CN1544473A - Recombinant fusion protein of mycobacterium tuberculosis Ag85B antigen and human IL-2, and its uses - Google Patents
Recombinant fusion protein of mycobacterium tuberculosis Ag85B antigen and human IL-2, and its uses Download PDFInfo
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- CN1544473A CN1544473A CNA2003101110091A CN200310111009A CN1544473A CN 1544473 A CN1544473 A CN 1544473A CN A2003101110091 A CNA2003101110091 A CN A2003101110091A CN 200310111009 A CN200310111009 A CN 200310111009A CN 1544473 A CN1544473 A CN 1544473A
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Abstract
The invention provides an Ag85B/IL-2 fusion protein through gene engineering recombination, wherein the fusion protein is fused from M. tuberculosis Ag85B antigen or Ag85B antigen in other mycobacterium with human IL-2. The invention also provides the genes for encoding the recombination fusion protein, which possesses the functions of activating immunocyte and improving body antineoplastic immunological reaction, thus can be used in preparing medicament for immunization therapy of tumor, especially suitable for preparing medicament or protein vaccine for treating bladder tumor and preventing its recurrence.
Description
Technical field
The present invention relates to a kind of genetically engineered recombinant protein: Ag85B/IL-2 fusion rotein, and be used for immunotherapy of tumors, especially tumor of bladder treatment and the purposes of preventing the medicine or the protein vaccine of its recurrence in preparation.
Technical background:
Tumor of bladder is the urinary system most common tumor, has clinical characters such as sickness rate height, easy infiltration metastasis and easy recurrence.
Bacille Calmette-Guerin vaccine (BCG) is a kind of effective biological immune modifier as a kind of attenuation mycobacterium bovis of work.From (Morales A, et al.Intracavitary BacillusCalmette-Guerin in the treatment of superficial bladder tumors.J Urol 1976Aug after at first report is used BCG treatment bladder cancer such as Merales in 1976; 116 (2): 180-183), practice for many years confirms: bladder intra-bladder instillation BCG immunoprophylaxis is a kind of successful immunotherapy method with the treatment superficial bladder tumor, recur in prophylaxis of tumours, treating residual tumour compares with chemotherapy in the chamber with the carcinoma in situ aspect, demonstrated remarkable advantages, become the recurrence of prevention tumor of bladder, one line medicine and prefered method (Meyer JP, the et al.Use of baci lle Calmette-Guerin in superficial bladder cancer.Postgrad.MedJ.2002 Aug of treatment carcinoma in situ; 78 (922): 449-54).
Cytokine interleukin element-2 (IL-2) is produced by activated T lymphocyte, and it can not only promote T, B cell proliferation differentiation, strengthens the T cytoactive, can also activate the NK cell, induce the generation of other cytokine and LAK cell and til cell.(Den Otter W, et al.Intravesical interleukin-2 in T1 papillary bladdercarcinima:regression of marker lesion in 8 of 10 patients.J Urol 1998Apr such as Den Otter; 159 (4): 1183-1186) using IL-2 has obviously residual patient's row irrigation of bladder to 10 postoperatives, treatment was a course of treatment in 5 days continuously, treat carry out cystoscopy and residual position after 2 months the repetition biopsy with observe the curative effect, as a result 10 patients have 8 residual positions healings and repeatedly biopsy do not see tumour.The author thinks that row IL-2 perfusion therapy is a kind of effective means behind the TUR, can produce stronger anti-tumour effect.Although use IL-2 treatment tumor of bladder to obtain certain effect separately, but people also find to use IL-2 separately because the relation that is directly proportional with dosage by experiment, it is big more to exist using dosage, the problem that side effect is big more (Velotti F, et al.Local activation of immune response in bladder cancer patientstreated with intraarterial infusion of recombinant interleukin-2.Cancer Res 1991Mayl, 51 (9): 2456-2462).
Existing multinomial BCG of studies confirm that and IL-2 associating irrigation of bladder are better than single with BCG or IL-2 perfusion in the recurrence of prevention tumor of bladder.BCG associating IL-2 perfusion bladder can brought into play synergy mutually aspect prevention bladder tumor recurrence and the shallow type bladder cancer of treatment table.BCG can activate body NK cell, T cell and scavenger cell, and IL-2 helps the maturation of NK cell and cytotoxic T cell (CTL).
