CN102242150A - Heat shock protein-GP96 (HSP-GP96) recombinant adenovirus vector and construction method and application thereof - Google Patents
Heat shock protein-GP96 (HSP-GP96) recombinant adenovirus vector and construction method and application thereof Download PDFInfo
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Abstract
The invention provides a heat shock protein-GP96 (HSP-GP96) recombinant adenovirus vector. A construction method comprises the following steps of: inserting a human GP96 gene segment into a p-shuttle cytomegalovirus (CMV) shuttle vector by a genetic engineering technology, converting BJ5183-AD-1, screening a correct recombinant adenovirus plasmid, linearizing, transfecting an AD293 cell, packaging in the AD293 cell and massively amplifying and purifying. An HSP-GP96 leukemia peptide complex is obtained by infecting a leukemia cell with the HSP-GP96 recombinant adenovirus vector, so that the HSP-GP96 recombinant adenovirus vector is applied to the preparation of a tumor vaccine complex for treating leukemia; and the prepared complex can be applied to the elimination of leukemia minimal residual disease, is expected to achieve a treatment effect equivalent to that of allogeneic bone marrow transplantation, can save treatment cost, and obviously reduces medical treatment cost.
Description
Technical field
The invention belongs to biological technical field, relate to the structure and the application of HSP-GP96 recombinant adenovirus, be to utilize molecule clone technology, comprise that pcr amplification gene, enzyme are cut and insert to connect etc., the HSP-GP96 recombinant adenoviral vector that acquisition can the multiple leukemia cell of transfection.Then adopt the protein purification technology, extracted HSP-GP96 leukemia peptide complex from leukemia cell line K562, behind the loaded dendritic cell (DC), inquired into this mixture to leukemia cell K562, the influence of HL60 and U937 immunologic cytotoxicity and DC function has been established experiment basis for further developing the HSP tumor vaccine.
Background technology
Because the small residual existence of tumour makes tumour be difficult to radical cure, so begin to pay attention to the immunotherapy of tumour in the world.But how improving the specificity of tumor biotherapy, how to induce and activate the tumour-specific immunologic function, is the problem that presses for solution.At present, it is known having only the minority tumour specific antigen, as melanoma.Most of tumour does not find specific antigens, and tumour cell have highly heterogeneous, if only be difficult to bring into play best antitumor action at single antigenic determinat.(Heat shock protein HSP) is the class stress protein with high conservative to heat shock protein(HSP), and the HSP that extracts from tumor tissues can induce body to produce the restrictive immune response of cytotoxic T lymphocyte of antineoplastic mhc class i and reply.HSP is as whole antigens of " molecular chaperones " portability tumour cell, need not separation, purifying, the entrained tumour specific antigen of evaluation, have polyvalency, specificity, convenience with its tumor vaccine of making, and therefore become the focus of recent research.(dendritic cell DC) has vital role to dendritic cell in antigen presentation, it is to activate T cells and keep the most important antigen presenting cell of cellular immunization.At present few to the correlative study between HSP-GP96 peptide and the DC function, and the HSP-GP96 leukemia peptide complex that leukemia is originated does not appear in the newspapers especially to the influence of DC.We adopt the protein purification technology, from leukemia cell line K562, extracted HSP-GP96 leukemia peptide complex among HL60 and the U937, inquired into leukemia cellular immunization is killed and wounded influence with the DC function, for further development HSP tumor vaccine has been established experiment basis.
The present invention utilizes molecule clone technology, comprises that pcr amplification gene, enzyme are cut insert to connect etc.Acquisition can the multiple leukemia cell of transfection the HSP-GP96 recombinant adenoviral vector.Then adopt the protein purification technology, extracted HSP-GP96 leukemia peptide complex from leukemia cell line K562, inquired into this mixture to leukemia cell K562, the influence of HL60 and U937 immunologic cytotoxicity and DC function has been established experiment basis for further developing the HSP tumor vaccine.
