CN1431305A - Reorganization immunity toxin with high specificity and its preparing method - Google Patents
Reorganization immunity toxin with high specificity and its preparing method Download PDFInfo
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- CN1431305A CN1431305A CN03117130A CN03117130A CN1431305A CN 1431305 A CN1431305 A CN 1431305A CN 03117130 A CN03117130 A CN 03117130A CN 03117130 A CN03117130 A CN 03117130A CN 1431305 A CN1431305 A CN 1431305A
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Abstract
A high-specificity recombinant immunotoxin for preventing and treating autoimmunopathy, rejection reaction and tumors is prepared from IL-18 as target moleculae and the actigve fragment PE38 of pseudomonas aeruginosa's exotoxin through configuring recombinant plasmid, preparing engineering bacteria transfected by said plasmid, expressing immunotoxin IL-18-PE38 and purifying. Its advantage is high target kill to TH1 cells.
Description
One, technical field
The present invention relates to a kind of is target cell with the TH1 cell, and special attack also kills and wounds recombinant immunotoxin of activated T H1 cell and preparation method thereof.
Two, background technology
(Immunotoxins ITs) is the hybrid molecule that is made of jointly guide molecule and lps molecule to immunotoxin, claims biological missile again.Immunotoxin the earliest (being first-generation immunotoxin) is that carrier and lps molecule are formed by chemical coupling, and this para-immunity lps molecule amount is big, stability and homogeneity are relatively poor, and is difficult to scale operation, thereby has limited its application.Along with development of molecular biology, people are merged vector gene and toxin gene by engineered recombinant technology in recent years, through expressing and purifying preparation and get recombinant immunotoxin.The immunotoxin that this novel process produces can effectively overcome the defective of first-generation immunotoxin, has greatly promoted the further investigation of immunotoxin in association area.Relevant immunotoxin is in biomedical application, mainly all concentrate on to some malignant tumour and with the targeted therapy of Ia human diseases.Wherein, be that the immunotoxin of target cell has been obtained good efficacy (referring to Hu HZ, et al.CellImmunol.1997,177:26 at treatment T cellularity autoimmune disorder and graft versus host disease (GvHD) with the T cell; BrennerT, et al.Immunol lett.1999,68:403).The action target spot of these immunotoxins mainly is to select at t cell surface antigen or cytokine receptor, as CD3, CD5, IL-2 etc., after the treatment of accepting these immunotoxins, though having, the corresponding autoimmune disorder or the GvHD state of an illness go down, but toxic side effect is also more remarkable, even makes clinical treatment be forced to stop.The one of the main reasons of these toxic side effect is because the selected guide molecule specificity of this para-immunity toxin is not good enough, kills all T cells with often causing non-selectivity.Along with the progress of basic immunology, people recognize the autoreactive T cell of the various autoantigens of identification, are actually by CD
4 +The TH1 cell is born, and CD
4 +TH2 only has certain regulating effect (referring to LiblauRS, et al.Immunol.Today.1995,16:34; Costa GL, et al.J.Immunol.2000,164:3581).Prompting thus if can utilize the higher immunotoxin of specificity only these activatory autoreactivities TH1 cell to be killed, then might remedy above-mentioned defective, sets up corresponding specificity autoimmunization tolerance, realizes the purpose of treatment autoimmune disorder.Autoimmune disorder (autoimmune disease) is because a variety of causes causes immunity system that autoantigen is launched a offensive, and causes autologous tissue's damage and handicapped one big class disease.Present treatment measure mainly is to adopt various non-specific immunosuppressor, but often make patient's immunity system be subjected to ubiquity suppress and cause infecting, the generation of tumour and bone marrow depression etc., and the recurrence that can not ward off disease effectively.Thereby impel the angle of the investigator of association area from immunotherapy, seek a kind of special, safe and effective method of treatment: as MHC blocked method, CD
4Molecule blocked method, TCR blocked method, cytokine therapy, T cell inoculation and oral antigen are induced tolerance or the like, but these methods or because of specificity is not high, curative effect is unstable, or, the toxic side effect that be difficult to overcome big because of technical difficulty etc. can not successfully carry out the transition to clinical (referring to Waldor MK, et al.Science.1985,227:415; ChenY, et al.Nature.1995,376:177; Weiner HL, Immunology Today.1997,18:355), thereby the treatment of autoimmune disorder remains the clinical a great problem of puzzlement so far.
