaga?tgttcccaga?agtcaaggag?aaaggaatgg?ctgctcttcc?caggctcatt 840

gccttcacgt?ctgaacatag?tcatttttct?ctcaagaagg?gagctgcagc?cttagggatt 900

ggaacagaca?gcgtgattct?gattaaatgt?gatgagagag?ggaaaatgat?tccatctgat 960

cttgaaagaa?ggattcttga?agccaaacag?aaagggtttg?ttcctttcct?cgtgagtgcc 1020

acagctggaa?ccaccgtgta?cggagcattt?gaccccctct?tagctgtcgc?tgacatttgc 1080

aaaaagtata?agatctggat?gcatgtggat?gcagcttggg?gtgggggatt?actgatgtcc 1140

cgaaaacaca?agtggaaact?gagtggcgtg?gagagggcca?actctgtgac?gtggaatcca 1200

cacaagatga?tgggagtccc?tttgcagtgc?tctgctctcc?tggttagaga?agagggattg 1260

atgcagaatt?gcaaccaaat?gcatgcctcc?tacctctttc?agcaagataa?acattatgac 1320

ctgtcctatg?acactggaga?caaggcctta?cagtgcggac?gccacgttga?tgtttttaaa 1380

ctatggctga?tgtggagggc?aaaggggact?accgggtttg?aagcgcatgt?tgataaatgt 1440

ttggagttgg?cagagtattt?atacaacatc?ataaaaaacc?gagaaggata?tgagatggtg 1500

tttgatggga?agcctcagca?cacaaatgtc?tgcttctggt?acattcctcc?aagcttgcgt 1560

actctggaag?acaatgaaga?gagaatgagt?cgcctctcga?aggtggctcc?agtgattaaa 1620

gccagaatga?tggagtatgg?aaccacaatg?gtcagctacc?aacccttggg?agacaaggtc 1680

aatttcttcc?gcatggtcat?ctcaaaccca?gcggcaactc?accaagacat?tgacttcctg 1740

attgaagaaa?tagaacgcct?tggacaagat?ttataatcta?gagcggccgc?tc 1792

<210>2

<211>585

<212>PRT

＜213〉people source GAD65 aminoacid sequence

<400>2

MetAlaSerProGlySerGlyPheTrpSerPheGlySerGluAspGlySerGlyAspSer

5 10 15 20

GluAsnProGlyThrAlaArgAlaTrpCysGlnValAlaGlnLysPheThrGlyGlyIle

25 30 35 40

GlyAsnLysLeuCysAlaLeuLeuTyrGlyAspAlaGluLysProAlaGluSerGlyGly

45 50 55 60

SerGlnProProArgAlaAlaAlaArgLysAlaAlaCysAlaCysAspGlnLysProCys

65 70 75 80

SerCysSerLysValAspValAsnTyrAlaPheLeuHisAlaThrAspLeuLeuProAla

85 90 95 100

CysAspGlyGluArgProThrLcuAlaPhcLcuGlnAspValMctAsnIlcLcuLcuGln

105 110 115 120

TyrValValLysSerPheAspArgSerThrLysValIleAspPheHisTyrProAsnGlu

125 130 135 140

LeuLeuGlnGluTyrAsnTrpGluLeuAlaAspGlnProGlnAsnLeuGluGluIleLeu

145 150 155 160

MetHisCysGlnThrThrLeuLysTyrAlaIleLysThrGlyHisProArgTyrPheAsn

165 170 175 180

GlnLeuSerThrGlyLeuAspMetValGl?yLeuAlaAlaAspTrpLeuThrSerThrAla

185 190 195 200

AsnThrAsnMetPheThrTyrGluIleAlaProValPheValLeuLeuGluTyrValThr

205 210 215 220

LeuLysLysMetArgGluIleIleGlyTrpProGlyGlySerGlyAspGlyIlePheSer

225 230 235 240

ProGlyGlyAlaIleSerAsnMetTyrAlaMetMetIleAlaArgPheLysMetPhePro

245 250 255 260

GluValLysGluLysGlyMetAlaAlaLeuProArgLeuIleAlaPheThrSerGluHis

265 270 275 280

SerHisPheSerLeuLysLysGlyAlaAlaAlaLeuGlyIleGlyThrAspSerValIle

285 290 295 300

LeuIleLysCysAspGluArgGlyLysMetIleProSerAspLeuGluArgArgIleLeu

305 310 315 320

GluAlaLysGlnLysGlyPheValProPheLeuValSerAlaThrAlaGlyThrThrVal

325 330 335 340

TyrGlyAlaPheAspProLeuLeuAlaValAlaAspIleCysLysLysTyrLysIleTrp

345 350 355 360

