CN1131314C - Recombination eukaryotic vector containing sensitinogen gene and application thereof - Google Patents

Abstract

The present invention provides a recombinant vendor capable of being used for preventing and treating anaphylactic diseases, which comprises a eukaryotic expression vendor and an allergen gene. When the recombinant vendor is taken by individuals in the mode of intramuscular injection, intranasal conveying, endotracheal conveying or intradermal conveying, the generation of allergen specific IgE is inhibited. The present invention also provides a medicament compound containing the recombinant vendor, and the medicament compound can be used for preventing and treating anaphylactic diseases and inhibiting the generation of allergen specific IgE.

Description

Contain the reorganization eukaryotic vector and the application thereof of anaphylactogen gene

The present invention relates to a kind of recombinant vectors that can be used for preventing and treating anaphylactic disease, the invention still further relates to the pharmaceutical composition that comprises described recombinant vectors.

Anaphylactic disease (AD) comprises allergic rhinitis, allergic asthma and atopic dermatitis, influences about 20% population, and is one of principal element of disease and death.Recent findings, most of AD are relevant with the immediacy high susceptibility at the suction anaphylactogen, and family's tendency (this phenomenon is commonly referred to as atopy) is arranged.The nosetiology of AD it be unclear that, yet, find the defective that it has a lot of cellularitys and body fluid at present, comprise the IgE total amount of rising and the specific IgE antibody of multiple positive anaphylactogen.Though inquired into out many kinds of pathogeny ideas at present, the general treatment of AD still can't be satisfactory.Immunosuppressor such as steroid and S-Neoral is applied to existing improvement of clinical effectiveness on the patient, yet, owing to having toxicity to liver and renal system, it has reduced it in children's purposes on one's body.Have effective therapy of remarkable side effect even have some now, still some patient all has resistance to the medicine of form of ownership.

The heterogeneous principal character of supersensitivity is the tendency that develops to lasting immunity sphaeroprotein E (IgE) reaction of environmental antigens.The production of IgE is highly to depend on IL-4 and be subjected to the powerful inhibition of IFN-γ.Other cytohormones such as IL-5, IL-6, IL-8, IL-10 and IL-13 and cell surface molecule such as CD40 and CD23 etc. also have participation.Recently with studies show that bronchoalveolar lavage fluid is carried out, being suppressed property of the production T cell of IgE is regulated.Moreover one of factor of immunotherapy success is relevant with the generation of suppressor T cell, and it can downgrade anaphylaxis.Also evidence show has the defective of suppressor T cell function in the atopy individuality, is particularly having on one's body the children of allergic asthma.Recently some animal experimental datas also show the CD8 of some hypotype ⁺The T cell may be played the part of important role in IgE produces, and can suppress the respiratory tract overreaction (AHR) that allergy is brought out.Therefore, may be by the suppressor T cell that produces antigen-specific to regulate IgE antibody response and the AHR among the atopy patient.

Purified protein or virus vector have been used in previous subunit's preventive vaccination.But some substantial restriction, if can make immunity use protein at host cell inner expression, then this restriction can be overcome.This respect, gene vaccine have been represented the developmental a kind of new orientation of subunit vaccine.Shown in intramuscular injection DNA and can cause coded protein expression by this DNA, be described in as Wolff, J.A.et al., Science 247,1465-1468 (1990), Tang, D.L.et al., Nature 356,152-154 (1992) and Ulmer, J.B.et al., Science 259,1745-1749 (1994).Moreover the result shows that plasmid DNA independently is retained in beyond the karyomit(e), does not duplicate in the genome of also not inserting host cell, as Wolff J.A.etal., described in the Hum.Mol.Genet.1:363-369 (1994).To so far, do not observe serious inflammatory response or other complication as yet at inoculation position.In addition, now fully understand, normally be derived from the victory peptide of intracellular antigen and pass CD8 by main tissue compatible complex body (MHC) I quasi-molecule ⁺The T cell, this MHC I quasi-molecule all has expression on nearly all somatocyte, and is to be by MHC II quasi-molecule to pass CD4 derived from the antigenic victory peptide in extracellular ⁺The T cell, this MHC II quasi-molecule is normally expressed by special antigen presentation cell.Therefore, anaphylactogen being sent into internal antigens processing approach may be a kind of antigen-specific CD8 that impels ⁺The method that the T cell produces, and then the IgE of adjusting allergen specificity is synthetic.