Summary of the invention
The purpose of this invention is to provide the recombinant adenoviral vector of a kind of heat shock protein(HSP)-GP96(HSP-GP96), the nucleotide sequence of described carrier such as SEQ ID NO:1.
Another object of the present invention provides above-mentioned construction of carrier, obtains by following steps:
(1) primer, pcr template and amplification gene sequence
Design upstream primer P1(SEQ ID NO:2): 5 '-GCAGATCT atgaagttgatgtgg atg-3 '; Downstream primer P2(SEQ ID NO:3): 5 '-CGAAGCTT ttacaattcatctttttC-3 ', upstream primer 5 ' end comprises the BgLII enzyme and cuts sequence A GATCT, and downstream primer 5 ' end comprises Hind III enzyme and cuts sequence A AGCTT.Amplification HSP-GP96 gene order (being primer sequence) is got the 5ml amplified production, and electrophoresis confirms to have expection length fragment (about 2420bp) in 1% sepharose;
(2) make up PSC-GP96 shuttle vectors
With BgLII and Hind III double digestion PSC plasmid and GP96 gene PCR purifying thing, after cutting connection, enzyme obtains reorganization PSC-GP96 shuttle vectors, carry out the amplification and the evaluation of plasmid;
A. use BgLII and Hind III double digestion PSC plasmid and HSP-GP96 gene PCR purifying thing, after enzyme is cut connection, obtain reorganization PSC-GP96 shuttle vectors;
B. prepare DH
The competence bacterium, get 10 ml connection product and 1ml control plasmid and add 100 ml DH5a competent cells, purchase a uncommon lucky Bioisystech Co., Ltd in Shanghai, conversion through PSC-GP96 recombinant plasmid and control plasmid PSC-LacZ, carry out the screening of positive colony and recombinant plasmid enzyme cutting method then and identify, have and insert the fragment person of discharging about 2420bp and be considered as positive recombinant chou;
(3) structure of adenovirus carrier HSP-GP96 and adenovirus carrier LacZ
The A.PmeI enzyme is cut and is made PSC-GP96 and PSC-LacZ linearizing, respectively get 3 ml1,0.75% agarose gel electrophoresis, conclusive evidence PSC-GP96 and PSC-LacZ linearizing fully, surplus enzyme is cut product StrataPrep PCR purification kit purifying, last respectively with 20 ml water elution DNA, the row electricity transforms again;
The evaluation of B. positive recombinant adenoviral vector plasmid, the Ad-GP96 recon through kalamycin resistance,
The AdLacZ plasmid obtains positive colony after kantlex and Lan Bai bacterium colony preliminary screening, with amount extracting recombinant adenovirus plasmid DNA in the alkaline lysis, the plasmid that extracting is obtained is transformed into respectively in common DH5a competence bacterium and the XL10-Gold competence bacterium, extract plasmid DNA with in a small amount ultrapure plasmid DNA extraction agent box, 0.75% gel electrophoresis shows AdGP96 and Lamda/HindIII Marker relatively, and main fragment is done PacI single endonuclease digestion, BgLII and HindIII double digestion and PCR evaluation the above person of 23kb; AdLacZ only makes the PacI enzyme and cuts evaluation;
C. the recombinant adenovirus plasmid enzyme cutting method is identified, with Hind III and BgLII double digestion 7 ml recombinant adenovirus plasmid DNA, enzyme is cut after product and got 8ml electrophoresis in 0.75% sepharose, and having has the fragment person of discharging to be considered as positive recombinant chou about 2420bp; Pac I enzyme is cut the 7ml recombinant adenovirus plasmid, gets 8ml electrophoresis in 0.75% sepharose after enzyme is cut, if having 4.5kb or the 3.0kb fragment person of discharging to be considered as positive recombinant chou;
The preliminary evaluation of D.HSP-GP96 recombinant adenoviral vector plasmid packing and s-generation infective virus, HSP-GP96 recombinant adenoviral vector plasmid is packaged in the AD293 cell, the plasmid DNA cell transfecting with former generation virus infection AD293 cell, obtains a large amount of s-generation viruses.
