CN100352512C - Recombinant human co-stimulatory molecule bacilli-calmette-guerin strain and process for making same - Google Patents
Recombinant human co-stimulatory molecule bacilli-calmette-guerin strain and process for making same Download PDFInfo
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Abstract
The present invention discloses a bacilli-calmette-guerin strain of a recombinant human co-stimulatory molecule and a preparation method thereof, which belongs to gene engineering techniques. The strain has the preservation number of CGMCC No. 1120. The method of the present invention comprises the following steps: firstly, polymerase chain reaction is carried out to a hB7-2(IgC+IgV) segment, and a plasmid pYL-hB7-2 is established; the plasmid is converted, an E. Coli-pYL-hB7-2 monoclonal bacterial colony is amplified, and the plasmid is extracted and purified; cataphoresis, enzyme cutting and PCR reaction are carried out to identify the pYL-hB7-2 plasmid; the bacilli-calmette-guerin strain is electrotransformed, and the rBCG-hB7-2 monoclonal bacterial colony is selected, amplified and identified. Compared with a wild type bacille calmette-guerin strain, the bacilli-calmette-guerin strain of the present invention reduces dosages under the condition that the same immune effect is realized or even immune effect is increased, so toxic and side effect is reduced. The present invention avoids the disadvantages of high cost and repeated perfusion brought by the direct use of a cell factor. The strain can secrete cell factors at the most appropriate time and in the most appropriate position. Strong and persistent anti-tumor immunity can be obtained by one-time inoculation. The present invention provides a new formulation for the clinical treatment of bladder tumors.
Description
Technical field
The present invention relates to technique for gene engineering, specifically is a kind of recombined human costimulatory molecules strain of BCG vaccine and preparation method thereof.
Background technology
Bacillus calmette-guerin vaccine (BCG) is the biological immunomodulator that the strong mycobacterium bovis BCG attenuation of a kind of virulence forms, and has the safety of height and few severe complication, and the whole world is widely used in prevention tuberculosis.
Intravesical perfusion wild type bacillus calmette-guerin vaccine has been obtained gratifying clinical efficacy at aspects such as prophylaxis of tumours recurrence, treatment tumors remaining and cancer in situ, but still have the patient of 30-45% reactionless to intracavity bacillus calmette-guerin vaccine perfusion therapy, in addition, the perfusion of intracavity bacillus calmette-guerin vaccine can cause bladder part and general reaction, severe complication can appear in 5% patient, but 0.5% even threat to life, make its prevention and treat shallow table tumor of bladder to be subjected to certain limitation.
Chinese patent 1353183 discloses " a kind of recombinant strain of BCG vaccine is set up and used ", this invention is by making up a kind of rBCG that to carry two kinds of exogenous genes be IL-2 gene and GFP gene, make original BCG have the function of IL-2 and GFP, can utilize the action principle of this rBCG-GFP-IL-2 bacterial strain research BCG or recombinant BCG, remedy the deficiency of biological preparation simultaneously with polyporusum bellatus, thereby set up a kind of method of effective elimination tumor cell.The technical characterictic of this invention is to utilize green fluorescent protein (GFP) as reporter gene, inserts the IL-2 genetic fragment in shuttle plasmid, constitutes compound expressing green fluorescent protein and interleukin-22 recombinant BCG (rBCG-GFP-IL-2) bacterial strain.
At present international and domestic have the research that the gene of other cytokine is changed over to BCG, do not adopt costimulatory molecules to form the strain of BCG vaccine that the double stimuli signal is treated but still have.
Summary of the invention
The present invention is reactionless to the treatment of intravesical perfusion wild type bacillus calmette-guerin vaccine in order to solve the part patients with bladder cancer, and the wild type bacillus calmette-guerin vaccine produces serious toxicity to the part patient, and provide a kind of efficient, low side effect, immunoprophylaxis and the special-purpose gene recombinaton strain of BCG vaccine of treatment, i.e. recombined human costimulatory molecules strain of BCG vaccine and preparation method thereof
Theoretical foundation of the present invention: mainly by the T cell mediated, the latter's activation and propagation need two signals to body to the immunne response of tumor: antigenic specificity first signal and by the secondary signal of the non-antigenic specificity of costimulatory molecules mediation.The stimulation that lacks costimulatory signal can cause the tolerance of T cellular immunization or cause the inductive T cell death of activation.Costimulating factor comprises bone-marrow-derived lymphocyte activation antigen B7 molecule etc., and the B7 molecule is wherein most important a kind of, does not express or weak expression in most of tumors, and this is to cause one of major reason that tumor is escaped.