Three, summary of the invention
The present invention is directed to the deficiency that prior art exists, provide a kind of recombinant immunotoxin of novel special attack TH1 cell, for clinical urgency treatment of autoimmune diseases to be solved provides a kind of new departure, for a new way is opened up in the application of recombinant immunotoxin.
Technical scheme of the present invention is: a kind of plasmid for preparing recombinant immunotoxin is provided, and this kind plasmid has accompanying drawing 1 described IL-18cDNA base sequence (from gene bank) and accompanying drawing 2 described Pseudomonas aeruginosas ectotoxic active fragments PE38cDNA base sequences (from gene bank) are formed by connecting.A kind of recombinant immunotoxin of high degree of specificity is provided, this kind recombinant immunotoxin is formed by above-mentioned plasmid expression, purifying, be guide molecule with IL-18, be lps molecule, have special attack and kill and wound the characteristic of activated T H1 cell with the ectotoxic active fragments PE38 of Pseudomonas aeruginosa.IL-18 is a kind of novel cytokine of finding in recent years, and in the process of immunne response, the activated T cell will be expressed multiple acceptor, as acceptor and the number of chemical chemokine CCR-5 of cytokine IL-2, IL-12, IL-18, the acceptor of CXCR-3 etc.In the membrane receptor of these T cells, confirmed that now the IL-18 receptor expression only limits to activated T H1 cell, and the TH2 cell is not expressed, and the expression of this membrane receptor be stable and continue (referring to Okamura H, et al.Nature.1995,378:88; Chan WL, et al.J.Immunol.2001,167:1238; Nakanishi K, et al.Annu.Rev.Immunol.2001,19:423), therefore, the IL-18 acceptor not only can be used as the sign of identification TH1 cell and TH2 cell, also provides an ideal target position for relevant immunotherapy.It is guide molecule that the present invention selects IL-18 for use, the ectotoxic active fragments PE38 of Pseudomonas aeruginosa is a lps molecule, preparation IL-18-PE38 recombinant immunotoxin, special attack also kills and wounds activated T H1 cell, induce the autoimmunization tolerance, thereby solved the not good enough problem of original immunotoxin specificity, realized the effectively purpose of treatment autoimmune disorder.
The construction of recombinant plasmid method may further comprise the steps successively:
1, the IL-18 gene with animal increases with RT-PCR;
2, the IL-18 gene fragment of above-mentioned amplification is pressed the TA cloning and realize and the plasmid vector reorganization that transfection is increased again, filters out then to contain the segmental clone of correct insertion to the competence bacterium;
3, restriction endonuclease map is analyzed recombinant plasmid, and carries out dna sequence analysis, to guarantee that IL-18 inserts segmental exactness in the recombinant plasmid;
4, restriction enzyme digests the plasmid DNA of the IL-18 and the PE38 of correct sequence respectively, and purifying DNA fragment is connected into IL-18 and PE38 fragment in the plasmid vector, promptly is built into recombinant plasmid;
5, measure the expression activity of recombinant plasmid with the external translation kits of TNT.
The available plasmid vector of construction recombination plasmid has pET27, PRKL459K, PGEX-2T.
The preparation method of recombinant immunotoxin IL-18-PE38 may further comprise the steps successively:
1, the preparation of the engineering bacteria of above-mentioned recombinant plasmid transfection;
2, the expression and purification of immunotoxin.
The concrete operations of above steps are as follows:
1, the engineering bacteria of preparation recombinant plasmid transfection
(1) preparation competence bacterium;
(2) above-mentioned recombinant plasmid is changed in the cell of competence bacterium;
(3) by the synthetic expression activity that suppresses the measuring engineering bacteria of albumen.
2, the expression and purification of immunotoxin
(1) engineering bacteria of recombinant plasmid transfection is inoculated in the substratum cultivates;
(2) collect the engineering bacteria bacterium liquid efficiently express and break the bacterium processing, obtain the crude extract of immunotoxin IL-18-PE38;
(3) with Glutahione Sepharose4B or affinity column immunotoxin IL-18-PE38 crude extract is carried out purification process;
(4) by the synthetic expression activity that suppresses the measuring immunotoxin of albumen.
The available competence bacterium of engineering bacteria of preparation recombinant plasmid transfection has E.coli.BL21, DH5 α, JM109.
Novel immunotoxin involved in the present invention and preparation method thereof has the following advantages and positively effect:
1, selecting novel cytokine IL-18 is guide molecule, because the IL-18 acceptor only is expressed in activated T H1 cell surface, the TH2 cell surface is not expressed, thereby by the recombinant immunotoxin of IL-18 guiding only at the TH1 cell, thereby make vectored attacks more special, more effective, reduce the toxic side effect of clinical application.
