CN102168074B - Recombinant adenovirus and application thereof - Google Patents
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Abstract
The invention discloses a recombinant adenovirus and application thereof. The recombinant adenovirus is obtained by transferring an encoding gene of an interleukin 17 receptor D extracellular region Fc fusion protein to a host cell, wherein the amino acid sequence of the interleukin 17 receptor D extracellular region Fc fusion protein is a sequence 2 in a sequence table; and through a recombinant vector, the interleukin 17 receptor D extracellular region Fc fusion protein is transferred to the host cell. The experiment in the invention proves that a CIA (collagen in-duced arthritis) mice model is constructed by using an adenovirus vector of constructed and purified Ad/Hil-17RD-ECD-Fc, the prevention and treatment on the CIA mice are achieved by using the Ad/Hil-17RD-ECD-Fc, and finally, the alleviation degrees of CIA mice arthritis and bone injury are estimated and analyzed through He dyeing. Thus, a novel and effective method is provided for treating rheumatoid arthritis.
Description
Technical field
The present invention relates to the genetically engineered field, relate in particular to a kind of recombinant adenovirus and application thereof.
Background technology
Rheumatoid arthritis (RA) is a kind of chronic autoimmunity system disorders disease, and its clinical manifestation is mainly symmetry arthralgia, aggressiveness synovitis, even carrying out property joint injury, deformity, permanent disability, can involve internal organs outside the joints such as eye, lung sometimes.This disease is perplexing the crowd of whole world 1-2%, and wherein great majority are middle-aged womens, and Most patients has the chronic course of disease of outbreak repeatedly.The disability rate of rheumatoid arthritis is very high, and its treatment is the focus that people pay close attention to always.At present, the treatment to rheumatoid arthritis mainly comprises like NSAIDs, acts on slowly that antirheumatic, glucocorticosteroid, biotechnological formulation, autologous stem cell are transplanted and several different methods such as gene therapy.Along with to the pathogenetic further investigation of rheumatoid arthritis, people have proposed the New Policy of increasing treatment rheumatoid arthritis, many conditions of patients are eased, but still do not have the radical cure method at present.Therefore, the medicine of the new treatment rheumatoid arthritis of research is imperative.
Therapeutic strategy for rheumatoid arthritis mainly is the balance of control relevant inflammatory factors.Attach most importance to the activity that suppresses proinflammatory factor TNF-α and IL-1 β at present; Utilize to the antibody of these two kinds of proinflammatory factors and in clinical application, also obtained certain effect with its receptor fusion protein; But some patient is insensitive for the treatment of these two kinds of factors of blocking-up.Therefore, to this a part of patient's treatment, need to study other pharmacological agent strategy.A large amount of researchs show; Contain a large amount of Th17 cells and high expression level IL-17 in rheumatoid arthritis people's the synovial membrane liquid, this proinflammatory factor works at the upper reaches of arthritic pathologic process, can stimulate scavenger cell TNF secretion-α and IL-1 β; Also can induce the synovial cell to produce IL-6; IL-8, GM-CSF, the PSE2 factors such as (prostaglandin E2).There are some researches show that with in the IL-17 antibody and behind the IL-17, the early stage arthritis of CIA mouse alleviates or disappears, late period, the bone injury of CIA mouse can have recovery to a certain degree.
The IL-17 of Th17 emiocytosis comprises IL-17A and IL-17F.These two inflammation molecules all are to transmit signal through the IL-17 receptor family, thereby influence downstream gene expression.
Summary of the invention
An object of the present invention is to provide a kind of recombinant adenovirus and application thereof.
Recombinant adenovirus provided by the invention, for the encoding sox with interleukin-17 acceptor D extracellular region Fc fusion rotein changes host cell over to, the recombinant adenovirus that assembling obtains;
The aminoacid sequence of said interleukin-17 acceptor D extracellular region Fc fusion rotein is the sequence 2 in the sequence table.