Therefore study a kind of efficient, low side effect, the special-purpose gene recombinaton bacillus calmette-guerin vaccine vaccine of immunoprophylaxis and treatment has become the key subjects of basis and clinical research.Recombinant bacillus Calmette-Guerin vaccine be with bacillus calmette-guerin vaccine as engineering bacteria, utilize technique for gene engineering that exogenous gene is imported in the bacillus calmette-guerin vaccine, rely on bacillus calmette-guerin vaccine in the host, to duplicate, express exogenous antigen, to induce specificity humoral and cellular immunization to multiple disease.
The present invention realizes by following technical scheme.
A kind of recombined human costimulatory molecules strain of BCG vaccine, escherichia coli-mycobacterium shuttle expression carrier of its expressing human costimulatory molecules B7-2, in bacillus calmette-guerin vaccine, express, obtain having the recombinant strain of BCG vaccine rBCG-hB7-2 of secretion activity costimulatory molecules B7-2 function.
A kind of preparation method of recombined human costimulatory molecules strain of BCG vaccine, its rebuild escherichia coli-mycobacterium shuttle expression plasmid, with electroporation with the recombinant plasmid transformed bacillus calmette-guerin vaccine.
Described preparation method, its hB7-2 (IgC+IgV) fragment polymerase chain reaction (PCR) be adopt forward primer 5 '-gctcctctgaagattc-3 ', downstream primer 5 '-agctgtaatccaaggaatg-3 '.
Described preparation method, be established as pYL-hIFN-α-2B plasmid and hB7-2 (IgC+IgV) polymerase chain reaction (PCR) product of plasmid pYL-hB7-2 are connected with behind BamH I and the Xho I enzyme action.
Described preparation method, the conversion of its plasmid are that every pipe adds the competence antibacterial, add the pYL-hB7-2 plasmid, mix the back and transform, and are inoculated on the LB solid medium of kanamycin, select positive bacterium colony.
Described preparation method, the amplification of its monoclonal bacterium colony are picking monoclonal bacterium colonies, and it is inoculated in the LB fluid medium of kanamycin, and plasmid extraction kit extracts plasmid.
Described preparation method, the electricity of its bacillus calmette-guerin vaccine transforms: get centrifugal behind the bcg bacteria liquid ice bath, abandon supernatant; Pre-cooling glycerol washing precipitation secondary; Plasmid DNA is dissolved in liquid, then competence bcg bacteria liquid is poured into, fully mix the back electricity and transform, be applied to again on the 7H10 solid culture ware that contains kanamycin, choose the positive bacterium colony of kanamycin after 3～4 weeks.
Described preparation method, the selecting of the positive bacterium colony of its rBCG-hB7-2, amplification and the order-checking of PCR product are correct, and the recombinant bacillus Calmette-Guerin vaccine expression product is verified through enzyme linked immunosorbent assay (ELISA) and lymphocyte proliferation assay.
Zhi Bei recombined human costimulatory molecules strain of BCG vaccine and preparation method thereof like this, its culture presevation number: CGMCC No.1120.
Recombinant bacillus Calmette-Guerin vaccine of the present invention is compared with the wild type bacillus calmette-guerin vaccine, 1. because recombinant bacillus Calmette-Guerin vaccine can secrete cytokines, is reaching same even is increasing under the condition of immune effect and can reduce consumption than the wild type bacillus calmette-guerin vaccine, thereby reducing toxic and side effects; 2. recombinant bacillus Calmette-Guerin vaccine high cost and the dabbling repeatedly shortcoming of can secrete cytokines having avoided direct use cytokine to bring; 3. recombinant bacillus Calmette-Guerin vaccine can be at the most suitable time and position secrete cytokines.
This genetic engineering strain of BCG vaccine is compared with the mixed vaccine that mycobacterial adjuvants add the pathogen antigen preparation, and rBCG integrates adjuvant and carrier, and the various exogenous genes of holding concurrently and live strain are in one, and once inoculation can obtain strong and persistent antineoplastic immune.Strain of BCG vaccine rBCG-hB7-2 wild type vaccine than before has the advantage of high-efficiency low-toxicity side effect.