No matter be that BCG or IL-2 all exist shortcomings such as dosage is big, the course of treatment is long, toxic side effect is big in the clinical treatment bladder cancer.BCG is as a kind of attenuation mycobacterium bovis of work, and its biological activity still has very strong antigenicity, sensitization and residual toxicity.Clinical application causes urocystitis, exothermic reaction, bladder contractura often, occurs general BCG infection and septicemia and then threat to life when serious.Heavy dose of IL-2 uses and can cause that often vascular permeability increases, ypotension, kidney function damage, marrow hemopoiesis inhibition etc.
Ag85B is called α-Ag or MPB59 again, be BCG, tubercule bacillus and the topmost secretory protein of other mycobacterium, account for 1/4 (Harth G of mycobacterium exocrine protein total amount, Lee BY, Wang J, et al.Novel insights into thegenetics, biochemistry, and immunocytochemistry of the 30-kilodalton majorextracellular protein of mycobacterium tuberculosis.Infect Immun, 1999,64 (8): 3038-3047).In different mycobacteriums, Ag85B all has the homology of height at amino acid and dna level.Ag85B gene length 858bp, 285 amino-acid residues of encoding, the about 30KD of molecular weight.Ag85B is the main component that BCG brings into play biological action in vitro and in vivo, also is to be distributed widely in the albumen that has cross reactivity in mycobacterium tuberculosis and other anonymous mycobacteria, can induce the immune response of body intensive Th1 type.Utilize the immunogenicity of mycobacterium Ag85B, the applied research in bladder cancer and other oncotherapy comes into one's own in recent years.Ag85B albumen can be induced the proliferative response of bladder cancer patients peripheral blood mononuclear cell (PBMC), and can induce cytokine (Zlotta AR such as Th1 emiocytosis cytokine IFN-γ and IL-12, Drowart A, Vooren JV, et al.Superficial bladder tumors and increased reactivity againstmycobacterial antigens before bacillus calmette-guerin therapy.J Urol, 1998,159,1885-1891); (Kuromatsu I, Matsuo K, Takamuta S, et al.Induction of effective antitumorimmune responses in a mouse bladder tumor model by using DNA of α antigen frommycobacteria.Cancer Gene Therapy, 2001,8 (7): 483-490) utilize the tumor of bladder cell vaccine immune animal of transfection Ag85B gene after, antigen presenting cell (APC) is respectively by MHC I class and II quasi-molecule, can submission CTL epitope peptide, again can submission Th epitope peptide, thus bigger antitumor advantage had than other the tumour-cell vaccine of genetic modification.
The deficiency when treating its recurrence of tumor of bladder and prevention in view of BCG and IL-2, we utilize genetic engineering means at imagination, with main secretion antigen gene A g85B and the human IL-2's gene fusion in the mycobacterium, make up the Ag85B/IL-2 fusion gene, prokaryotic expression obtains the Ag85B/IL-2 fusion rotein.The Ag85B/IL-2 fusion rotein is used for the treatment of tumor of bladder and prevents its recurrence, has both brought into play both immune synergisms, can reduce the toxic side effect that BCG and IL-2 cause again.
Summary of the invention:
The purpose of this invention is to provide a kind of recombinant protein, it is with the main secretion antigen Ag85B of mycobacterium tuberculosis or other mycobacterium secretes Ag85B antigen and the human IL-2 is merged and the genetically engineered recombination fusion protein of formation.
Another object of the present invention provides a kind of gene order of code book invention recombination fusion protein.
Another order of the present invention has provided this recombination fusion protein and has been used for immunotherapy of tumors in preparation, and especially the medicine of its recurrence or the purposes in the protein vaccine are treated and prevented to tumor of bladder.
In the recombination fusion protein provided by the invention, the main secretion antigen Ag85B of mycobacterium tuberculosis or other mycobacterium secretion Ag85B antigen can be positioned at the aminoterminal of this fusion rotein, also can be positioned at the carboxyl terminal of this fusion rotein, can there be connexon between Ag85B and the IL-2, also can connect by the connexon that is rich in glycine (Linker), thereby increase the snappiness of fusion rotein, help the correct folding of two kinds of protein monomers.The connexon aminoacid sequence can be SEQ ID NO:1, also can be SEQ ID NO:2.