In the present invention, the inventor finds that the plasmid (pCMVD) of direct inoculation coding dermatophagoides pteronyssinus (Dermatophagoides pteronyssinus) the 5th group of anaphylactogens (Der p5) is to the musculus quadriceps of mouse, can when accepting Der p5 again and excite, produce suppress that Der p5 specific IgE in vivo is synthetic, the effect of AHR and respiratory inflammation, use the pCMVD of various dose to treat the mouse of sensitization, find that curative effect and dosage have dependency.The inventor finds that also the plasmid DNA (pCMVG) of direct injection coding recombination Japanese schistosomicide (Schistosoma japonicum) protein 26 (rSj26) can downgrade the allergic immune response that rSj26 brings out with dosage dependence form effectively, comprises the synthetic and dermatitis of rSj26 specific IgE.Therefore, to implant be a kind of prevention and the treatment method by IgE institute mesomeric anaphylactic disease for inference direct gene of the present invention.More specifically, the present invention relates to a kind of recombinant vectors, it comprises carrier for expression of eukaryon and anaphylactogen gene, the invention still further relates to the pharmaceutical composition that comprises aforementioned recombinant vectors, it can be used for prevention and treatment anaphylactic disease and suppresses allergenic specific IgE producing, and it is fit to offer medicine in mode such as carry in conveying in intramuscularly, the nose, intradermal or the tracheae.PCMVD and pCMVG are preserved in Hsinchu, Taiwan Province, China Foodstuff Industrial and Development Inst. (FIRDI); It is numbered FIRDI 940114 and 940115, and preservation day is on April 2nd, 1996, and is preserved in American type culture collection (ATCC), and it is numbered ATCC97499 and preservation on April 1 in 97498,1996 year.

Description of drawings

Fig. 1 detects the expression of Derp5 for using muscle original position cytochemical staining method.The thick freezing microtome section of the muscle that is taken out 12 days the time behind injection 100 microgram pCMVD DNA is monoclonal antibody (mAb) dyeing with Derp5 (A) or Der p1 (B), and optics amplifies 100 times.Section is made counterstaining with hematoxylin.All obtain similar result at six groups of discrete injection positions.Arrow is pointed out the positive staining cell.The control group muscle of injecting blank carrier does not then show the muscle cell that is colored.

Fig. 2 is (A) immune response behind intramuscular injection pCMVD.The serum of BALB/c mouse of having accepted pCMVD injection is through collecting weekly, and measures tiring of anti-Derp5 IgG1, IgG2a and IgE in the mode described in the embodiment.In this group, detect less than Der p5 specific IgE.The mouse of injecting blank carrier does not have Der p5 specific immune response.One unit antibody is equivalent to 1 microgram IgG1/ milliliter and 1 microgram IgG2a/ milliliter.(B) the in vitro proliferative response of the mouse that crosses by the pCMVD immunity splenocyte subgroup of taking out.Described in the removal such as embodiment of subgroup.Analyzed by FAScam, purified cell subsets shows to have and is less than 0.5% contamination of cells.To derive from the CD4 of natural B ALB/c mouse spleen then ⁺Or CD8 ⁺Cell adds this cell group to replace removed cell.Cell was cultivated 72 hours with Der p5 (15 mcg/ml).Propagation be with ³Mixing of H-thymidine analyzed.Shown data are mean value ± SD of three subcultures.Cell is not to the stimulation of Der p1 and react propagation.

Fig. 3: (A) pCMVD injection (intramuscular) is to the restraining effect of main IgE reaction, and it is the specific restraining effect of Ag.Mouse (every group of n=6) carries out immunity with pCMVD or blank carrier (pCMV) as shown.The immunity back is during three weeks, and mouse is accepted Der p5 (10 micrograms add aluminium hydroxide) or Der p1 (10 micrograms add aluminium hydroxide) respectively.Shown in data are mean value ± SD (every group of n=6) when exciting back 21 days.To measure tiring of antigen-specific IgG2a and IgE as the described mode of embodiment.* represent p＜0.01.(B) through the adoptive transfer of the splenocyte of pCMVD immune mouse restraining effect to the reaction of Der p5 specific IgE.Shown in FAScam analyzes, used CD4 ^-And CD8 ⁺Group is contained＜0.5% contamination of cells.Undecomposed splenocyte with the pCMVD mice immunized of must hanging oneself is organized in contrast.Shown in the mean value ± SD (every group n=6) of data when being the 21st day, anti-Der p5 IgG2a and IgE measure in the described mode of embodiment.* represent p＜0.01.1 unit antibody is equivalent to 1 microgram IgG2a/ milliliter and 100 nanogram(ng) IgE/ milliliters.

Fig. 4 carries out the over-reactive immunoprophylaxis of respiratory tract for changeing the mode of planting with the anaphylactogen gene.By intramuscular injection 100 microgram pCMV or pCMVD in mouse, and 3 week the back with Der p5 or salt solution sensitization.After three weeks of sensitization, the suction that mouse is accepted Der p5 or salt solution excites.Sucked back 18 hours, and measured lung's resistance in the mode described in the embodiment.* represent p＜0.01.

Fig. 5 suppresses the respiratory tract overreaction that anaphylactogen brings out for changeing the mode of planting with the anaphylactogen gene.And with mouse by in addition sensitization of peritoneal injection Der5, then after two weeks of sensitization, with the pCMVD plasmid DNA treatment of various dose (3 micrograms, 30 micrograms, 100 micrograms).After treating for 1 week with synthetic plasmid, mouse sucks anaphylactogen, and measures the variation of lung's resistance.* represent p＜0.01.

Fig. 6 suppresses the respiratory inflammation that anaphylactogen brings out for changeing the mode of planting with the anaphylactogen gene.

Fig. 7 is the photo with the mouse of pCMVG (left side) and pCMV (right side) processing.