A further object of the present invention provides the application of described carrier in the preparation knurl seedling mixture residual at leukemia.Be to utilize the HSP-GP96 recombinant adenoviral vector to infect the leukemia cell, obtain HSP-GP96 leukemia peptide complex, thus the residual knurl seedling mixture of preparation treatment leukemia.Realize by following steps:
(1)
The purifying amplification of HSP-GP96 recombinant adenovirus: behind people GP96 gene fragment insertion p-shuttle CMV shuttle vectors, transform BJ5183-AD-1 (pre-inversion adenovirus skeleton plasmid pAdEasy-1), make it that homologous recombination take place in thalline, screen correct recombinant adenovirus plasmid and with transfection AD293 cell after its linearizing, in AD293 cell internal packing and a large amount of amplification, behind the cesium chloride ultracentrifugation, obtain the AD-GP96 recombinant adenovirus of a large amount of high titres;
(2)
The preparation of HSP-GP96 leukemia peptide complex and leukemia peptide and evaluation:AD-GP96 is infected 1 * 10 with MOI=100
10Human leukemia cell line K562, HL-60, U937, another group does not add AD-GP96 and infects, and after 1 week the leukemia cell is added the abundant mixing of lysate, after the lysis in 4 ℃, centrifugal 30 min of 2 000 * G, the sucking-off supernatant, 4 ℃, centrifugal 15 min of 15000G; The sucking-off supernatant is through 50%, 70% saturated ammonium sulphate; Throw out filters with concanavalin A affinity chromatography column chromatography, then with containing 10% after the balance liquid of 5 times of column volumes is resuspended
The balance liquid of-methyl sweet dew pyrans carries out wash-out, collects elutriant; By the DEAE ion exchange column, use the balance liquid wash-out of 300-1000mMol/L NaCl salt ion then respectively, collect the elutriant of each ion gradient, obtain GP96-leukemia antigen peptide and leukemia antigen peptide respectively, carry out quantitatively with the uv-spectrophotometric instrument after the filtration sterilization;
(3)
Human dendritic cell (DC) is cultivated and is identified:Isolate peripheral blood mononuclear cell (PBMNC) with normal people's lymphocyte separation medium, suspend with RPMI 1640 complete culture solutions, the adjustment cell concn is 1 * 106/ml, be seeded in the culture dish of the RPMI 1640 complete culture solution 5ml that contain 10% FCS, get adherent cell after in 37 ℃, 5 % CO2 incubators, hatching 2 hours and add GM-CSF 500 U/ml, IL-2 500 U/ml, cultivate 2 groups of the 7th natural gift and carry out DC and cultivate: (1) leukemia antigen peptide-DC group: add leukemia antigen peptide 5 μ g/ml; (2) HSP GP96-leukemia antigen peptide-DC group adds the HSP GP96 5 μ g/ml that extract.More than two groups all put into 37 ℃, 5 % CO2 incubators and hatch cultivation, amount was changed liquid 1 time in per 4 days half, cultivated to add TN F-on the 7th day
50 U/ml, the 10th day collection suspension cell.Use CD80, CD86, marks such as CD1a and HLA-DR carry out identification and analysis;
(4)
Stimulate allosome T lymphocyte proliferation assay:Peripheral blood mononuclear cell is cultivated the T lymphocyte that obtains in 96 orifice plates, cultivate (containing the IL-2 that final concentration is 200 U/ml in the nutrient solution), be provided with 5 groups: leukemia cell's group; Leukemia peptide complex group; HSP-GP96 leukemia peptide complex group; Leukemia peptide complex-DC group; HSP-GP96 leukemia peptide complex-DC group.Each group is all with 5