MetHisValAspAlaAlaTrpGlyGlyGlyLeuLeuMetSerArgLysHisLysTrpLys

365 370 375 380

LeuSerGlyValGluArgAlaAsnSerValThrTrpAsnProHisLysMetMetGlyVal

385 390 395 400

ProLeuGlnCysSerAlaLeuLeuValArgGluGluGlyLeuMetGlnAsnCysAsnGln

405 410 415 420

MetHisAlaSerTyrLeuPheGlnGlnAspLysHisTyrAspLeuSerTyrAspThrGly

425 430 435 440

AspLysAlaLeuGlnCysGlyArgHisValAspValPheLysLeuTrpLeuMetTrpArg

445 450 455 460

AlaLysGlyThrThrGlyPheGluAlaHisValAspLysCysLeuGluLeuAlaGluTyr

465 470 475 480

LeuTyrAsnIleIleLysAsnArgGluGlyTyrGluMetValPheAspGlyLysProGln

485 490 495 500

HisThrAsnValCysPheTrpTyrIleProProSerLeuArgThrLeuGluAspAsnGlu

505 510 515 520

GluArgMetSerArgLeuSerLysValAlaProValIleLysAlaArgMetMetGluTyr

525 530 535 540

GlyThrThrMetValSerTyrGlnProLeuGlyAspLysValAsnPhePheArgMetVal

545 550 555 560

IleSerAsnProAlaAlaThrHisGlnAspIleAspPheLeuIleGluGluIleGluArg

565 570 575 580

LeuGlyGlnAspLeu

585

<210>3

<211>494

<212>DNA

＜213〉GDNF artificial sequence

<400>3

atgtacagga?tgcaactcct?gtcttgcatt?gcactaagtc?ttgcacttgt?cacaaacagt 60

ggatcaccag?ataaacaaat?ggcagtgctt?cctagaagag?agcggaatcg?gcaggctgca 120

gctgccaacc?cagagaattc?cagaggaaaa?ggtcggagaa?gccagagggg?caaaaaccgg 180

ggttgtgtct?taactgcaat?acatttaaat?gtcactgact?tgggtctggg?ctatgaaacc 240

aaggaggaac?tgatttttag?gtactgcagc?ggctcttgcg?atgcagctga?gacaacgtac 300

gacaaaatat?tgaaaaactt?atccagaaat?agaaggctgg?tgagcgacaa?agtagggcag 360

gcatgttgca?gacccatcgc?ctttgatgat?gacctgtcgt?ttttagatga?taacctggtt 420

taccatattc?taagaaagca?ttccgctaaa?aggtgtggat?gtatctgaaa?gcttgcatca 480

ctagtgaatt?cgcg 494

<210>4

<211>206

<212>PRT

＜213〉GDNF artificial sequence

<400>4

MetTyrArgMetGlnLeuLeuSerCysIleAlaLeuSerLeuAlaLeuValThrAsnSer

5 10 15 20

GlySerProAspLysGlnMetAlaValLeuProArgArgGluArgAsnArgGlnAlaAla

25 30 35 40

AlaAlaAsnProGluAsnSerArgGlyLysGlyArgArgSerGlnArgGlyLysAsnArg

45 50 55 60

GlyCysValLeuThrAlaIleHisLeuAsnAlaThrAspLeuGlyLeuGlyTyrGluThr

65 70 75 80

LysGluGluLcuIlcPhcArgTyrCysScrGlyScrCysAspAlaAlaGluThrThrTyr

85 90 95 100

AspLysIleLeuLysAsnLeuSerArgAsnArgArgLeuValSerAspLysValGlyGln

105 110 115 120

AlaCysCysArgProIleAlaPheAspAspAspLeuSerPheLeuAspAspAsnLeuVal

125 130 135 140

TyrHisIleLeuArgLysHisSerAlaLysArgCysGlyCysIle

1451 50 155

Claims

1, a kind of coexpression of anthropogenic glutamate takes off shuttle enzyme (GAD, down with) and the recombinant virus of human cell factor, especially coexpression of anthropogenic GAD65 and glial cell line-derived neurotrophic factor (GDNF, recombinant adeno-associated virus (AAV) down together), be primarily characterized in that, recombinant virus contains the expression cassette structure of coexpression human GAD65 and human cell factor, described expression cassette structure is made up of pCMV promotor, beta-globin intron, people source GAD, internal ribosome binding site (IRES, down together), the human archeocyte factor and human growth hormone poly VITAMIN B4 signal.