SEQ?ID?NO：1?GGGGS
SEQ?ID?NO：2?GGGGSGT
Ag85B antigen of the present invention can also be the Ag85B antigen in bacille Calmette-Guerin vaccine, the M. smegmatics.
The present invention also provides a kind of gene of the recombination fusion protein of the present invention of encoding.This gene can have the nucleotide sequence shown in SEQ ID NO:3.
SEQ?ID?NO：3
TTCTCCCGGCCGGGGCTGCCGGTCGAGTACCTGCAGGTGCCGTCGCCGTCGATGGGCCGCGACATCAAGGTTCAGTTCC
AGAGCGGTGGGAACAACTCACCTGCGGTTTATCTGCTCGACGGCCTGCGCGCCCAAGACGACTACAACGGCTGGGATAT
CAACACCCCGGCGTTCGAGTGGTACTACCAGTCGGGACTGTCGATAGTCATGCCGGTCGGCGGGCAGTCCAGCTTCTAC
AGCGACTGGTACAGCCCGGCCTGCGGTAAGGCTGGCTGCCAGACTTACAAGTGGGAAACCTTCCTGACCAGCGAGCTGC
CGCAATGGTTGTCCGCCAACAGGGCCGTGAAGCCCACCGGCAGCGCTGCAATCGGCTTGTCGATGGCCGGCTCGTCGGC
AATGATCTTGGCCGCCTACCACCCCCAGCAGTTCATCTACGCCGGCTCGCTGTCGGCCCTGCTGGACCCCTCTCAGGGG
ATGGGGCCTAGCCTGATCGGCCTCGCGATGGGTGACGCCGGCGGTTACAAGGCCGCAGACATGTGGGGTCCCTCGAGTG
ACCCGGCATGGGAGCGCAACGACCCTACGCAGCAGATCCCCAAGCTGGTCGCAAACAACACCCGGCTATGGGTTTATTG
CGGGAACGGCACCCCGAACGAGTTGGGCGGTGCCAACATACCCGCCGAGTTCTTGGAGAACTTCGTTCGTAGCAGCAAC
CTGAAGTTCCAGGATGCGTACAACGCCGCGGGCGGGCACAACGCCGTGTTCAACTTCCCGCCCAACGGCACGCACAGCT
GGGAGTACTGGGGCGCTCAGCTCAACGCCATGAAGGGTGACCTGCAGAGTTCGTTAGGCGCCGGCGGTGGCGGAGGCTC
TGGTACCGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATT
TTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCA
CAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAA
CTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTC
ATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCT
CAACACTGACTTGA
Recombination fusion protein provided by the invention has the aminoacid sequence as SEQ ID NO:4.This fusion rotein is that mycobacterium tuberculosis secretory antigen A g85B and human IL-2 are merged the fusion rotein that forms, after being fed into tumor of bladder patient intravesical, can be by Ag85B and The bladder wall fiber adhesion albumen (FN) bonded characteristic (Naito M, et al.The novel fibronectin-binding motifand key residues of mycobacteria.J Bio Chem, 1998,273 (5), 2905-2909), mediating this fusion rotein and The bladder wall adheres to, Ag85B processes through MHC II classpath and offers, can induce the Th1 type cell immune response of body, secretion Th1 cytokines IFN-γ and IL-12 etc., and the proliferative response of stimulation bladder cancer patients peripheral blood mononuclear cell (PBMC); IL-2 can promote the differentiation of T, B cell proliferation, strengthens the T cytoactive, activation NK cell, induces the generation of other cytokine and LAK cell and til cell.Both are inducing performance synergy aspect the organism immune response, and tumour cell is killed in immune response local by body and general, reaches prevention of recurrence and therapeutic action.
SEQ?ID?NO：4
MFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSS
FYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDP
SQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFV
RSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAGGGGGSGTAPTSSSTKKTQLQLEHLLL
DLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK
GSETTFMCEYADETATIVEFLNRWITFCQSIISTLT
Recombination fusion protein of the present invention can be used known gene engineering method, obtains by prokaryotic expression or eukaryotic expression.
The purposes of invention:
Be applicable to that preparation is used for the medicine of immunotherapy of tumors, the medicine or the protein vaccine that are particularly useful for preparing the tumor of bladder treatment and prevent its recurrence.