To suppress the IgE that anaphylactogen brings out synthetic in order to change the mode of planting with the anaphylactogen gene for Fig. 8.Mouse is to add that with 10 microgram rSj26 aluminium hydroxide comes sensitization by peritoneal injection, after two weeks of sensitization, handles with the pCMVD plasmid DNA of various dose (3 micrograms, 30 micrograms, 100 micrograms) then.Shown in data for handling the mean value ± SD (every group n=6) of back in the time of the 7th day, and measure tiring of anti-rSj26 IgG2a and IgE in the described mode of embodiment.* represent p＜0.01.

The immune response that downgrades avirulence antigen is to keep respiratory tract and stomach and intestine The homeostatic key of road mucomembranous surface immunity, and existing people thinks it The inefficacy of controlling mechanism is the crucial paathogenic factor of anaphylactia. This process A key factor be Th2 dependence to suck or eat antigen The selective inhibition of IgE reaction, and this inhibitory action is by antigen-specific Property CD8⁺The T cell mediates. The inventor's result of study show by The modes such as intramuscular injection, the interior conveying of nose or the interior conveying of tracheae were encoded The anaphylactogen that the DNA expression vector of quick crude protein can come continuing swashs Send out and produce significant " divergence type tolerance (split tole Rance) ".

Inventor's recent findings will be encoded plasmid (pCMVD) direct inoculation of the 5th group of anaphylactogens of dermatophagoides pteronyssinus (Sermatophagoides pteronyssinus) (Der p5) to the musculus quadriceps of mouse the time, can cause Der p5 specific T-cells and IgG reaction.When the practice that this kind gives mouse with plasmid DNA caused and in vivo excites with Der p5 again, the Der p5 specific IgE that is taken place is synthetic to be had more than 90% and is suppressed, and AHR also is suppressed.This effect can be by the CD8 through the pCMVD mice immunized ⁺Splenocyte and being transferred in other mouse.The main immune response feature of the mouse of handling through pCMVD is to produce Der p5 specific C D8 ⁺T cell, these cells can suppress the synthetic of allergenic specific IgE and reduce AHR.The inventor also finds that the plasmid DNA (pCMVG) of direct injection coding rSj26 can be effectively downgrades in the mouse by the allergic immune response that rSj26 was brought out with dosage dependence form, comprises the synthetic of dermatitis and rSj26 specific IgE.Previous report shows, always is considered to the just CD8 of cytotoxic cell ⁺In fact the T cell is played the part of one and is had more active role in immunoreactive adjusting.CD8 ⁺The T cell can be regulated the generation of IgE, be via IFN-γ B cell inhibiting effect to be suppressed IgE to synthesize, and/or the class Th2 CD4 that IgE produces is supported in the mat influence ⁺The differentiation and the function of T cell are reached.The another kind of explanation is possible CD8 ⁺T cell and B cell or CD4 ⁺The T cell has the exchange interaction of physical property, and may provide the inhibition signal via the homology interaction.

Based on above-mentioned experimental result, the invention provides the recombinant vectors that can be used for preventing and treating anaphylactic disease, it comprises carrier for expression of eukaryon and anaphylactogen gene, as cDNA.The prevention of anaphylactic disease is meant the inflammation phenomenon in the synthetic and Target organ of IgE that the immunoprophylaxis anaphylactogen brings out.The treatment of anaphylactic disease be meant treatment through the individuality of anaphylactogen sensitization with the inflammation phenomenon in the synthetic and Target organ that downgrades IgE.This carrier for expression of eukaryon is to be selected from by the carrier of the carrier that contains the CMV promotor, RSV promotor or to contain one group that the carrier of SV40 promotor is formed, and wherein is preferably pCMV.This anaphylactogen comprises any human anaphylactoid environmental antigens that causes, as glutathione S-transferase, house dust, animal scurf, pollen and the peanut etc. of mite class anaphylactogen, Schistosoma japonicum.

The present invention also provides a kind of pharmaceutical composition and a kind of pharmaceutical composition that is used to suppress the allergenic specific IgE generation that is used to prevent and treat anaphylactic disease, and it comprises recombinant vectors of the present invention and pharmaceutically acceptable carrier.This anaphylactic disease comprises as allergic asthma, allergic rhinitis, atopic dermatitis and allergy.The administering mode of this pharmaceutical composition is preferably to be carried in intramuscular injection, the nose and the interior conveying of tracheae.This pharmaceutically acceptable carrier can be any known carrier that is applicable to that conveying in intramuscular injection, the nose, the interior conveying of tracheae or intradermal are carried as known in the art, for example, and physiologically acceptable buffered soln, physiological saline, gold bead or liposome.

When handling patient according to the present invention, the using dosage of recombinant vectors is looked speciality and progress and other factors of the disease of desire prevention or treatment about 0.01 between about 1.0 mg/kg body weight, for example patient's age and physical state and change to some extent.

Recently, the research in this field of atopy nearly all focuses on the treatment of the anaphylactic disease of having set up.In specific words, treatment is to concentrate on the various media that the terminal irritated function cells of the immune inflammation phase of control is discharged, as cytokine.Genetic immunization acts on generation immune response aspect and can save time and manpower, and a kind of preventive vaccination method of uniqueness can be provided.The new discovery of this research can cause " immune response departs from " to anaphylactogen for the genetic immunization effect, and the invasion and attack of the immune hypersensitivity asthma that watches for animals.The immigration of plasmid DNA is suppressing to have surprising high-level efficiency on the allergic immune response, and expression the inventive method provides a kind of simple, lasting again effectively immunoprophylaxis of safety and treated the anaphylactoid mode of the 1st type.