The encoding sox of said interleukin-17 acceptor D extracellular region Fc fusion rotein changes host cell over to and realizes through recombinant vectors;
Said recombinant vectors is the carrier that Ad-Easy and recombinant shuttle plasmid obtain through homologous recombination;
The plasmid of said recombinant shuttle plasmid for obtaining between the Hind IIII that the encoding sox of interleukin-17 acceptor D extracellular region Fc fusion rotein is inserted into carrier A d-Track-CMV and EcoR V recognition site.
The nucleotides sequence of said interleukin-17 acceptor D extracellular region Fc fusion rotein encoding sox is classified the sequence 1 in the sequence table as.
Said host cell is an eukaryotic cell.
The mammalian cell of said eukaryotic cell for exsomatizing is preferably the HEK-293 cell.
Another object of the present invention provides a kind of recombinant vectors.
Recombinant vectors provided by the invention, the carrier that obtains through homologous recombination for Ad-Easy and recombinant shuttle plasmid;
The plasmid of said recombinant shuttle plasmid for obtaining between the Hind III that the encoding sox of interleukin-17 acceptor D extracellular region Fc fusion rotein is inserted into carrier A d-Track-CMV and EcoR V recognition site.
Said recombinant adenovirus or said recombinant vectors also are the scopes that the present invention protects in the application that preparation prevents and/or treats in the autoimmune disease product.
Said autoimmune disease is a rheumatoid arthritis.
Said product is a medicine.
The 3rd purpose of the present invention provides a kind of vaccine that prevents and/or treats rheumatoid arthritis.
A kind of vaccine that prevents and/or treats rheumatoid arthritis provided by the invention, said recombinant adenovirus of its activeconstituents or said recombinant vectors.
Experiment of the present invention proves; The adenovirus carrier of structure of the present invention and purifying Ad/hIL-17RD-ECD-Fc; Make up the CIA mouse model; The CIA mouse is prevented and treat with Ad-hIL-17RD-ECD-Fc, the degree that alleviates through He dyeing evaluation analysis CIA mouse arthritis disease and bone injury at last, thus a kind of new effective means is provided for the treatment of rheumatoid arthritis; And the negative two mutants Ad/hIL-17RD-ECD-Fc of the dominance of utilizing adenovirus mediated hIL-17RD acceptor, exploitation can be conducted new candidate's medicine of treating rheumatoid arthritis through suppressing IL-17 family signal.
Description of drawings
Fig. 1 cuts evaluation for the enzyme of Ad-hIL-17RD-ECD-Fc
Fig. 2 is recombinant adenovirus Ad-hIL17RD-ECD-Fc and the expression of contrast adenovirus Ad-GFP in 293 cells
Fig. 3 detects reorganization gland virus expression thing for Western blot
Fig. 4 is the prophylactic effect of Ad-IL17RD-ECD-Fc for the CIA mouse arthritis
Fig. 5 is the therapeutic action of Ad-IL17RD-ECD-Fc for the CIA mouse arthritis
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The structure of embodiment 1, recombinant adenovirus
1, the formation of human interleukin-11 7 acceptor D extracellular region Fc antigen-4 fusion protein genes
Artificial synthesized sequence 1 is as template; With Sense primer:5 '-ATAAAGCTTATGGCCCCGTGGCTGC-3 ' Antisense primer:5 '-ATTCTCGAGTCATTTACCCGGGAGACAG-3 ' is primer; With Hind III and EcoRV it being carried out enzyme cuts; Glue reclaims the purpose band of 1536bp, and the fragment that reclaims is connected among the Ad-Track-CMV (available from Stratagene, catalog number #240009) with same restriction enzymes double zyme cutting; Transform bacteria Top10 (invitrogen Catalog#C404003) obtains transformant.Transformant is extracted plasmid; Send to order-checking; The result is for containing the positive plasmid of Nucleotide shown in the sequence 1 (Nucleotide of hIL-17RD-ECD-Fc) in the ordered list; This plasmid is the carrier that obtains between the Hind III of the 1 insertion Ad-Track-CMV of the sequence in the sequence table and EcoR V restriction enzyme site, with this carrier called after Ad-Track-CMV-hIL17RD-ECD-Fc.