The advantage of invention:
The present invention compares with IL-2 with BCG, when treatment tumor of bladder and the recurrence of prevention tumor of bladder following advantage is arranged: 1. have the difunctional activity of Ag85B and IL-2, can bring into play both immunizations simultaneously; 2.Ag85B be the main secreted protein of mycobacterium, have the immunogenicity of BCG, but do not have the toxic side effect of BCG live bacterial vaccines; 3.Ag85B merge with IL-2, both have immune synergism, thus the clinical consumption of corresponding minimizing IL-2, and its toxic side effect also reduces; 4.Ag85B have and The bladder wall fiber adhesion albumen (FN) bonded characteristic, thereby the guiding fusion rotein of target adheres to The bladder wall, the action time of prolong drug.
Description of drawings
The present invention further specifies by embodiment, and embodiment comprises following accompanying drawing:
Accompanying drawing 1 is an Ag85B pcr amplification product DNA electrophorogram;
Accompanying drawing 2 is cut electrophorogram for the restriction enzyme of plasmid pUC18-Ag85B;
Accompanying drawing 3 is a hIL-2 pcr amplification product DNA electrophorogram;
Accompanying drawing 4 is cut electrophorogram for the restriction enzyme of plasmid pUC18-IL-2;
Accompanying drawing 5 is cut electrophorogram for the restriction enzyme of plasmid pUC-Ag85B/IL-2;
Accompanying drawing 6 is cut electrophorogram for the restriction enzyme of plasmid pG Ag85B/IL-2;
The sequencer address of the part sequence of accompanying drawing 7 Ag85B/IL-2 fusion genes;
Accompanying drawing 8 is the SDS-PAGE electrophorogram that plasmid pG-Ag85B/IL-2 recombination bacillus coli BL21 expresses;
Accompanying drawing 9 is the Western blot figure of pG Ag85B/IL-2 recombination bacillus coli BL21 abduction delivering product;
Accompanying drawing 10 is Ag85B/IL-2 fusion rotein SDS-PAGE electrophorogram behind the RP-HPLC purifying;
Accompanying drawing 11 is Ag85B/IL-2 fusion rotein RP-HPLC purity check figure;
Accompanying drawing 12 is the IFN-γ content comparison diagram in the human peripheral blood single nucleus cell culture supernatant under the Ag85B/IL-2 fusion rotein hormesis;
Accompanying drawing 13 is the IL-12 content comparison diagram in the human peripheral blood single nucleus cell culture supernatant under the Ag85B/IL-2 fusion rotein hormesis;
Accompanying drawing 14 is the cytotoxicity comparison diagram of Ag85B/IL-2 fusion rotein activated killer cell to the tumor of bladder cell.
Embodiment
Embodiment 1
Obtain the antigenic encoding gene of mycobacterium tuberculosis (Edman strain) Ag85B
Mycobacterium tuberculosis (Edman strain) derives from Basic Medical Science Inst., Shandong Prov. Academy of Medical Science.Adopt the 7H9 liquid nutrient medium to cultivate tubercule bacillus, 37 ℃ of culture temperature, the centrifugal culture in 2 week backs is collected thalline, therefrom extracts tubercle bacillus gene group DNA.
The method of extracting the mycobacterium tuberculosis genomic dna is with reference to Molecular clonging one book (J.Sambrook, Isolation of high-molecular-weight DNA from mammaliancells, 9.16-9.22, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989).
Adopt the PCR method Ag85B structure gene that in tubercle bacillus gene group DNA, increases.The 5` end primer sequence that adopts is; 5`CGGAATTCTTCTCCCGGCCG 3` (SEQ ID NO:5); 3` end primer sequence is 5`GGGGTACCAGAGCCTCCGCCACC GCCGGCGCCTAACG 3` (SEQ ID NO:6).
Described PCR schedule of operation is: add following reaction reagent in a 50ul Eppendorf tube:
Template DNA 2ul
10 * PCR damping fluid (containing magnesium chloride) 5ul
dNTPs(10mmol/L)4ul
5` end and each 1ul of 3` end primer (0.01mmol/L)
TaqDNA polymkeric substance (5u/ul) 1ul
Add deionized water to final volume 50ul
Reaction conditions: 94 ℃, 5min; 94 ℃, 30sec; 55 ℃, 30sec; 72 ℃, 90sec; After 30 circulations, 72 ℃ are fully extended 10min.
Ag85B PCR result is referring to accompanying drawing 1.