The following example further specifies the present invention, but unrestricted scope of the present invention, and substituting and change known to any those skilled in the art all still is covered by in the scope of the present invention, and do not depart from spirit of the present invention and purpose.

Embodiment 1A materials and methods

1, animal

Use big female BALB/c of 6 to 8 weeks, its derive from medical college of Taiwan Univ. animal center (be from the Jackson laboratory at first, Bar Harbor, ME).In each group experiment, mouse is matched on age and sex.

2, the molecular cloning of pCMVD recombinant plasmid

Der p5 cDNA is by Lin, K.L.et al., and J.Allergy Clin, the described clone of Immunol.94:989 (1994) WM obtains through PCR amplification.The sequence of 5 ' and 3 ' primer is respectively 5 '-AAAA AGATCTATCAT GAAATTCATC-3 ' (drawing the bottom line place is Bgl II site) and 5 '-ATT AAGCTTAACTTCAATCTTTTTA-3 ' (drawing the bottom line place is Hind III site) is contained whole Der p5 sequence.The PCR product is cloned among the carrier for expression of eukaryon pCMV2 with BglII and Hind III digestion again, and pCMV2 is derived from pcDNA3 (Invitrogen), called after pCMVD then at first.The plasmid breeding is carried out in the SURE bacterial strain earlier, then with Wizard ^TMDna purification system (prestige state Mai Dixun Promega company) carries out the purifying of plasmid according to manufacturer's indication.With 260 and 280nm extinction and agarose gel electrophoresis come the quality and quantity of analyzing DNA.

3, the immunohistochemical staining of Der p5 anaphylactogen

Immunostaining is according to Histomouse-SP ^TMThe indication of test kit (south, California San Francisco Zymed company) is carried out.In brief, refrigerated muscle section (5 μ m) at room temperature after the drying, is fixed in pure cold acetone (10 minutes, 4 ℃), handles 45 seconds to stop inner Peroxidase activity with Peroxo-Block (Zymed company) earlier.Blockaded 1 hour with 10% NIS then.After blockading, under 1 mcg/ml, cultivated 1 hour with suitably section is common with the mAb (deriving from doctor Lin of Taiwan Univ.) of Der p5.Organize antibody in contrast with Der p1 mAb (4CI derives from Mr. M.Chapman of the U.S.).Inject the mouse of blank carrier as negative control group with the same manner.All cultivations are to carry out in humidistat under 25 ℃.The second antibody and the streptavidin-peroxidase conjugated body that add biotin-conjugated after the cultivation again.Add 3-amino-9-ethyl card azoles to present signal.At last, with slide with hematoxylin solution counterstaining.

4, the mensuration of Der p5 specific IgG 1, IgG2a and IgE

The amount of Der p5 specific IgG 1, IgG2a and IgE is to measure with ELISA.The high board of protein is diluted in bag with 100 microlitres and is cushioned liquid (0.1M NaHCO ₃, pH 8.2) and concentration is the purified Derp5 bag quilt of 5 mcg/ml.After 4 ℃ cultivation is spent the night down, clean plate 3 times, and under 25 ℃, blockaded 2 hours with 3% (weight/volume) BSA-PBS damping fluid.When measuring IgG, used serum is dilution in 1: 100, and when measuring IgE, used serum is dilution in 1: 10, and mensuration is to carry out secondary to repeat.After 4 ℃ cultivation is spent the night down, adding is diluted in mono-clonal rat anti-mouse IgEmAb in the 0.05% gelatin damping fluid and biotin-conjugated (San Diego, California Phar Minigen company) or rat anti-mouse IgG mAb (Phar Minigen company), cultivates 1 hour again.Add antibiotin-alkaline phosphatase (Saint Louis, Missouri State Sigma chemical company) then, cultivated 1 hour down, then clean 6 times at 25 ℃.Add phosphoesterase substrate p-nitrophenyl phosphate disodium salt (Sigma chemical company).With plate in little test panel automatic reading instrument (Taiwan counting technology (Metertech) company) in the 405nm reading of data.Reading is to be reference value with commercially available radiation isotope, and this standard is mouse anti-TNPmAb, IgG1 (107.3), and IgG2a (G155-178) and IgE (IgE-3) are (PharMinigen).

5, lymphocytic preparation and cell transfer step

The CD4 of purifying ^-And CD8 ^-Splenocyte derives from magnetic active cells classification " MASC " (German Bergisch Gladbach, Miltenyi Biotec).In brief, taking from the splenocyte of pCMVD immunity and control group mice after immunity 3 weeks cultivates jointly with the super table magnetic particle that is coated with anti-CD4 or anti-CD8 monoclonal antibody, 4 ℃ following 30 minutes, cultivated jointly 30 minutes with the streptoavidin particulate (Miltenyi Biotec) that closes that stops again.Insert the magnetic field of 0.6Tesla with cell and unlabelled cellular segregation with " MACS " steel suede tubing string with mark.Splenocyte (10 with desire concentration ⁶/ acceptor) resuspending is in PBS, and final volume is 0.1 milliliter.This cell suspending liquid is injected in the tail vein of the homogenic acceptor that age and sex match.Acceptor is subsequently by by the in addition sensitization of peritoneal injection 10 microgram Der p5 and 4 milligrams of aluminium hydroxides (huge uncle Wyeth pharmaceutical factory, Australia).Under the anesthesia situation, take out venous blood weekly from tail vein.