2, make up the adenovirus shuttle vector that contains human interleukin-11 7 acceptor D extracellular region Fc
Cut carrier A d-Track-CMV-hIL17RD-ECD-Fc 6h with Pme1 at 37 ℃ of following enzymes, transform to get into then in the BJ5183 competent cell that contains Ad-Easy that (Ad-Easy is available from Stratagene, catalog number #240009; The BJ5183 competence is available from the outstanding U.S. gene in Shanghai Pharmaceutical Technology Co., Ltd, production number GMS12205.3; The BJ5183 competent cell that contains Ad-Easy is for to be transformed into the cell that the BJ5183 competent cell obtains with the Ad-Easy electricity.), cultivate 3h for 170rpm37 ℃, it is dull and stereotyped that card-coating is received the LB of resistance, makes it cultivate 16h at 37 ℃.The picking mono-clonal, 170rpm cultivates 20h for 37 ℃.
Extract the plasmid of positive colony undetermined, the Pac1 enzyme is cut the back and is identified with 0.8% DNA race glue, obtains the positive plasmid of 4.5kb fragment.Change positive plasmid over to TOP 10 competence, 170rpm cultivated 18 hours for 37 ℃, extracted plasmid; With the checking of Pme1 enzyme, the result is as shown in Figure 1, and wherein 1 is plasmid; 2 cut product for enzyme reclaims the back small segment; As can be seen from the figure, obtain 1536bp hIL-17RD-ECD-Fc fragment, prove the positive plasmid of this plasmid.Send to order-checking once more, the result is the carrier of this plasmid for the sequence in the sequence table 1 is obtained to the Ad-easy through homologous recombination, and note is made Ad-hIL-17RD-ECD-Fc.
Adopting uses the same method contains empty carrier Ad-Track-CMV conversion entering in the BJ5183 competence of Ad-Easy; Cultivate; Extract plasmid; Send to order-checking, the result is the carrier of this plasmid for empty carrier Ad-Track-CMV is obtained to the Ad-easy through homologous recombination, and note is made reorganization empty carrier Ad-Track-CMV.
3, the recombinant adenovirus of expressing human interleukin-17 acceptor D extracellular region Fc fusion rotein
Cut Ad-hIL-17RD-ECD-Fc with the Pac1 enzyme; Ethanol sedimentation reclaims and in transfection on same day HEK-293 cell (Beijing consonance medical university), receives cell, 4 cracking of multigelation in liquid nitrogen; Infect 100mm petridish (dish) degrees of fusion with lysate and reach 80% cell amplification virus; When cell rounding is thyrsiform, collect all cells, the 500g rotating speed is centrifugal, abandons supernatant.With the resuspended deposition of sterilization PBS, 4 freezing-thawing and cracking cells in liquid nitrogen are collected supernatant, are Ad/hIL-17RD-ECD-Fc.Ad-Track-CMV is contrast with the reorganization empty carrier, collects supernatant, is Ad/GFP.
Under fluorescent microscope, observe after the 10th day in transfection, the result is as shown in Figure 2, and wherein left side figure is an Ad-hIL-17RD-ECD-Fc transfection HEK-293 cell; Right figure is an Ad-Track-CMV transfection HEK-293 cell; Can find out that green fluorescence appears in cell, and (GFP that carries on the adenovirus carrier Ad-easy is after reorganization is good; Can go out GFP on the expression vector); Explain that virus packets dresses up merit, Ad-hIL-17RD-ECD-Fc transfection HEK-293 cell obtains virus of A d/hIL-17RD-ECD-Fc, and reorganization empty carrier Ad-Track-CMV transfection HEK-293 cell obtains virus of A d/GFP.