After/ml concentration stimulates 48h, cell piping and druming is suspended, 100 μ l cell suspensions are taken out in every hole, and adding 100 μ l again, to contain volume fraction be that RPMI 1640 substratum of 10% calf serum, 200 U/ml IL-2 continue to go down to posterity cultivations in 96 orifice plates, detects with mtt assay behind cultivation 5 d that go down to posterity;
(5)
NK cellular segregation purifying and active the detection:Per 10
7Mononuclearcell add 80 μ l damping fluids and the anti-CD56 immunomagnetic beads of 20 μ l, after 4 ℃ of lucifuges are hatched 15 minutes, centrifugal 10 minutes of 300 r/min, remove supernatant, add and carry out magnetic bead after the damping fluid of 500 μ l and separate, and the cell of crossing post acquisition CD3-CD56+ is the NK cell, the action effect cell.5 groups is target cell with K562, HL-60, three kinds of leukemia cell lines of U937 respectively.Respectively the effector cell: target cell is 20:1, and 10:1 during 5:1, adopts LDH release to birth ratio color method to detect the NK cell activity, and the result gets the mean value of three experiments;
(6)
CTL is active to be detected:With above-mentioned 5 groups with
/ ml concentration, cultivated for 1 week altogether with the CTL precursor cell respectively after, collect CTL cell action effect cell, each group is all with k562, HL-60, three kinds of leukemia cell lines of U937 are target cell.Respectively the effector cell: target cell is 40:1,20:1, and 10:1 during 5:1, adopts LDH release to birth ratio color method to detect the activity of CTL.Simultaneously, be the effector cell with the HSP-GP96 leukemia peptide complex-DC that derives from the K562 cell, same effect target ratio is down, to K562, HL-60 and three kinds of leukemia cells' of U937 CTL effect, the specificity of observing lethal effect, result are got the mean value of three experiments.
The invention has the advantages that: (1) utilizes genetic engineering technique, behind people GP96 gene fragment insertion p-shuttle CMV shuttle vectors, transform BJ5183-AD-1, screen correct recombinant adenovirus plasmid and with transfection AD293 cell after its linearizing, in AD293 cell internal packing and a large amount of amplification purification.(2) established experiment basis for preparation ideal leukemia knurl seedling, can be applicable to eliminate the leukemia minimal residual disease, be expected to reach the suitable curative effect of allogeneic bone marrow transplantation, estimate that every routine patients ' expenses reduces more than 200,000 yuan than allogeneic bone marrow transplantation, can obviously reduce medical expense.
Description of drawings
Fig. 1 is reorganization adenovirus vector construct synoptic diagram.
Fig. 2 cuts back gel electrophoresis result for the recombinant adenoviral vector enzyme and identifies.
Fig. 3 derives from K562, the comparison of respectively organizing peptide complex stimulation PBMNC ability of HL-60 and U937 cell.
Fig. 4 is for deriving from K562 (a), HL-60 (b) and U937 (c) cell respectively organize peptide complex different effect targets than under to the influence of NK cytoactive.
Fig. 5 a: for the peptide complex of respectively organizing that derives from the K562 cell is imitated under the target ratio, to the CTL effect of K562 cell in difference.
5 b: for the peptide complex of respectively organizing that derives from the HL-60 cell is imitated under the target ratio, to the CTL effect of HL-60 cell in difference.
5 c: for the peptide complex of respectively organizing that derives from the U937 cell is imitated under the target ratio, to the CTL effect of U937 cell in difference.