2, according to claim 1, described recombinant virus, the recombinant adeno-associated virus (AAV) of preferred coexpression of anthropogenic GAD65 and glial cell line-derived neurotrophic factor (GDNF, together following) is characterized in that the expression cassette structure of structure is as follows:

Upstream, beta-globin intron that a, pCMV promotor are positioned at the beta-globin intron splice in the N-end of people GAD65 gene with restriction endonuclease BamH1 at its C-end;

The C-end of b, people GAD65 gene splices the segmental N-end in IRES with restriction endonuclease Xbal1;

The segmental C-end of c, IRES splices the end in the N-of people's gdnf gene with restriction endonuclease Smal1;

The C-end of d, people's gdnf gene splices in the upstream of human growth hormone poly VITAMIN B4 signal segment with restriction endonuclease Bgl11.

3, according to claim 1, described recombinant virus, especially the people source GAD65 of recombinant adeno-associated virus institute coexpression and the Nucleotide and the encoding amino acid sequence thereof of gdnf gene:

A, people source GAD65 for the parent come from the people gene, be coding GAD65 full length gene amino acid whose nucleotide sequence;

B, people source GDNF for the parent come from people's gene, for the amino acid whose nucleotide sequence of encoding mature type GDNF, and its N-does not hold and has merged the secretion leader that 20 amino acid by the Human Inter Leukin-2 constitute, substituted preceding 58 aminoacid sequences of proteic first amino acid to the of GDNF, its C-terminal aminoacid sequence is constant.

4, according to claim 1, described recombinant virus, especially recombinant adeno-associated virus (AAV) contains the expression cassette structure of coexpression of anthropogenic GAD65 and the combination of people GDNF cytokine gene, this expression cassette structure expressed genes combination can be GAD and other neuroprotectives, short nervous cell regenerating, prevent the cytokine of apoptosis function, as a kind of formed assortment of genes in the genes such as transcription factor Nurr1 and pitx3, midbrain stellate cell derived neurotrophic factor (MANF), CDNG, Neurturin (NTN) and BDNF; The GAD and the assortment of genes anti-oxidant, Green Tea Extract have also been comprised, as SOD1; Can also be GAD cytokine tyrosine hydrolysis enzyme (TH) gene synthetic relevant or the coexpression of a kind of formed assortment of genes in the GTP cyclohydrolase gene with Dopamine HCL.