The EcoRI+KpnI double digestion is used in the purified back of Ag85B goal gene, and (available from Promega company) is connected with same pUC18 plasmid with the EcoRI+KpnI double digestion, and enzyme is cut, connected, the screening of conversion and recombinant clone is undertaken by Molecular clonging.Structure contains the plasmid pUC18-Ag85B of tubercule bacillus Ag85B structure gene.
The restriction enzyme of plasmid pUC18-Ag85B is cut qualification result referring to accompanying drawing 2.
This embodiment successfully obtains the antigenic encoding gene of mycobacterium tuberculosis (Edman strain) Ag85B, and constructs the plasmid pUC18-Ag85B that contains tubercule bacillus Ag85B encoding gene.
Obtain human IL-2's encoding gene
Extract total RNA in the people Jurkat of mitogenstimulated T lymphoma cell (from cell institute of the Chinese Academy of Sciences), extracting method is given birth to the worker RNA of company extraction test kit by Shanghai and is carried out.Adopt the RT-PCR method to obtain people Jurkat T glucagonoma cDNA.
In above-mentioned cDNA, use PCR method amplification human IL-2's encoding gene.The 5` end primer sequence that adopts is: 5`GG GGTACCGCACCTACTTC 3` (SEQ ID NO:7); 3` end primer sequence is 5`CGGGATCCTCAAGTCAGTGT 3` (SEQ ID NO:8)
Described PCR schedule of operation is: add following reaction reagent in a 50ul Eppendorf tube:
Template DNA 1ul
10 * PCR damping fluid (containing magnesium chloride) 5ul
dNTPs(10mmol/L)4ul
5` end and each 1ul of 3` end primer (0.01mmol/L)
TaqDNA polymkeric substance (5u/ul) 1ul
Add deionized water to final volume 50ul
Reaction conditions: 94 ℃, 5min; 94 ℃, 30sec; 55 ℃, 30sec; 72 ℃, 90sec; After 30 circulations, 72 ℃ are fully extended 10min.
HIL-2 pcr amplification result is referring to accompanying drawing 3.
The KpnI+BamHI double digestion is used in the purified back of human IL-2's goal gene, and (available from Promega company) is connected with same pUC18 plasmid with the KpnI+BamHI double digestion, and enzyme is cut, connected, the screening of conversion and recombinant clone is undertaken by Molecular clonging.Structure contains the plasmid pUC18-IL-2 of human IL-2's encoding gene.
The restriction enzyme of plasmid pUC18-IL-2 is cut qualification result referring to accompanying drawing 4.
This embodiment successfully obtains human IL-2's encoding gene, and constructs the plasmid pUC18-IL-2 that contains human IL-2's encoding gene.
Make up Ag85B-IL-2 prokaryotic expression plasmid: pG-Ag85B/IL-2.
Adopt KpnI+BamHI double digestion pUC18-IL-2, glue reclaims the IL-2 gene fragment, is connected with same pUC18-Ag85B plasmid with the KpnI+BamHI double digestion, and enzyme is cut, connected, the screening of conversion and recombinant clone is undertaken by Molecular clonging.Structure contains the plasmid pUC-Ag85B/IL-2 of Ag85B/IL-2 fusion gene.
The restriction enzyme of plasmid pUC-Ag85B/IL-2 is cut qualification result referring to accompanying drawing 5.
Adopt EcoRI+SalI double digestion plasmid pU-Ag85B/IL-2, glue reclaims Ag85B/IL-2 fusion gene fragment, (available from Promega company) is connected with same pGEX4T-1 plasmid with the EcoRI+SalI double digestion, makes up the prokaryotic expression plasmid pG-Ag85B/IL-2 that contains the Ag85B/IL-2 fusion gene.
The restriction enzyme of plasmid pG-Ag85B/IL-2 is cut qualification result referring to accompanying drawing 6.
Gene sequencing: the dna sequencing instrument with ABI company carries out.The result shows: the sequence of Ag85B/IL-2 fusion gene and design in full accord.The sequencing result of the part sequence of Ag85B/IL-2 fusion gene is referring to accompanying drawing 7.
This embodiment has successfully made up the prokaryotic expression plasmid pG-Ag85B/IL-2 that contains the Ag85B/IL-2 fusion gene.
Embodiment 4
Plasmid pG-Ag85B/IL-2 transformed into escherichia coli BL21 and abduction delivering.
1.BL21 the preparation of competence bacteria.