That 2, lps molecule is selected for use is the ectotoxic active fragments PE38 of Pseudomonas aeruginosa, thereby has avoided in the blood ubiquitous diphtheria extracellular toxin antibody to the bioactive interference of immunotoxin.
3, PE38 is the ectotoxic active fragments of Pseudomonas aeruginosa, and cell land and a disulfide linkage than PE40 has further removed the PE molecule can further reduce non-specific binding and reduce untoward reaction.
4, immunotoxin IL-18-PE38 is the recombinant immunotoxin preparation that obtains by the DNA recombinant technology, and it has been avoided the preparation technology of original chemical coupling immunotoxin complexity and has been difficult to the shortcoming of marking, and makes its easier industrialization.
5, immunotoxin IL-18-PE38 can pass through the multipath administration.1. directly utilize the IL-18-PE38 protein formulation; 2. utilize IL-18-PE38 DNA preparation, promptly adopt the method for IL-18-PE38 expression plasmid transfection live body muscle cell, the IL-18-PE38 expression plasmid is directly imported animal muscle, IL-18-PE38 is efficiently expressed in skeletal muscle and play therapeutic action.
6, immunotoxin IL-18-PE38 also can be used for the control of graft-rejection and some tumour.
Four, description of drawings
Fig. 1 is the base sequence of IL-18cDNA;
Fig. 2 is the base sequence of PE38cDNA.
Five, embodiment
Embodiment 1: the prokaryotic expression plasmid that makes up preparation recombinant immunotoxin IL-18-PE38
The base sequence of IL-18cDNA as shown in Figure 1, the base sequence of PE38cDNA as shown in Figure 2, carrier is selected pET27 for use.Concrete steps are as follows:
1, mouse IL-18 gene increases with RT-PCR
(1) the guanidinium isothiocyanate single stage method is extracted total RNA of activating macrophage, gets 10 μ g Oligo (dT) and mixes, the row reverse transcription.
(2) primer of pcr amplification is
The upstream: 5 '-CGGAATTCATAACTTTGCCGACTTCAGTGTAC
The downstream: 5 '-CGGAATTCACTAACTTTGATGTAAGTTAGTGAGAG
(3) PCR reaction conditions: 94 ℃ 0.5 minute, 56 ℃ 0.5 minute, 72 ℃ 1 minute, 30 circulations were extended 10 minutes for back 72 ℃.
(4) the capable 2% agarose gel electrophoresis analysis of amplified production.
2, the IL-18 gene segment of above-mentioned amplification is pressed the TA cloning and realized the reorganization with plasmid vector pET27, transfection is increased to the competence bacterium again, filters out to contain the segmental clone of correct insertion.
3, restriction endonuclease map is analyzed recombinant plasmid, and carries out dna sequence analysis, to guarantee that IL-18 inserts segmental exactness in the recombinant plasmid.
4, restriction enzyme digests the plasmid DNA of the IL-18 and the PE38 of correct sequence respectively, and purifying DNA fragment is connected into IL-18 and PE38 fragment respectively in the pET27 expression plasmid carrier, constitutes the prokaryotic expression plasmid pET27-IL-18-PE38 of IL-18-PE38.
5, the determination of activity of IL-18-PE38 prokaryotic expression plasmid, with the external translation kits of TNT, translate corresponding proteins matter with the pET27-IL-18-PE38 plasmid DNA as template, stimulate the mouse spleen cell with ConA again, activation T cell wherein, these activated T cells will be examined and determine the activity of IL-18-PE38 prokaryotic expression plasmid as target cell.
Embodiment 2: preparation recombinant immunotoxin IL-18-PE38
Processing step is as follows successively:
1, the engineering bacteria of preparation prokaryotic expression plasmid pET27-IL-18-PE38 transfection
(1) preparation competence bacterium
A. with intestinal bacteria E.coli BL21 streak inoculation on the nutrient agar flat board, cultivated 12-16 hour for 37 ℃.
B. take out single bacterium colony of 2-3mm size from flat board, move to and contain in the 3mlLB substratum, 37 ℃ of shaking culture are spent the night.Get next day bacterium liquid 1ml be seeded to contain the 100mlLB substratum triangular flask in, 37 ℃ of strong shaking culture of 300rpm 3 hours make cell concn reach 5 * 10
7Individual cell/ml, at this moment, the OD of bacterium
600Generally about 0.4.
C. the bacterium of cultivating was placed on ice 10 minutes, transfer to then in the ice-cold 50ml centrifuge tube that aseptically process crosses.