6, T analysis of cell proliferation

The purified back of cell resuspending wherein is supplemented with Streptomycin sulphate (100 mcg/ml), penicillin (100 units per ml), L-glutamic acid (5 mmoles/liter) and 10% heat-killed foetal calf serum in complete tissue culture medium (RPMI1640).Every hole has 1 * 10 ⁵Cell was cultivated 72 hours jointly with Der p5 (15 mcg/ml) in the flat tissue culturing plate in 96 holes.Cell is again with 1 μ Ci[ ³H] TdR pulse 18 hours, collect machine with cell then and collect cell.In liquid scintillation counter (the honest Beckman of California joy company), measure the thymidine incorporation.

7, derive from the cytohormone production of the splenocyte of pCMVD immune mouse

IL-4 and IFN-γ measure according to the step that the Phar Minigen of manufacturer provides with the ELISA method.IL-10 (R ﹠amp; D) and TGF-β (Promega) measure according to the step that manufacturer provides with the ELISA test kit.

8, the analysis of the suction of aerosol and lung's resistance

Mouse is come sensitization by peritoneal injection 10 microgram Der p5, and sensitization excited with the 0.1%Der p5 that is diluted among the PBS with ultrasonic sprayer after 21 days.It is to carry out between 1 lift-off that is connected on the De Vilbiss lung ultrasonic sprayer (Binzhou Marseille De Vilbiss company of Soviet Union, the 2512nd type) that suction excites, and it can produce aerosol.Be exposed under the aerosol after 8-18 hour, with anesthetized mice, and insert the trachea cannula of 20-specification by peritoneal injection Promaz.The change of measuring esophageal pressure with the conduit (PE60) that is full of salt solution and differential pressure transmitter (DP45-14 of this basic Validyne engineering corporation is moved in the California).Esophageal tube is stretched into the mouse esophagus up to knowing that the district discerns the influence of heart.(MP45-14, spirograph Validyne) (Fleisch 00000, Zabona, Basel, SUI) detects the respiratory capacity on the tracheae with being connected to differential pressure transmitter.Signal from transmodulator is connected on the computer, and with numerical digit electronics lung detecting system (PMS, Mumed, London), it can calculate lung's resistance (RL) and dynamic compliance (Ddyn) on the real time point.Experimental data is to store with telecommunication.Experimental tracking or treated data then optionally are imprinted on association and penetrate on the printer.Give vagusstoff (Ac) by intravenously, initial dose is 1.25 mg/kg.The average-volume of every Ac dosage is 10 microlitres.Approximately at interval after 5 minutes, and only slow lung pressure and volume reduce to last dosage reference line 10% in, and before next dosage gives, increase the Ac of 20 times of concentration.Before Ac first dosage, give 10 microlitre intravenously salt solutions to set up basic value.Individual animal on each Ac dosage, is calculated the mean value ± standard deviation to the change per-cent of reference line, to obtain the Ac dose-response curve of false agent (Sham) sensitization of Der p5 sensitization or PBS group.

9, bronchoalveolar lavage and cell counting

After measuring the pulmonary function parameter, with mouse with 0.9% aseptic salt solution of 5 * 0.5 milliliters of aliquots via the polyethylene tube lavation of going into tracheotomy.With irrigating solution centrifugal (4 ℃, 500g, 10 minutes), with the cell precipitation resuspending in 0.5 milliliter of Hank ' s balanced salt solution.Add in 10 microlitre cell suspending liquid to the 90 microlitre Kimura dyestuffs and in Neubauer case chamber, under opticmicroscope, calculate again to obtain total cellular score.Obtain the noble cells number by the cytospin prepared product through the May-Grunwald dyeing.Cell is identified and be distinguished as eosinophil with the standard type technology, lymph corpuscle, neutrophil and scavenger cell calculate 500 cells under 400 magnifications, and calculate the per-cent and the absolute number of each cell category.B, result

1, the immune response of plasmid pCMVD dna immunization

Whether muscle cell picked-up pCMVD DNA can produce immune response in order to inspect, and the pCMVD in PBS is injected in the musculus quadriceps of female BALB/c mouse with 100 micrograms.Control animals is injected PBS or is not had the suitable blank carrier that Der p5 gene inserts.After immunity 12 days, carry out original position cytochemistry immunostaining with anti--Der p5mAb, to determine to be transferred to the expression of gene (Fig. 1) in the muscle cell.In addition, the immune response of antigen-specific be with Der p5 specific IgG 1 and IgG2a production of antibodies as index, production of antibodies after immunity around the time peak, descend gradually then.Der p5 specific IgE antibody is detecting less than (Fig. 2 A) in mice immunized.