The present invention is the advantage in the bladder cancer immunization therapy in conjunction with bacillus calmette-guerin vaccine and costimulatory molecules: (1) bacillus calmette-guerin vaccine: strengthen the tumor cell immunogenicity, lure immunocytes such as lymphocyte, produce cytokine.(2) B7: provide costimulatory signal, activated lymphocyte, NK cell, killing tumor cell.Chong Zu treatment can be strengthened first signal with strain of BCG vaccine like this, and provides costimulatory signal, dual function to reach simultaneously to reduce the bacillus calmette-guerin vaccine consumption and reduce toxic and side effects, and can obviously improve the purpose of therapeutic effect.
Description of drawings
Accompanying drawing is reorganization PLY-hB7-2 plasmid figure.
1hB7-2 gene function region sequence, 2 kalamycin resistance genes, 3 escherichia coli origin of replications, 4 mycobacterium origin of replications, 5 heat shock proteins, 60 promoteres, 6 signal sequences among the figure.
The specific embodiment
One. material
(1) bacterial strain: escherichia coli are DH5 α, and this institute preserves.Bacillus calmette-guerin vaccine is available from Beijing Biological Product Inst., Denmark I type.
(2) plasmid: vector plasmid pYL-hIFN-α-2B plasmid is provided by YI professor LUO of American I WA university.HuB7-2 (the IgV+C)/pGEX-4T-3 plasmid that contains hB7-2cDNA comes precious elder teacher to provide by Xian Medical Univ.
(3) key instrument equipment
1. microplate reader: SUNRISE TECAN A-5082 Austria
2. constant incubator and desk centrifuge:
3. low-temperature and high-speed centrifuge: the BECKMAN J2-HS U.S.
4.PCR instrument: PERKIN ELEMER GeneAmp PCR System 2400 U.S.
5. electroporation: Genepulser Electroprotocol, the U.S.
(4) main agents and solution preparation
1. glue reclaims test kit: E.Z.N.A Gel Extraction Kit
2. purification kit: TaKaRa DNA Fragment Purification Kit Ver 2.0
3. plasmid extraction kit: E.Z.N.A Plasmid Midiprep Kit
4. enzyme linked immunosorbent assay (ELISA) test kit: ELISA for s-hCD86 DIACLONE company
5. culture medium: the Middlebrook 7H9 Broth DIFCO company U.S. Middlebrook 7H10 Agar DIFCO company U.S.
6.T
4DNA Lingase, BamH I, Xho I, Taq enzyme, DNA Marker DL2000, DNA Marker DL15000 are all available from TaKaRa company
7.Tween-80:0442-500ml AMRESCO packing
8. bovine serum albumin: grind scientific ﹠ technical corporation of institute available from Chinese Academy of Medical Sciences's blood
Two. method
(1) hB7-2 (IgC+IgV) fragment PCR reaction:
1. forward primer: 5 '-gctcctctgaagattc-3 '
Downstream primer: 5 '-agctgtaatccaaggaatg-3 ', 50 μ mol/l.
2. about 100 μ g/ml of template plasmid: hB7-2 (IgV+C)/pGEX-4T-3, PCR reaction system reaction cumulative volume 50 μ l, reaction condition: 94 ℃ of pre-degeneration 5 minutes, 94 ℃ of degeneration 30 seconds, 57 ℃ of annealing 50 seconds, 72 ℃ were extended 50 seconds, last 72 ℃ were extended totally 30 circulations 7 minutes.
(2) foundation of plasmid pYL-hB7-2:
1.pYL-hIFN-α-2B plasmid and hB7-2 (IgC+IgV) PCR product spend the night for 37 degrees centigrade with BamH I and Xho I enzyme, E.Z.N.A glue reclaims test kit and reclaims DNA by explanation behind the electrophoresis.
2. connect:
| 10×T 4PYL-hIFN-α-2B plasmid T behind hB7-2 (IgC+IgV) enzyme action behind the DNA Ligase Buffer enzyme action 4?DNA?Ligase | 2.5 μ l 0.3pmol 0.03pmol 1 μ l adds water to 25ml |
The connection of spending the night in 16 ℃ of water-baths adds the 3M NaAC of 2.5 μ l, adds the cold dehydrated alcohol of 62.5 μ l, places 30～60 minutes for-20 ℃, and centrifugal recovery precipitation precipitates vacuum drying with 70% cold washing with alcohol.The two disinfectants that steam of 25～50 μ l dissolve, and get 10～20 μ l and can be transformed in the 100 μ l competence escherichia coli.
(3) conversion of plasmid:
1. every pipe adds 100 μ l competence antibacterials, adds 1 μ l pYL-hB7-2 plasmid, fully mixes ice bath 30 minutes.