With test kit BIOMIGA ViraTrapTM Adenovirus Purification Miniprep Kit (V1160) (available from BIOMIGA and catalog number (V1160)) purifying Ad/hIL-17RD-ECD-Fc and Ad/GFP, obtain behind the purifying Ad/GFP behind the Ad/hIL-17RD-ECD-Fc and purifying.
Measure virus titer: will supply test agent and BUFFER (the Elution Buffer solution in BIOMIGA ViraTrapTM AdenovirusPurification Miniprep Kit (V1160) test kit.) with lysate 56 ℃ of water-bath cracking 10 minutes, be placed to room temperature (25 ℃), be blank with BUFFER, measure OD260, the OD280 of adenovirus sample, calculate titre VP by following formula: titre VP=OD260 * diluted sample multiple * 1.1 * 10
12, the numerical value that obtains is VP concentration (every ml).
The result is 1.3 * 10 for Ad/hIL-17RD-ECD-Fc titre behind the purifying
12V.p/ml, the titre of the Ad/GFP of purifying is 1.1 * 10
12V.p/ml.
It is subsequent use that packing is stored in-80 ℃ of refrigerators.
4, detect Ad/hIL-17RD-ECD-Fc with Western blot
Collect the lysate (representation of recombinant adenovirus Ad/hIL-17RD-ECD-Fc) of above-mentioned Ad-hIL-17RD-ECD-Fc cells infected; Through the SDS-PAGE electrophoresis; Be transferred on the NC film; (mouse hybridoma cell (mus musculus hybridoma) CGMCC No.4576 enlarges cell cultures and obtains to use the hIL-17RD monoclonal antibody.) (1: 10000) dilutes and the sheep anti-mouse igg (bse-0296G Shanghai biotechnology company of large alliance) of HRP mark detects.The result is as shown in Figure 3; 1 is the lysate of Ad-hIL-17RD-ECD-Fc cells infected; The lysate of 2 positive contrast Ad/hIL-17RD cells infecteds finds out that this antibodies specific is good from figure, can detect Ad/hIL17RD-ECD-Fc and express; And be the dimeric structure of the 120kd of expection; The lysate preparation method of the lysate of Ad/hIL-17RD cells infected and Ad-hIL-17RD-ECD-Fc cells infected is basic identical, and wherein, Ad/hIL-17RD construction process and top Ad/hIL17RD-ECD-Fc are basic identical; Different is that hIL17RD-ECD-Fc is replaced to IL-17RD, and the nucleotides sequence of hIL-17RD is classified the 1-2220 position Nucleotide of GenbankNM_017563.3 as.。
The application of embodiment 2, recombinant adenovirus
1, the structure of CIA mouse
Get 40 male DBA-1 mouse (Chinese Academy of Sciences Shanghai Experimental Animal Center DBA-1 mouse) (10-12 age in week) according to Erik Lubberts (IL-1-Independent Role ofIL-17 in Synovial Inflamation andJoint Destruction During Collagen-Induced Arthritis; J.Immunol.2001; The method that 167:1004-1013) waits is carried out sensitization, and: antigen Bovine type II collagen (CII) (ox two Collagen Type VI SIGMA C7806-10MG) uses 0.05M acetic acid to be diluted to concentration to be 2mg/ml, and the CII with this concentration carries out emulsification with isopyknic Fo Shi Freund's complete adjuvant (CFA) then.Only (every mouse body weight is about 25g at the CII/ of mouse tail subcutaneous injection 100ul after the emulsification.), obtain the mouse after the allergy.Promptly can be observed arthritic generation in back 21 days in injection, significantly rubescent or swelling can appear in mouse knee joint and ankle.
Arthritic scoring according to measuring mouse palm thickness, is estimated 0 fen＜3,0.5 fens 3-3.3 with the scoring of 0-3,1 fen 3.4-3.5 usually.1.5 divide 3.6-3.8,2 fens 3.9-4.1,2.5 fens 4.1-4.3,3 minutes＞4.3mm), adopt double blind experiment.