(1) incites somebody to action-20 ℃ of frozen e. coli bl21 streak inoculations in containing on penbritin (100ug/ml) the LB agar plate overnight incubation (16～18h) in 37 ℃ of incubators.
(2) get single colony inoculation and contain in the penbritin LB liquid nutrient medium, 37 ℃ of vibrations (200rpm/min) overnight incubation in 3～5ml.
(3) get 250 μ l bacterium liquid and add in the 25ml LB liquid nutrient medium, 37 ℃ of about 2～4h of shaking culture to OD600=0.4～0.5 o'clock, stop to cultivate.
(4) bacterium liquid is moved in the centrifuge tube ice bath 10min, the centrifugal 10min of 5000rpm.
(5) abandon nutrient solution, precipitation is iced the 0.1mol/LCaCl of precooling with 5ml
2After resuspended, 5000rpm, the 10min centrifuge washing is once.
(6) add the ice-cold 100mmol/L CaCl of 10ml in the above-mentioned precipitation
2, ice bath promptly can be used for transformation experiment in 2～4 hours.
2. the conversion of plasmid pG-Ag85B/IL-2
(1) in the sterilization EP pipe of the 1.5ml of 3 precoolings, adds competence bacterium and plasmid DNA by table.
EP pipe competence bacteria plasmid DNA
1. 95 μ l, 5 μ l are organized in conversion
2. negative control group 100 μ l-
3. positive controls 98 μ l 2 μ l
(2) with above-mentioned EP pipe ice bath 30min～1h, 42 ℃ of heat-shocked 90sec, ice bath 2min.
(3) each pipe adds and does not contain antibiotic LB liquid nutrient medium 400 μ l, and 37 ℃ of water-bath 5min put 37 ℃ of shaking culture 45min～1.5h of shaking table.
(4) draw the above-mentioned nutrient solution of 100 μ l with sterilization rifle head respectively, add the dull and stereotyped and common LB flat board of the LB that contains penbritin, liquid evenly is applied on the plate, after forward is placed 1h in 37 ℃ of incubators, be inverted incubated overnight with elbow sterilization glass rod.Identify recombinant clone with the Restriction Enzyme blanking method.
(5) preservation of plasmid and bacterial classification: the plasmid stored frozen is in-20 ℃.Bacterial classification is-70 ℃ of preservations in the nutrient solution that contains 20-50% glycerine.
3.Ag85B/IL-2 the prokaryotic expression of fusion gene
(1) draw plate with the good positive recombinant bacterium liquid of evaluation, single colony inoculation that picking is fresh is gone into the LB (containing Amp 100 μ g/mL) of 3ml, and 37 ℃ of joltings are spent the night.
(2) with the extent of dilution adding 5ml LB (contain Amp100 μ g/mL) of saturated bacterium liquid with 1: 100,37 ℃ of joltings are to OD
600Be about 0.6.
(3) add IPTG to final concentration 1mmol/L, continue 37 ℃ of joltings and cultivate 4h.4 ℃ of centrifugal 5min (5000rpm), collecting bacterial body, use or frozen immediately.
(4) get an amount of thalline, after adding 10 μ l sterilization deionized water and thoroughly suspending, add 10 μ l * SDS sample loading buffer in bacterium, 100 ℃ of heating 3min are so that protein denaturation.Handle high-molecular-weight protein Marker with quadrat method.Analyze above sample with SDS-PAGE (concentrating glue 5%, separation gel 11%), determine that fusion rotein has or not expression.
(5) the SDS-PAGE electrophoretic analysis detects recombinant protein.The result is referring to accompanying drawing 8.
Show among the figure: pG-Ag85B/IL-2 has obtained to efficiently express in the BL21 bacterium, expression amount about 40%.Owing to have gst gene after the expression vector pGEX4T-1 promotor, so Ag85B/IL-2 albumen is that molecular weight is about 71KD, and exists with the form of undissolved inclusion body with the formal representation of GST-Ag85B/IL-2 fusion rotein.
4.GST-Ag85B/IL-2 the Westrn blot of fusion rotein identifies
Behind the pG-Ag85B/IL-2 reorganization bacterium abduction delivering, row SDS-PAGE, electricity is transferred to pvdf membrane.Utilize the anti-human IL-2's monoclonal antibody immunity of rabbit to identify expression product, the result is referring to accompanying drawing 9.