2. sample was placed 42 ℃ of heat shocks 45 seconds, to increase the expression vector conversion ratio.
3. ice bath 5 minutes once more, every pipe adds 42 ℃ of LB culture medium of 0.9ml, mixing.
4. cultivated 1 hour 30 minutes 37 ℃ of water-baths.
5. 200 μ l are transformed the back sample and be inoculated on the LB solid medium that contains 30 μ g/ml kanamycin, hatched 24～48 hours for 37 ℃.Select positive bacterium colony.
(4) amplification of E.Coli-pYL-hB7-2 monoclonal bacterium colony, plasmid extract and purification:
1. the amplification of monoclonal bacterium colony.
Picking monoclonal bacterium colony is inoculated into 10ml with it and contains in the LB fluid medium of 30 μ g/ml kanamycin, and 37 ℃, 150rpm shaken cultivation 10 hours.
2.E.Z.N.A Plasmid Midiprep Kit plasmid extraction kit extracts plasmid, presses the test kit explanation and extracts plasmid, the resuspended DNA of 500 μ l high purity waters.The volume of water is decided according to DNA concentration in the experiment.
(5) electrophoresis, enzyme action and PCR identify the pYL-hB7-2 plasmid:
The PCR of pYL-B7-2 and sequencing primer:
Upstream-5 '----ATCTTACCCATACGACGTCC----3 '
Downstream-5 '-----GTTAACTACGTCGACATCG---3 '
1.PCR reaction system: reaction cumulative volume 50 μ l; PCR reaction system reaction cumulative volume 5 μ l, reaction condition: 94 ℃ of pre-degeneration 5 minutes, 94 ℃ of degeneration 30 seconds, 57 ℃ of annealing 50 seconds, 72 ℃ were extended 50 seconds, and last 72 ℃ were extended totally 30 circulations 7 minutes.1% sepharose electrophoresis, the observed result correct position.
2. order-checking:, cut glue purification and reclaim with the pYL-B7-2 plasmid amplification rear electrophoresis of above-mentioned structure.Above-mentioned primer send TaKaRa company to check order.
(6) electricity of bacillus calmette-guerin vaccine transforms:
1. get bcg bacteria liquid (OD of 600nm place value is about 0.5) 50ml, ice bath centrifugal 10 minutes of 4000rpm after 1.5 hours.
2. abandon supernatant, 10%4 ℃ of pre-cooling glycerol 20ml washing precipitation secondaries.
3. prepare: 5-15 μ g plasmid DNA (being dissolved in 10-2 μ l liquid) is placed the 1.5ml pipe.Then 100 μ l competence bcg bacteria liquid are poured into, made cumulative volume be no more than 150 μ l, fully mix.Join in the electric revolving cup of 0.1cm.
4. electric commentaries on classics condition: voltage 1.8KV, electric capacity 25 μ F, resistance 100 Ω, 0.1cm electricity revolving cup.
5. electricity changes post processing: the 7H9 culture medium that rapidly 1ml is not contained kanamycin after electricity changes joins in the electric revolving cup.Change the 7ml test tube behind the mixing over to, 180rpm shaken cultivation 5 hours.Get the above-mentioned culture fluid of 200ul and be applied on the 7H10 solid culture ware that contains the 30ug/ml kanamycin, seal film and tightly seal culture dish, choose positive bacterium colony after 3～4 weeks.
(7) preparation of the selecting of rBCG-hB7-2 monoclonal bacterium colony, amplification and polymerase chain reaction (PCR) template:
1. transfer single colony inoculation in the 10ml 7H9 fluid medium that contains 30 μ g/ml kanamycin, 150rpm, 37 ℃ of shaken cultivation.
2. when the OD of 600nm place value is 1.0 left and right sides, get 1ml bacterium liquid, centrifugal 10 minutes of 10000rpm.
3. remove supernatant, with distilled water washing precipitation three times, the centrifugal supernatant that goes, add 30 μ l distilled waters in precipitate, mixing boiled 10 minutes.
4.10000rpm after centrifugal 10 minutes, get supernatant 20 μ l and carry out the PCR reaction as template.