2, Ad/hIL-17RD-ECD-Fc is for the prevention and the treatment of CIA mouse arthritis
Mouse after the allergy is divided into following two groups of processing, every group of 8 mouse:
Prevention group: after 21 days, be the CII of 1mg/ml at mouse peritoneal injection injection Freund's incomplete adjuvant emulsive final concentration, while abdominal injection 10
8The Ad/hIL-17RD-ECD-Fc of pfu, injection in per 3 days 1 time totally 4 times,
Prevention negative control group: after 21 days, be the CII of 1mg/ml at mouse peritoneal injection injection Freund's incomplete adjuvant emulsive final concentration, while abdominal injection 10
8The Ad/GFP of pfu, injection in per 3 days 1 time totally 4 times;
The experiment triplicate, results averaged.
The injection back was observed at the 27th, 29,32,34 day:
Every mouse of prevention contrast group is respectively 2.6mm, 3.1mm, 3.7mm, 3.8mm at the 27th, 29,32,34 day mouse palm thickness; Arthritis score is respectively 0,0.6,1.3,1.5;
Every mouse of prevention group is respectively 2.6mm, 2.6mm, 2.7mm, 2.8mm at the 27th, 29,32,34 day mouse palm thickness; Arthritis score is respectively 0,0,0.6,0.5.
Each group is got after 1 mouse adopts disconnected neck to put to death, its knee joint is separated with ankle joint and with 10% (quality percentage composition) formalin fixed 4 days, after the formic acid decalcification with 5% (quality percentage composition); Sample carries out paraffin embedding, and tissue slice (7um) is according to the prevention result of HE staining analysis CIA mouse arthritis, and the result is as shown in Figure 4; A is mouse palm thickness (with above-mentioned numerical value mapping); B is arthritis score (with above-mentioned numerical value mapping), from figure C, can find out among the D; Inflammatory cell (bluish voilet in the prevention group Ad/IL-17RD-ECD-FcHE dyeing; The zone of arrow indication) obviously be less than prevention control group A d/GFP, explain that the prevention group state of an illness is better than the prevention control group, the mouse inflammation degree of prevention group is starkly lower than control group.
Divide into groups according to its state of an illness producing arthritic D B A-1 mouse after the above-mentioned secondary sensitization, carry out following packet transaction, (mouse of 1.25 fens state of an illness injects 10 to every group of 8 of mouse
8The pfuAd/IL-17RD-ECD-Fc treatment)
Treatment group: with state of an illness mouse peritoneal injection 10 in 1.25 fens
8The Ad/IL-17RD-ECD-Fc of pfu treats, injection in per 3 days 1 time, and totally 5 times,
Treatment control group: with state of an illness mouse peritoneal injection 10 in 1.25 fens
8The Ad/GFP of pfu treats, injection in per 3 days 1 time, and totally 5 times,
46th, 49,52,54,59,61 day the mouse palm thickness of every mouse of treatment contrast group behind injecting virus is respectively 3.6mm, 4.25mm, 4.1mm, 4.2mm, 4.15mm, 3.9mm; Arthritis score is respectively 1.25,2.25,2,2,2.25,1.7;
Every mouse of treatment group mouse palm thickness of the 46th day, 49 days, 52 days, 54 days, 59 days, 61 days behind injecting virus is respectively 3.6mm, 3.mm, 3.4mm, 3.35mm, 3.25mm, 3.2mm; Arthritis score is respectively 1.25,1.3,1.1,0.9,0.9,0.8.