2, intravital allergen specificity t cell responses

For inspecting the Der p5 specific T-cells reaction after the pCMVD injection, after immunity, during 3 weeks,, cultivate with Der p5 then by in mice immunized, extracting splenocyte.With [ ³H]-the thymidine incorporation analyzes its propagation.In 3 whens week, showed the specific t cell responses of Der p5 (Fig. 2 B) after immunity.Moreover the splenocyte of portioning can be because of CD4 to the proliferative response of Der p5Ag ⁺The removal of cell and suppressing, but because of CD8 ⁺The removal of cell and promoting shows CD4 ⁺The cell proliferation meeting is by CD8 ⁺Subgroup suppresses.

3, the in vivo inhibition of allergenic specific IgE reaction

Measure pCMVD DNA injection to regulating the in vivo effect of IgE reaction.In 3 whens week after immunity, the mouse that carrier and pCMVD handle all excites with peritoneal injection allergen Der p5.Excite the back during three weeks, at anaphylactogen with existing of anti-Der p5IgE in the elisa assay serum.In the group with vehicle treated, Der p5-specific IgE significantly increases; Relatively, the mouse of handling with pCMVD is then expressed and surpasses 90% Der p5 specific IgE synthetic inhibition (Fig. 3 A).PCMVDDNA injection to the IgE synthetic suppress be Der p5 is had specific because if the mouse of handling with pCMVD can produce the Derp1 specific IgE when exciting with another kind of house dust mite allergen Der p1.Therefore, direct gene shifts and can be effectively to suppress the synthetic of allergenic specific IgE in vivo in the mode of allergen specificity.

4, the T cell is to the influence of allergenic specific IgE reaction inhibition

Because CD8 is presented and driven to inner expressed antigen normally by MHC the 1st quasi-molecule ⁺The T cell, whether the inhibition in vivo of Derp5 specific IgE is by CD8 so study ⁺The T cell causes.CD8 does not hive off ⁺Remove or CD4 ⁺The splenocyte of removing moves in the natural receptor to interrupt shifting.Then acceptor is excited with Der p5 hydro-oxidation aluminium adjuvant, and measure the specific IgE reaction of Der p5-.CD4 ^-Cell and the group of not hiving off all show the remarkable inhibition to Der p5-specific IgE.Relatively, CD8 ^-Group does not then show the inhibition effect, expression CD8 ⁺The T cell can be adjusted the IgE that falls well afoot and produce (Fig. 3 B).

5, related cytohormone production in the restraining effect of allergenic specific IgE reaction

Owing to the previous CD8 that studies show that ⁺It is to produce soluble factor by killing reactive B cell of Ag or mat that the T cell suppresses antigen-specific antibody reaction, realize as IFN-γ, IL-4, IL-10 and TGF-β, therefore study the live body alienation and the cytohormone production of spleen t-cell of the pCMVD that the hangs oneself mouse of handling.With 3 whens week after the pCMVD immunity, take out the spleen of mouse and cultivate with Der p5.After cultivating 48 hours, collect supernatant liquor, and measure the concentration of IFN-γ, IL-4, IL-10 and TGF-β with ELISA.The not branch splenocyte of the mouse of dna immunization is at specificity Ag secretion a large amount IFN-γ, and this reacts because of CD8 ⁺Remove and significantly minimizing, but remove CD4 ⁺Cell does not then have this phenomenon.Simultaneously, CD8 ⁺Group produces IL-4 in a small amount.Relatively, IL-4 and IL-10's is created in CD4 ⁺Comparatively remarkable in the group.In these two groups, the generation of TGF-β and indifference, as shown in table 1.

Table 1 illustrates the cytohormone production of the splenocyte of the mouse of handling through pCMVD.With plasmid pCMVD immunity four groups of BALB/c mouse (every group of n=3).After 3 weeks of immunity, with shown in mode cultivate splenocyte with reorganization Der p5 (15 mcg/ml).For IFN-γ and IL-4, collected culture supernatants at 48 hours, and, in the time of 72 hours, collect culture supernatants TGF-β and IL-10, measure cytohormone concentration with ELISA.Shown in the result be the mean value of comprehensive secondary independent experiment, and represent with mean value ± SD.

The secretory cell IFN-γ IL-4 IL-10 TGF-β of cytohormone

(millimicro grams per milliliter) (grams per milliliter slightly) (units per ml) (grams per milliliter slightly) do not hived off 16.5 ± 2.3 870.4 ± 130.5 3.96 ± 0.4 362.0 ± 27.5 CD4 ^-11.41 ± 1.6 198.6 ± 46.3 1.08 ± 0.3 219.3 ± 21.4 CD8 ^-0.9 ± 0.5 719.1 ± 58.6 4.02 ± 0.5 365.2 ± 30.2 nutrient solution 0.7 ± 0.4 15.1 ± 8.0 0.39 ± 0.2 365.1 ± 20.4

Embodiment 2A. materials and methods

1, animal

Use big female BALB/c of 6 to 8 weeks, its be the animal center that derives from medical college of Taiwan Univ. (at first from the Jackson laboratory, Bar Harbor, ME).In each group experiment, select mouse for use with sex and age.