D.4 ℃ centrifugal, 4000g * 10 minute, abandoning supernatant reclaims cell.
E. ice-cold 0.1mol CaCl
2The resuspended thalline of 10ml was put ice bath 30 minutes.
F.4 ℃ centrifugal, 4000g * 10 minute, abandoning supernatant.The 0.1mol CaCl that adds the precooling of 4ml ice again
2Resuspended thalline, this is the competence bacterium.
(2) the prokaryotic expression plasmid pET27-IL-18-PE38 with embodiment 1 preparation changes in the cell of competence bacterium
A. get fresh competence bacterium 100 μ l under the sterile state and place sterile test tube, add the 10ngpET27-IL-18-PE38 plasmid DNA, rotate the mixing content gently, placed on ice 30 minutes.
B.42 in 90 seconds of ℃ heat-shocked, do not shake test tube, put back to rapidly in the ice, cell is cooled off 1-2 minute after, add 1000 μ lSOC substratum, 37 ℃ of shaking culture 45 minutes.
C. get 80 μ l transformation mixtures, be laid on L type glass rod and contain on the antibiotic LB agar plate of proper concn, placed 20 minutes under the room temperature, treat that solution is absorbed by agar after, is inverted 37 ℃ of cultivations 16-20 hour; After treating that bacterium colony grows, aseptic picking list bacterium colony carries out enlarged culturing.
(3) by the synthetic expression activity that suppresses the measuring engineering bacteria of albumen
ConA activated T cell inoculation is cultivated (about 5 * 10 in 96 porocyte plates
4Individual cells/well), add and not contain leucic RPMI1640 substratum and engineering bacteria culture supernatant 20 μ l, hatch jointly behind the mixing 20 hours (37 ℃, 5%CO
2), add H again
3Leucine (4.5 μ ci/ml) 10 μ l, the synthetic percentage that suppresses of albumen is measured and calculated to collecting cell after 4 hours, determines the expression activity of engineering bacteria.
2, the expression and purification of recombinant immunotoxin IL-18-PE38
(1) will be inoculated in the engineering bacteria of pET27-IL-18-PE38 plasmid and contain in the antibiotic LB substratum of finite concentration, 30 ℃ of shaking tables are cultured to OD
600Reach 0.5, go to 42 ℃ and continue to cultivate 5 hours.
(2) collect the engineering bacteria bacterium liquid of cultivating that efficiently expresses, 4 ℃ of centrifugal 5000rpm 15 minutes, abandon supernatant.
(3) the wet bacterium of every gram adds the 3mlTE damping fluid, and high strength supersonic (20kHz) breaks bacterium and handles.
(4) centrifugal 10000g 30 minutes, collects supernatant liquor.
(5) get 10 μ l supernatants and 10 μ l2 * SDS sample buffer mixing, in boiling water, boiled 5 minutes, go up sample then and carry out 10%SDS-PAGE electrophoresis (200V, 50-60 minute), gel coomassie brilliant blue staining.
(6) the IL-18-PE38 protein crude extract administration is carried out purifying with 50%Glutahione Sepharose 4B or affinity column, the recombinant immunotoxin behind the purifying is done further identification and analysis by SDS-PAGE electrophoresis and Western blot again.
(7) the bioactive mensuration of recombinant immunotoxin IL-18-PE38
The bioactive mensuration of recombinant immunotoxin IL-18-PE38 adopts the synthetic inhibition test of albumen to carry out, promptly by ConA activated T cell inoculation 96 porocyte plates are cultivated (about 5 * 10
4Individual cells/well), add the IL-18-PE38 recombinant protein sample 10 μ l (control group is the irrelevant cell of IL-18 receptor negative) do not contain leucic RPMI1640 substratum and serial dilution, hatch jointly behind the mixing 20 hours (37 ℃, 5%CO
2), add H again
3Leucine (4.5 μ ci/ml) 10 μ l, collecting cell after 4 hours is measured radioactivity on the Beckman scintillation counter, calculate the synthetic percentage that suppresses of albumen, to determine the biological activity of IL-18-PE38 recombinant immunotoxin.
Claims (8)
1, a kind of plasmid for preparing recombinant immunotoxin is characterized in that it has accompanying drawing 1 described IL-18cDNA base sequence and the ectotoxic active fragments PE38cDNA of accompanying drawing 2 described Pseudomonas aeruginosas base sequence is formed by connecting.
2, a kind of recombinant immunotoxin of high degree of specificity is characterized in that this recombinant immunotoxin is formed by the described plasmid expression of claim 1, purifying, has special attack and kills and wounds the characteristic of activated T H1 cell.