(8) evaluation of the positive bacterium colony of rBCG-hB7-2:
The PCR of pYL-B7-2 and sequencing primer:
Upstream-5 '----ATCTTACCCATACGACGTCC----3 '
Downstream-5 '-----GTTAACTACGTCGACATCG---3 '
1. polymerase chain reaction (PCR) reaction: reaction cumulative volume 50 μ l; PCR reaction system reaction cumulative volume 50 μ l, reaction condition: 94 ℃ of pre-degeneration 5 minutes, 94 ℃ of degeneration 30 seconds, 57 ℃ of annealing 50 seconds, 72 ℃ were extended 50 seconds, and last 72 ℃ were extended totally 30 circulations 7 minutes.1% sepharose electrophoresis, the observed result correct position.
2. order-checking:
With above-mentioned PCR product electrophoresis, cut glue purification and reclaim, make sequencing template after surveying its content, send TaKaRa company to check order.。
(9) evaluation of rBCG-hB7-2 expression product
1. enzyme connects immunoabsorption (ELISA) mensuration:
Take out the above-mentioned reorganization rBCG-hB7-2 bacterium liquid of 1ml, centrifugal 10 minutes of 10000rpm through identifying.Get supernatant as being secreted into the sample that antibacterial is expressed hB7-2 product assay outward.The precipitation of above-mentioned centrifugal back bacterium liquid is washed three times with distilled water, and centrifugal back adds 100u l distilled water, ultrasonication behind the mixing in precipitate.Centrifugal 10 minutes of broken back 10000rpm gets its supernatant as sample in the antibacterial, measures hB7-2 content.
The ELISA assay method: get above-mentioned sample 100 μ l, be added in 96 orifice plates of coated antibody, press the test kit description operation, the 490nm place measures.Every number specimen is got three parts.Every duplicate samples is surveyed two holes.Calculate sample size according to standard curve, average as final result.The inside and outside sample size of antibacterial is respectively 10.5 and 3.8U/ml.
2. lymphocyte proliferation assay:
Extract healthy human peripheral blood, obtain lymphocyte with lymphocyte separation medium, with the wild type bacillus calmette-guerin vaccine as first signal, with wild type bacillus calmette-guerin vaccine supernatant is contrast, above-mentioned rBCG-hB7-2 supernatant is an experimental group, carry out mtt assay after 72 hours with the lymphocyte Mixed culture and detect, absorbance is measured at the 490nm place.The lymphopoiesis exponential average is 3.5.The pYL-B72 sequencing result:
atggctcctctgaagattcaagcttatttcaatgagactgcagacctgccatgccaatttgc
aaactctcaaaaccaaagcctgagtgagctagtagtattttggcaggaccaggaaaacttgg
ttctgaatgaggtatacttaggcaaagagaaatttgacagtgttcattccaagtatatgggc
cgcacaagttttgattcggacagttggaccctgagacttcacaatcttcagatcaaggacaa
gggcttgtatcaatgtatcatccatcacaaaaagcccacaggaatgattcgcatccaccaga
tgaattctgaactgtcagtgcttgctaacttcagtcaacctgaaatagtaccaatttctaat
ataacagaaaatgtgtacataaatttgacctgctcatctatacacggttacccagaacctaa
gaagatgagtgttttgctaagaaccaagaattcaactatcgagtatgatggtattatgcaga
aatctcaagataatgtcacagaactgtacgacgtttccatcagcttgtctgtttcattccct
gatgttacgagcaatatgaccatcttctgtattctggaaactgacaagacgcggcttttatc
ttcacctttctctatagagcttgaggaccctcagcctcccccagaccacattccttggatta
cagcttag
Claims (1)
1. recombined human costimulatory molecules strain of BCG vaccine, the bacillus coli-mycobacteria shuttle expression carrier that it is characterized in that expressing human costimulatory molecules B7-2, in Denmark I type bacillus calmette-guerin vaccine, express, obtain having the recombinant strain of BCG vaccine rBCG-hB7-2 of secretion activity costimulatory molecules B7-2 function, this strain classification called after mycobacterium tuberculosis (Mycobacteriumtuberculosis); CGMCC No.1120 was numbered in culture presevation by China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation on 03 23rd, 2004.
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| Recombinant bacille Calmette-Guerin (BCG) expressinghuman interferon-alpha 2B demonstrates enhancedimmunogenicity. Y. LUO,X. CHEN,R. HAN & M. A. O'DONNELL.Clin Exp Immunol,Vol.123. 2001 * |
| 卡介苗BCG治疗膀胱移行细胞癌的研究进展 李志等.中国老年学杂志，第24卷 2004 * |
| 卡介苗腔内灌注治疗浅表性膀胱肿瘤 汪东亚等.世界临床药物，第24卷第6期 2003 * |
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