Fig. 6 is that the peptide complex of respectively organizing that derives from the K562 cell is imitated under the target ratio in difference, to K562, and HL-60, the CTL effect of U937 cell.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
The construction process of embodiment 1. HSP-GP96 recombinant adenoviral vectors
(1) design and preparation primer, pcr template and amplification gene sequence,
According to America NI H(National institute of Health) the sequences Design primer (GenBank:BC066656.1) that provides of Nucleotide.Upstream primer P1:5 '-GCAGATCT atgaagttgatgtgg atg-3 '; Downstream primer P2:5 '-CGAAGCTT ttacaattcatctttttC-3 '.Upstream primer 5 ' end comprises the BgLII enzyme and cuts sequence A GATCT, and downstream primer 5 ' end comprises Hind III enzyme and cuts sequence A AGCTT.Amplification heat shock protein(HSP) GP96 gene order (SEQ ID NO:1) is got the 5ml amplified production, and electrophoresis confirms to have expection length fragment (about 2420bp) in 1% sepharose;
(2) make up the PSC-GP96 shuttle vectors, referring to Fig. 1
A. use BgLII and Hind III double digestion PSC plasmid and heat shock protein(HSP)-GP96 gene PCR purifying thing, after enzyme is cut connection, obtain reorganization PSC-GP96 shuttle vectors;
B. prepare PSC-GP96 recombinant plasmid.Get 10 ml connection product and 1ml control plasmid and add 100 ml competence bacterium (DH5a competent cells, purchase a uncommon lucky Bioisystech Co., Ltd) in Shanghai, conversion through PSC-GP96 recombinant plasmid and control plasmid PSC-LacZ, carry out the screening of positive colony and recombinant plasmid enzyme cutting method then and identify, have and insert the fragment person of discharging about 2420bp and be considered as positive recombinant chou;
(3) structure of adenovirus carrier HSP-GP96 and adenovirus carrier LacZ
The A.PmeI enzyme is cut and is made PSC-GP 96 and PSC-LacZ linearizing, respectively get 3 ml1% agarose gel electrophoresis, conclusive evidence PSC-GP 96 and PSC-LacZ linearizing fully, surplus enzyme is cut product StrataPrep PCR purification kit purifying, last respectively with 20 ml water elution DNA, the row electricity transforms again;
The evaluation of B. positive recombinant adenoviral vector plasmid, Ad-GP 96 recons obtain positive colony through kalamycin resistance, AdLacZ plasmid after kantlex and Lan Bai bacterium colony preliminary screening.With amount extracting recombinant adenovirus plasmid DNA in the alkaline lysis, the plasmid that extracting is obtained is transformed into respectively in common DH5a competence bacterium and the XL10-Gold competence bacterium, extract plasmid DNA with in a small amount ultrapure plasmid DNA extraction agent box, 0.75% gel electrophoresis shows AdGP96 and Lamda/HindIII Marker relatively, and main fragment is done PacI single endonuclease digestion, BgLII and HindIII double digestion and PCR evaluation the above person of 23kb; AdLacZ only makes the PacI enzyme and cuts evaluation;
C. the recombinant adenovirus plasmid enzyme cutting method is identified, with Hind III and BgLII double digestion 7 ml recombinant adenovirus plasmid DNA, enzyme is cut after product and got 8ml electrophoresis in 0.75% sepharose, and having has the fragment person of discharging to be considered as positive recombinant chou about 2420bp.The PacI enzyme is cut the 7ml recombinant adenovirus plasmid, gets 8ml electrophoresis in 0.75% sepharose after enzyme is cut, and finds to have 4.5kb or 3.0kb fragment to discharge, and turns out to be positive recombinant chou.Referring to Fig. 2: Lane1:500bp Marker; Lane2:PSC-GP96 BglII, HindIII double digestion product; Lane3:PCR amplification GP96 gene is a template with the Ad-GP96 plasmid; The Lane4:AdLacZ plasmid; The complete plasmid of Lane5:AdGP96; Lane6:AdGP96 PacI enzyme is cut product, discharges the fragment of 3.0kb; Lane7:AdGP96 PacI enzyme is cut product, discharges the fragment of 4.5kb; Lane8:Lamda/HindIII Marker.
(4) preliminary evaluation of HSP-GP96 recombinant adenoviral vector plasmid packing and s-generation infective virus, HSP-GP96 recombinant adenoviral vector plasmid is packaged in the AD293 cell.The plasmid DNA cell transfecting with former generation virus infection AD293 cell, obtains s-generation virus, and carries out purifying and amplification.