2, the molecular cloning of pCMVG recombinant plasmid

RSj26 cDNA is obtained through the amplification of polymerase chain amplified reaction by pGEX2.5 ' and 3 ' primer sequence is respectively 5 '-TAAC AGATCTATGTCCCCTATACTAGG-3 ' (drawing the bottom line place is Bgl II site) and 5 '-TAAT AAGCTTTGGAGGATGGTC-3 ' (drawing the bottom line place is Hind III site) is contained whole rSj26 sequence.This PCR product digests with Bgl II and Hind III and is cloned among the carrier for expression of eukaryon pCMV2, and pCMV2 is at first derived from pcDNA3 (Invitrogen), and called after pCMVG.The propagation of plasmid is carried out in the SURE bacterial strain at first, then with Wizard ^TMDna purification system (prestige state Madison Promega company) carries out the purifying of plasmid according to manufacturer's indication.With 260 and 280nm extinction and agarose gel electrophoresis come the quality and quantity of analyzing DNA.

3, research step

Originally mouse carries out sensitization with 10 microgram rSj26 and 4 milligrams of aluminium hydroxides (dragon uncle Wyeth pharmaceutical factory, Australia) by peritoneal injection.After the sensitization 14 days the time, by 0.1 milligram of PromAce of injection in the abdomen ^R(Ayerst laboratory, New York, New York) is with anesthetized mice.Lower floor's muscle cuts 10 centimeters of skins so that can be directly visible.With syringe needle insert in the musculus quadriceps 0.2 centimeter dark, use the pin of the 27-specification that is connected to 1 milliliter of syringe then, will be injected into mouse respectively in the pCMVG or the pCMV of 100 micrograms among 0.1 milliliter of PBS, 30 micrograms or 3 micrograms.Gene changes when planting back 7 days, and mouse is accepted exciting of 10 microgram rSj26 once again.When exciting back 7 days, kill mouse to carry out Skin biopsy and blood sampling.

4, the purifying of reorganization Sj26

Reorganization Sj26 is come out by purifying in the intestinal bacteria that contain guiding rSj26 synthetic plasmid (pSj26).Intestinal bacteria under 37 ℃, have grow overnight under the situation of vigorous stirring simultaneously in the Luria substratum that contains 100 microgram penicillin.Adding IPTG to 0.1mM also cultivated 3 hours again.At room temperature, cleaning cell precipitation, resuspending was in the 10mM Tris-HCl that contains 5mM EDAT centrifugal 30 minutes of 10000g, 150mM NaCl (TBS, PH7.5) in.3TIU press down proteolytic enzyme phthalein (Sigma), 0.5mM PMSF (Sigma) and 20 mcg/ml DNase (Boehringer) in the presence of, use Braun clarifixator (German B.Braun company) is broken cell with 0.1 millimeter granulated glass sphere.Triton X-100 (1%) is added in the bacterial lysate, again with centrifugal (40000rpm, 20 minutes) predefecation, with the supernatant liquor of lysate by gsh agar tubing string (Sigma).After fully cleaning with the TBS damping fluid, with 5mM dilution to go back the solution of ortho states gsh in 50mM Tris-HCl (pH8.0) molten from rSj26 protein.

5, the mensuration that rSj26 specific IgE and IgG tire in the serum

The amount of rSj26 specific IgE, IgG1 and IgG2a is measured with ELISA.The high board of Costar is with 100 microlitres purified rSj26 or Der p5 bag quilt, and it is to be diluted in bag to be cushioned liquid (0.1M NaHCO ₃, pH8.2) in to concentration be 5 mcg/ml.After 4 ℃ of following overnight incubation, clean plate 3 times and blockaded 2 hours at room temperature with 3% (weight/volume) BSA-PBS damping fluid.Serum sample is diluted in the gelatin damping fluid, when measuring IgG, diluted 1: 100, when measuring IgE, diluted 1: 10, repeat to add in the entering plate with secondary.When the specific IgE in the mensuration serum, use commercially available standard (PharMinigen), and reading is with reference to this standard.Serum sample and standard are 4 ℃ of following overnight incubation.Adding is diluted in the mono-clonal rat anti-mouse IgG Mo Ab (PharMinigen) that the vitamin H in the 0.05% gelatin damping fluid stops and closes, and cultivates 1 hour again.Add antibiotin-alkaline phosphatase (Sigma) (1: 1000) and at room temperature cultivating 1 hour then, clean again 6 times.Add phosphoesterase substrate p-nitrophenyl phosphate disodium salt (Sigma) to carry out color reaction.In little test panel automatic reading instrument (Metertech), under 405nm, read the data of plate.Reading is that the standard serum that merges with 4 mouse is reference, and this mouse adds aluminium hydroxide by peritoneal injection 10 microgram rSj26 or Der p5 at first, again with same dose injection 21 days.Standard serum is calculated as the 100ELISA units per ml.

6, change the AHR that plants the immunoprophylaxis anaphylactogen to bring out with the anaphylactogen gene

Because pCMVD suppresses Der p5 specific IgE synthetic effect and specificity, to mouse, can suppress the AHR that anaphylactogen brings out so whether inventor's test inoculates pCMVD.Carry out measuring the resistance (R of lung again after Der p5 aerosol excites with ultrasonic sprayer _L).In the pCMV mice immunized, the mouse of Der p5 sensitization shows the vagusstoff dosage (14.6 ± 0.5) of remarkable minimizing in PC100, with respect to the pCMVD mice immunized (31.9 ± .2) and the mouse (30.2 ± 1.3) of false agent (sham) sensitization (Fig. 4).Comprehensive these results as can be known, direct gene is changeed and is planted that not only can to suppress the IgE that anaphylactogen brings out synthetic, also can suppress the AHR that anaphylactogen brings out.