Each group is got after 1 mouse adopts disconnected neck to put to death, its knee joint is separated with ankle joint and with 10% (quality percentage composition) formalin fixed 4 days, after the formic acid decalcification with 5% (quality percentage composition); Sample carries out paraffin embedding, and tissue slice (7um) is according to the treatment result of HE staining analysis CIA mouse arthritis, and it is as shown in Figure 5 to map; A is mouse palm thickness (with above-mentioned numerical value mapping), and B is arthritis score (with above-mentioned numerical value mapping), from C; Can find out among the D figure; Inflammatory cell (bluish voilet, the zone of arrow indication) obviously is less than treatment control group A d/GFP in the treatment group Ad/IL-17RD-ECD-FcHE dyeing, explains that the treatment group state of an illness is better than the treatment control group.
Claims (9)
1. a recombinant adenovirus for the encoding sox with interleukin-17 acceptor D extracellular region Fc fusion rotein changes host cell over to, assembles the recombinant adenovirus that obtains;
The aminoacid sequence of said interleukin-17 acceptor D extracellular region Fc fusion rotein is the sequence 2 in the sequence table;
The encoding sox of said interleukin-17 acceptor D extracellular region Fc fusion rotein changes host cell over to and realizes through recombinant vectors;
Said recombinant vectors is the carrier that Ad-Easy and recombinant shuttle plasmid obtain through homologous recombination;
Said recombinant shuttle plasmid is inserted into the plasmid that obtains between the MCS of carrier A d-Track-CMV for the encoding sox with interleukin-17 acceptor D extracellular region Fc fusion rotein.
2. recombinant adenovirus according to claim 1 is characterized in that:
The nucleotides sequence of said interleukin-17 acceptor D extracellular region Fc fusion rotein encoding sox is classified the sequence 1 in the sequence table as.
3. recombinant adenovirus according to claim 1 and 2 is characterized in that: said host cell is an eukaryotic cell.
4. recombinant adenovirus according to claim 3 is characterized in that: the mammalian cell of said eukaryotic cell for exsomatizing.
5. recombinant adenovirus according to claim 4 is characterized in that: said eukaryotic cell is the HEK-293 cell.
6. recombinant vectors, the carrier that obtains through homologous recombination for Ad-Easy and recombinant shuttle plasmid;
Said recombinant shuttle plasmid is inserted into the plasmid that obtains between the MCS of carrier A d-Track-CMV for the encoding sox with interleukin-17 acceptor D extracellular region Fc fusion rotein;
The interleukin-17 acceptor D extracellular region Fc fusion rotein that said encoding sox encoding amino acid sequence is a sequence 2 in the sequence table.
7. arbitrary said recombinant adenovirus or the said recombinant vectors of claim 6 prevent and/or treat the application in the autoimmune disease product in preparation among the claim 1-5;
Said autoimmune disease is a rheumatoid arthritis.
8. application according to claim 7 is characterized in that: said product is a medicine.
9. vaccine that prevents and/or treats rheumatoid arthritis, its activeconstituents is arbitrary said recombinant adenovirus or the said recombinant vectors of claim 6 among the claim 1-5.
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| CN1467222A (en) * | 2002-07-10 | 2004-01-14 | 清华大学 | Human interleukin-17 receptor sampling molecule fusion protein and coding gene and expression cell line |
| CN1757651A (en) * | 2005-11-14 | 2006-04-12 | 清华大学 | Interleukin-17 acceptor, and its coding gene and application |
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| CN1467222A (en) * | 2002-07-10 | 2004-01-14 | 清华大学 | Human interleukin-17 receptor sampling molecule fusion protein and coding gene and expression cell line |
| CN1757651A (en) * | 2005-11-14 | 2006-04-12 | 清华大学 | Interleukin-17 acceptor, and its coding gene and application |
Non-Patent Citations (2)
| Title |
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| Zhili Rong et al..IL-17RD (Sef or IL-17RLM) interacts with IL-17 receptor and mediates IL-17 signaling.《Cell Research》.2009,第19卷(第2期), * |
| 夏宗敬, 孔祥丽, 张 平.重组IL-15/Fc融合蛋白治疗EAU的实验研究.《四川大学学报(医学版)》.2008,第39卷(第6期),944-949. * |
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