The HSP-GP96 recombinant adenoviral vector infected person leukemia cell that the present invention makes up brings into play leukemia resisting action then.
(1) preparation and the evaluation of HSP-GP96 leukemia peptide complex and leukemia peptide: AD-GP96 is infected 1 * 10 with MOI=100
10Human leukemia cell line K562, HL-60, U937, another group does not add AD-GP96 and infects.After 1 week the leukemia cell added the abundant mixing of lysate, after the lysis in 4 ℃, centrifugal 30 min of 2 000 * G, sucking-off supernatant, 4 ℃, centrifugal 15 min of 15000G; The sucking-off supernatant is through 50%, 70% saturated ammonium sulphate; Throw out filters with concanavalin A affinity chromatography column chromatography, then with containing 10% after the balance liquid of 5 times of column volumes is resuspended
The balance liquid of-methyl sweet dew pyrans carries out wash-out, collects elutriant; By the DEAE ion exchange column, then through the protein purification technology, use the balance liquid wash-out of 300-1000mMol/L NaCl salt ion respectively, collect the elutriant of each ion gradient, obtain GP96-leukemia antigen peptide and leukemia antigen peptide respectively, carry out quantitatively 1 * 10 after the filtration sterilization with the uv-spectrophotometric instrument
10The extracted amount of K562 cell GP96 is about 800 μ g.
(2) after HSP-GP96 leukemia peptide complex stimulates, strengthen the single nuclear breeding ability of allogeneic peripheral blood: HSP-GP96 leukemia peptide complex is compared with the leukemia cell with the leukemia peptide, can obvious stimulation T lymphopoiesis, three kinds of cell strains are after DC induces, compare with same antigen peptide, the ability that stimulates proliferation all strengthens, and statistical significance (P〉0.05) is arranged.Referring to Fig. 3, among Fig. 3 group be that the leukemia cell organizes respectively, leukemia peptide complex group, HSP-GP96 leukemia peptide complex group, leukemia peptide complex-DC group, HSP-GP96 leukemia peptide complex-DC group.The result shows that HSP-GP96 leukemia peptide complex group stimulates the ability of PBMNC obviously to strengthen for more preceding two groups, after cultivating altogether with DC, though stimulate the ability of PBMNC enhancing to be arranged, no difference of science of statistics.
(3) after HSP-GP96 leukemia peptide complex stimulates, strengthened the ability of three strain cell-stimulating NK cells.When the effect target is relatively lower (5:1), K562 cell and HL-60 cell, and U937 cell, HSP-GP96 leukemia peptide complex is compared with the leukemia peptide complex with the leukemia cell, can strengthen the NK cytoactive, after DC induced, the active increase of its NK was not obvious.And when imitating target when increasing, HSP-GP96 leukemia peptide complex and leukemia peptide complex, and the leukemia cell compares, and can strengthen the NK cytoactive, and significant difference is arranged.HSP-GP96 leukemia peptide complex can strengthen NK cell activity (P＜0.05) after DC induces, and the white corpuscle peptide complex is after DC induces, and the NK activity only slightly strengthens, and the result is referring to Fig. 4.
(4) after HSP-GP96 leukemia peptide complex stimulates, strengthened the CTL effect of T lymphocyte to three kinds of leukemia cells.Lower effect target than the time, no significant difference between leukemia cell and the leukemia peptide complex.But compare with HSP-GP96 leukemia peptide complex, show that the CTL effect strengthens (P＜0.05).And along with the increase of imitating the target ratio, the leukemia peptide complex is compared with the leukemia cell, and enhanced CTL effect also has significant difference (P＜0.05), and the result is referring to Fig. 5.