5, according to claim 1 and claim 4, recombinant virus, the assortment of genes of preferred recombinant adeno-associated virus (AAV) institute coexpression, this assortment of genes expression cassette structure that both the present invention narrated, promptly connect the expression cassette structure that described coexpression of anthropogenic glutamate takes off shuttle enzyme GAD and above-mentioned cytokine coexpression through internal ribosome binding site IRES by a promoters driven, also can be the genetic expression that in same recombinant virus structure, drives the said gene combination respectively by two promotors identical or inequality, these promotors both can be the promotors that comes from virus, also can be the gene promoters that stems from the people.

6, according to claim 1, described recombinant virus is not limited only to AAV virus, also comprised using other viruses, as adenovirus, slow virus, herpes virus and retrovirus as the constructed coexpression GAD of carrier and the recombinant virus of above-mentioned cytokine.

7, according to claim 1 and claim 2, human cloning applied method of GA65 gene and design of primers, the two-step pcr method that promptly adopts human cloning GA65 gene and design of primers.

Concrete operations are as follows: as template, using artificial synthetic Auele Specific Primer carries out PCR reaction, produces two PCR fragments that vary in size with human brain tissue cDNA, and pcr amplification product is cloned and tested after the tailing reaction, splices after correctly.

The primer of design is as follows:

A: the N-end that produces GAD65 is to the segmental primer in the terminal restriction endonuclease Stu1 site of C-

5’-ccggatccg atggcatctccgggctctggcttttgg-3’

5’-tgtaaggccttgtctccagtgtcatag-3’

B. produce the primer of the restriction endonuclease Stu1 site of GAD65 to the synthetic terminator of C-end amino acid

5’-gacaaggccttacagtgcggacgc-3’(stu1)

5’-gagcggccgctctagattataaatcttgtccaaggcgttctatt-3’。

8, according to claim 1 and claim 2, clone and terminal applied method of reconstruction people gdnf gene N-and design of primers, the method that promptly adopts two-step pcr method clone and reconstruct people's gdnf gene N-end.

Concrete operations are as follows: use 2 forward primers of synthetic, promptly (1), contain the amino acid whose nucleotide primer of Human Inter Leukin-2 guide of partly encoding; (2), contain the partly primer of the nucleotide sequence of the-terminal amino acid of the amino acid whose nucleotide sequence of Human Inter Leukin-2 guide and people GDNF; And 1 reverse primer, promptly people source gdnf gene C-terminal nucleotide sequence is that template is carried out pcr amplification with human brain cDNA, adopts two to go on foot PCR methods human cloning source gdnf gene, and finishes its N and do not hold the reconstruction of merging with Human Inter Leukin-2's leader.

Two forward primer designs are as follows:

a、5’-tcgagctcaggaggaaacgccatggagaaagaaacctggtgggaaacctggtggaccg-3’

b、5′-cctggtggaccgaatggtctcagccgaaaaaaaaacgtaaagtgtcaccagataaacaaatgg-3′

The reverse primer design is as follows:

c、5’-tcaagcttcagatacatccacaccttt-3’

For the second time pcr amplification product reacts through tailing, add VITAMIN B4 (T) after, be connected to the T-easy carrier, behind the transform bacteria, amplification, extracting, purifying are determined through dna sequencing.

9, according to claim 1, described coexpression of anthropogenic glutamate takes off the recombinant virus of shuttle enzyme GAD and cytokine, the recombinant adeno-associated virus that preferred coexpression of anthropogenic glutamate takes off shuttle enzyme GAD65 and GDNF is especially treated the application in the parkinsonian gene therapy medicament being used for the treatment of the central nervous system relative disease.

10, coexpression of anthropogenic glutamate according to claim 9 takes off the recombinant virus of shuttle enzyme GAD and cytokine, the application of preferred recombinant adeno-associated virus in the gene therapy medicament of treatment central nervous system relative disease is characterized in that: the effective active composition behaviour source L-glutamic acid of its treatment central nervous system relative disease takes off the marking protein of shuttle enzyme GAD and cytokine gene.