7, suppress the AHR that anaphylactogen brings out with direct immigration of anaphylactogen gene

Because all through the anaphylactogen sensitization, the inventor studies whether direct gene is changeed the AHR that plants in the mouse that can downgrade the anaphylactogen sensitization to most of individualities before treatment.Mouse is to add that with 10 microgram Der p5 aluminium hydroxide carries out sensitization by intraperitoneal at first, after 2 weeks of sensitization, with the pCMVD plasmid DMA treatment of various dose.The anaphylactogen gene changes plants the back during one week, and mouse is accepted to suck and excites and the lung functions test.Demonstration is planted in the commentaries on classics of anaphylactogen gene has remarkable effect to AHR, compared to the mouse of handling with false agent.Therefore, directly the AHR (Fig. 5) that can downgrade anaphylactogen and bring out is planted in the commentaries on classics of anaphylactogen gene.

8, suppress the respiratory inflammation that anaphylactogen brings out with direct immigration of anaphylactogen gene

Except measuring lung functions, the inventor carries out the pathogenic course of the AHR that bronchoalveolar lavage brings out with the research anaphylactogen, and measures the anaphylactogen gene and change the influence of planting the respiratory tract cellular infiltration.After measuring lung functions, mouse is accepted bronchoalveolar lavage.Compare with the mouse of handling with false agent, in the mouse of handling with pCMVD, neutrophil significantly reduces (Fig. 6).

9, Histological assessment

Each biopsy sample is again with paraffin embedding with 4% formaldehyde fixed.Cut out 5 microns sections for dyeing with hematoxylin-Yihong (eosin) staining.B. result

1, the anaphylactogen gene changes the minimizing of the rSj26 specific IgE of planting

Can to downgrade in vivo ongoing rSj26 specific IgE synthetic in order whether to study direct injection rSj26 gene, injection by the pCMVG plasmid of various dose between 3 microgram to 100 micrograms in the mouse of rSj26 sensitization.In the mouse that pCMVG handled, the rSj26 specific IgE significantly reduces.Relatively, rSj26 specific IgG 2a does not then have significant difference.In addition, the pCMVGDNA injection can suppress the synthetic of rSj26 specific IgE, and has specificity.Because when the rat of pCMVG processing excites with Derp5, can produce Der p5 specific IgE.Therefore, the direct gene commentaries on classics is planted and can be suppressed in vivo synthetic (Fig. 8) of allergenic specific IgE effectively.

2, the attenuating of inflammatory response in the skin

For research anaphylactogen gene changes the effect of planting treatment dermatitis, injection pCMVG plasmid DNA is to the mouse of rSj26 sensitization.7 days the time, all mouse are done the skin histology inspection after the booster injection.Histological assessment shows in the mouse that pCMV (blank carrier) handled, have significant inflammatory cells to soak into, and in the mouse that pCMV handled, then do not have tangible cellular infiltration.The mouse that pCMVG handled is the skin of showed smooth also, and relatively, and the mouse of handling with pCMVG shows the skin (Fig. 7) of erythema and rare book.Therefore, direct injection pCMVG plasmid can be regulated the inflammation phenomenon of skin.

Claims (11)

1, a kind of recombinant plasmid, it comprises carrier for expression of eukaryon and anaphylactogen gene, and described anaphylactogen is the glutathione S-transferase of dermatophagoides pteronyssinus (Dermatophagoides pteronyssinus) anaphylactogen, Schistosoma japonicum (Schistosoma japonicum).

2, recombinant plasmid according to claim 1, wherein this carrier for expression of eukaryon is to be selected from by the carrier that contains the CMV promotor, to contain the carrier of RSV promotor or contain one group that the carrier of SV40 promotor is formed.

3, recombinant plasmid according to claim 1, it can be used for prevention and treatment allergic asthma and atopic dermatitis.

4, recombinant plasmid according to claim 1, it can be used for suppressing the generation of allergenic specific IgE.

5, a kind of pharmaceutical composition that can be used for preventing and treating allergic asthma and atopic dermatitis, it comprises according to the recombinant plasmid of claim 1 and pharmaceutically acceptable carrier.

6, pharmaceutical composition according to claim 5, it is to offer medicine in the mode of carrying in conveying, the tracheae in intramuscular injection, the nose or intradermal is carried.

7, pharmaceutical composition according to claim 5, wherein this pharmaceutically acceptable carrier is physiological saline, gold bead or liposome

8, a kind of can be used for, suppressed the pharmaceutical composition that allergenic specific IgE produces, and it comprises the recombinant vectors and the pharmaceutically acceptable carrier of claim 1.

9, pharmaceutical composition according to claim 8, it is used for prevention and treatment allergic asthma and atopic dermatitis.

10, pharmaceutical composition according to claim 8, it is to offer medicine in the mode of carrying in conveying, the tracheae in intramuscular injection, the nose or intradermal is carried.

11, pharmaceutical composition according to claim 8, wherein this pharmaceutically acceptable carrier is physiological saline, gold bead or liposome.

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