(5) HSP-GP96 leukemia peptide complex has the HSP-GP96 leukemia peptide complex in specific CTL effect: K562 source, under same effect target ratio, the K562 cell is had very strong lethal effect.Simultaneously, HL60 and U937 cell are also had certain lethal effect, but its ability weak (P＜0.05).And to CTL effect indifference between U937 and the HL-60 cell strain.The result is referring to Fig. 6, and is strong than HL-60 and U937 at the CTL effect of K562.
Should understand, the present invention is described in conjunction with most preferred embodiment, yet after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims institute restricted portion equally.
＜110〉Zhejiang University
＜120〉HSP-GP96 recombinant adenoviral vector and structure and application
<160>?3
<210>?1
<211>?2412
<212>?DNA
＜213〉artificial sequence
<220>
<221>?CDS
<222>
＜223〉n=a or g or c or t
<400>?1
ATGAGGGCCCTGTGGGTGCTGGGCCTCTGCTGCGTCCTGCTGACCTTCGGGTCGGTCAGAGCTGACGATGAAGTTGATGTGGATGGTACAGTAGAAGAGGATCTGGGTAAAAGTAGAGAAGGATCAAGGACGGATGATGAAGTAGTACAGAGAGAGGAAGAAGCTATTCAGTTGGATGGATTAAATGCATCACAAATAAGAGAACTTAGAGAGAAGTCGGAAAAGTTTGCCTTCCAAGCCGAAGTTAACAGAATGATGAAACTTATCATCAATTCATTGTATAAAAATAAAGAGATTTTCCTGAGAGAACTGATTTCAAATGCTTCTGATGCTTTAGATAAGATAAGGCTAATATCACTGACTGATGAAAATGCTCTTTCTGGAAATGAGGAACTAACAGTCAAAATTAAGTGTGATAAGGAGAAGAACCTGCTGCATGTCACAGACACCGGTGTAGGAATGACCAGAGAAGAGTTGGTTAAAAACCTTGGTACCATAGCCAAATCTGGGACAAGCGAGTTTTTAAACAAAATGACTGAAGCACAGGAAGATGGCCAGTCAACTTCTGAATTGATTGGCCAGTTTGGTGTCGGTTTCTATTCCGCCTTCCTTGTAGCAGATAAGGTTATTGTCACTTCAAAACACAACAACGATACCCAGCACATCTGGGAGTCTGACTCCAATGAATTTTCTGTAATTGCTGACCCAAGAGGAAACACTCTAGGACGGGGAACGACAATTACCCTTGTCTTAAAAGAAGAAGCATCTGATTACCTTGAATTGGATACAATTAAAAATCTCGTCAAAAAATATTCACAGTTCATAAACTTTCCTATTTATGTATGGAGCAGCAAGACTGAAACTGTTGAGGAGCCCATGGAGGAAGAAGAAGCAGCCAAAGAAGAGAAAGAAGAATCTGATGATGAAGCTGCAGTAGAGGAAGAAGAAGAAGAAAAGAAACCAAAGACTAAAAAAGTTGAAAAAACTGTCTGGGACTGGGAACTTATGAATGATATCAAACCAATATGGCAGAGACCATCAAAAGAAGTAGAAGAAGATGAATACAAAGCTTTCTACAAATCATTTTCAAAGGAAAGTGATGACCCCATGGCTTATATTCACTTTACTGCTGAAGGGGAAGTTACCTTCAAATCAATTTTATTTGTACCCACATCTGCTCCACGTGGTCTGTTTGACGAATATGGATCTAAAAAGAGCGATTACATTAAGCTCTATGTGCGCCGTGTATTCATCACAGACGACTTCCATGATATGATGCCTAAATACCTCAATTTTGTCAAGGGTGTGGTGGACTCAGATGATCTCCCCTTGAATGTTTCCCGCGAGACTCTTCAGCAACATAAACTGCTTAAGGTGATTAGGAAGAAGCTTGTTCGTAAAACGCTGGACATGATCAAGAAGATTGCTGATGATAAATACAATGATACTTTTTGGAAAGAATTTGGTACCAACATCAAGCTTGGTGTGATTGAAGACCACTCGAATCGAACACGTCTTGCTAAACTTCTTAGGTTCCAGTCTTCTCATCATCCAACTGACATTACTAGCCTAGACCAGTATGTGGAAAGAATGAAGGAAAAACAAGACAAAATCTACTTCATGGCTGGGTCCAGCAGAAAAGAGGCTGAATCTTCTCCATTTGTTGAGCGACTTCTGAAAAAGGGCTATGAAGTTATTTACCTCACAGAACCTGTGGATGAATACTGTATTCAGGCCCTTCCCGAATTTGATGGGAAGAGGTTCCAGAATGTTGCCAAGGAAGGAGTGAAGTTCGATGAAAGTGAGAAAACTAAGGAGAGTCGTGAAGCAGTTGAGAAAGAATTTGAGCCTCTGCTGAATTGGATGAAAGATAAAGCCCTTAAGGACAAGATTGAAAAGGCTGTGGTGTCTCAGCGCCTGACAGAATCTCCGTGTGCTTTGGTGGCCAGCCAGTACGGATGGTCTGGCAACATGGAGAGAATCATGAAAGCACAAGCGTACCAAACGGGCAAGGACATCTCTACAAATTACTATGCGAGTCAGAAGAAAACATTTGAAATTAATCCCAGACACCCGCTGATCAGAGACATGCTTCGACGAATTAAGGAAGATGAAGATGATAAAACAGTTTTGGATCTTGCTGTGGTTTTGTTTGAAACAGCAACGCTTCGGTCAGGGTATCTTTTACCAGACACTAAAGCATATGGAGATAGAATAGAAAGAATGCTTCGCCTCAGTTTGAACATTGACCCTGATGCAAAGGTGGAAGAAGAGCCTGAAGAAGAACCTGAAGAGACAGCAGAAGACACAACAGAAGACACAGAGCAAGACGAAGATGAAGAAATGGATGTGGGAACAGATGAAGAAGAAGAAACAGCAAAGGAATCTACAGCTGAAAAAGATGAATTGTAA
<210>?2
<211>?26
<212>?DNA
＜213〉primer sequence
<220>
<221>?CDS
<400>?2
GCAGATCT?atgaagttgatgtgg?atg
<210>?3
<211>?26
<212>?DNA
＜213〉primer sequence
<220>
<221>?CDS
<400>?3
CGAAGCTT?TTacaattcatctttttC
Claims (3)
1. heat shock protein(HSP)-GP96 recombinant adenoviral vector, the nucleotide sequence of described carrier is shown in SEQ ID NO:1.
2. the construction process of a kind of heat shock protein(HSP) according to claim 1-GP96 recombinant adenoviral vector is characterized in that, realizes by following steps:
(1) design upstream primer SEQ ID NO:2:5 '-GCAGATCT atgaagttgatgtgg atg-3 '; Downstream primer SEQ ID NO:3:5 '-CGAAGCTT ttacaattcatctttttC-3 ', upstream primer 5 ' end comprises the BgLII enzyme and cuts sequence A GATCT, downstream primer 5 ' end comprises Hind III enzyme and cuts sequence A AGCTT, amplification heat shock protein(HSP)-GP96 gene order;
(2), after cutting connection, enzyme obtains reorganization PSC-GP96 shuttle vectors with BgLII and Hind III double digestion PSC plasmid and GP96 gene PCR purifying thing;
(3) the PmeI enzyme is cut and is made PSC-GP96 and PSC-LacZ linearizing, the evaluation of positive recombinant adenoviral vector plasmid, and fragment is done PacI single endonuclease digestion, BgLII and HindIII double digestion and PCR evaluation the above person of 23kb.
3. the application of a kind of heat shock protein(HSP) according to claim 1-GP96 recombinant adenoviral vector in the preparation knurl seedling mixture residual at leukemia.
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Application publication date: 20111116 |