3, the described construction of recombinant plasmid method of a kind of claim 1 is characterized in that may further comprise the steps successively:
(1) the IL-18 gene with animal increases with RT-PCR,
(2) the IL-18 gene fragment of above-mentioned amplification is pressed the TA cloning and realize and the plasmid vector reorganization that transfection is increased again to the competence bacterium, filter out then to contain the segmental clone of correct insertion,
(3) restriction endonuclease map is analyzed recombinant plasmid, and carries out dna sequence analysis, inserts segmental exactness with IL-18 in the assurance recombinant plasmid,
(4) restriction enzyme digests the plasmid DNA of the IL-18 and the PE38 of correct sequence respectively, and purifying DNA fragment is connected into IL-18 and PE38 fragment in the plasmid vector, promptly is built into recombinant plasmid,
(5) use the external translation kits of TNT to measure the expression activity of recombinant plasmid.
4, construction of recombinant plasmid method according to claim 3 is characterized in that the available plasmid vector of construction recombination plasmid has pET27, PRKL459K, PGEX-2T.
5, the preparation method of the recombinant immunotoxin of the described high degree of specificity of a kind of claim 2 is characterized in that may further comprise the steps successively:
(1) preparation of the engineering bacteria of the described recombinant plasmid transfection of claim 1,
(2) expression and purification of immunotoxin.
6, the preparation method of the recombinant immunotoxin of high degree of specificity according to claim 5 is characterized in that:
(1) concrete steps of the engineering bacteria of preparation recombinant plasmid transfection
A. prepare the competence bacterium,
B. the described recombinant plasmid of claim 1 is changed in the cell of competence bacterium,
C. synthesize the expression activity that suppresses the measuring engineering bacteria by albumen,
(2) concrete steps of immunotoxin expression and purification
A. the engineering bacteria of recombinant plasmid transfection is inoculated in the substratum and cultivates,
B. collect the engineering bacteria bacterium liquid that efficiently expresses and break the bacterium processing, obtain the crude extract of immunotoxin,
C. with Glutahione Sepharose4B or affinity column the crude extract of immunotoxin is carried out purification process,
D. by the synthetic expression activity that suppresses the measuring immunotoxin of albumen.
7, the preparation method of the recombinant immunotoxin of high degree of specificity according to claim 6, it is characterized in that the synthetic expression activity that suppresses the measuring immunotoxin of albumen is by ConA activated T cell inoculation is cultivated in cell plate, add the immunotoxin recombinant protein sample do not contain leucic RPMI1640 substratum and serial dilution, behind the mixing at 37 ℃, 5%CO
2Hatched jointly 16～20 hours, and added the H of 3～4.5 μ ci/ml again
3Leucine, collecting cell after 2～4 hours is measured radioactivity on the Beckman scintillation counter, calculate the synthetic percentage that suppresses of albumen.
8, according to the preparation method of the recombinant immunotoxin of claim 6 or 7 described high degree of specificity, it is characterized in that the available competence bacterium of engineering bacteria for preparing the recombinant plasmid transfection has E.coli.BL21, DH5 α, JM109.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1318591C (en) * | 2004-07-08 | 2007-05-30 | 杨安钢 | Immune tbid gene and its coded protein and application |
| CN100444895C (en) * | 2006-12-05 | 2008-12-24 | 广州市恺泰生物科技有限公司 | Immunomodulator for treating malignant tumor |
| CN110423273A (en) * | 2017-11-01 | 2019-11-08 | 深圳市国创纳米抗体技术有限公司 | High neutralization activity anti Bacillus pyocyaneu Flugge exotoxin A nano antibody and its application |
-
2003
- 2003-01-10 CN CN03117130A patent/CN1431305A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1318591C (en) * | 2004-07-08 | 2007-05-30 | 杨安钢 | Immune tbid gene and its coded protein and application |
| CN100444895C (en) * | 2006-12-05 | 2008-12-24 | 广州市恺泰生物科技有限公司 | Immunomodulator for treating malignant tumor |
| CN110423273A (en) * | 2017-11-01 | 2019-11-08 | 深圳市国创纳米抗体技术有限公司 | High neutralization activity anti Bacillus pyocyaneu Flugge exotoxin A nano antibody and its application |
| CN110423273B (en) * | 2017-11-01 | 2022-03-11 | 深圳市国创纳米抗体技术有限公司 | Anti-pseudomonas aeruginosa exotoxin A nano antibody and application thereof |
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