Showing among the figure: an obvious band appears at the 71KD place, and consistent with the theoretical size of GST-Ag85B/IL-2 fusion rotein.
Embodiment 5
The purifying of Ag85B/IL-2 fusion rotein.
1. the extraction of bacterium collection and inclusion body
(1) engineering bacteria of collection 1L abduction delivering, 4 ℃ of centrifugal 10min of 5000rpm collect and express the back thalline.
(2) add 10mmol/L PBS (pH7.5), magnetic agitation 30min, 4 ℃ of centrifugal 10min of 5000rpm, collecting precipitation.
(3) the same repeated washing is twice.
(4) carrying out ultrasonic bacteria breaking 2min/ time in ice bath, totally 10 times, 10sec at interval, output rating 70W.Suspend with the bacterium lysis buffer.
(5) 4 ℃ of centrifugal 20min of 8000rpm, collecting precipitation.
The preparation of bacterium lysis buffer
PBS(pH7.5) 10mmol/L
EDTA 1mmol/L
2. washing inclusion body
(1) by the inclusion body washings of 4 ℃ of the new precoolings of preparing of every gram inclusion body adding 10ml, suspends it with the magnetic force vortex.
(2) 4 ℃, the centrifugal 20min of 8000rpm, supernatant discarded.
(3) the same processing repeats 5 times.
The preparation of inclusion body washings
Triton?X-100 0.5％(V/V)
PBS(pH7.5) 10mmol/L
Urea 1mmol/L
3. dissolving inclusion body
In the inclusion body of washing back, add the solubilization of inclusion bodies liquid of the precooling of the new preparation of 30ml, softly stir, be positioned over 4 ℃ of refrigerator dissolvings and spend the night with magnetic force.Next day, under 4 ℃,, carefully draw supernatant with the centrifugal 20min of 15000rpm, it is standby to be positioned over 4 ℃ of preservations.
The preparation of solubilization of inclusion bodies liquid
Urea 8mol/L
Beta-mercaptoethanol 0.1mmol/L
4. the renaturation of inclusion body
Add the renaturation buffer of 9 times of volumes in the dissolved inclusion body, room temperature is placed 30min, keeps pH to 10.7 with KOH; Transfer pH to 8.0 with HCL, place 30min in room temperature at least.10000rpm, 4 ℃ of centrifugal 15min collect supernatant, and (molecular weight cut-off 50～80kD) is collected protein solution in the dialysis tubing with deionized water dialysis 48h after dialysis is finished to the dialysis tubing of packing into, and is concentrated freeze-dried.Put-70 ℃ of preservations.
The preparation of renaturation buffer
KH
2PO
4(pH10.7) 50mmol/L
EDTA(pH8.0) 1mmol/L
Reduced glutathion 2mmol/L
Sleep-promoting factor B 1mmol/L
5.Ag85B/IL-2 the Glutathione Sepharose affinity chromatography of fusion rotein and zymoplasm (Thrombin) enzyme are cut
Adopt Pharmacia AKTA explore 100 instrument that renaturing inclusion bodies liquid is carried out Glutathione Sepharose affinity chromatography.Chromatography column is XK 2b, column volume 50ml.Sample flow rate 10ml/min.Use 10mMolPBS pH7.5,10mMol gsh GSH eluant solution GST-Ag85B/IL-2 fusion rotein.The ultraviolet monitoring wavelength is 280nm, collects protein peak.The recombinant protein sample is diluted to 1mg/ml with 10mMolPBS pH7.5, adds zymoplasm (sigma) 0.2u mixing in every ml sample solution, act on 5h under the room temperature, enzymatic hydrolyzation is greater than 90%.
5.Ag85B/IL-2 the ion exchange chromatography of fusion rotein
The fusion rotein of the Q Sepharase Fast Flow medium that adopts Pharmacia company after to affinity chromatography is further purified.Pre-install the long Q Sepharase of a 20cm Fast Flow post, 10mMol PBS pH7.5 washes chromatography column, 1Mol Nacl, upper prop after the 10mMolPBS pH7.5 balance.Flow velocity 2.5ml/min.Take off condition with the continuous concentration gradient wash-out of 0-100%Nacl with the suitableeest definite Xian earlier, capable again Nacl stepwise elution is collected the ultraviolet absorption peak sample.
6.Ag85B/IL-2 the RP-HPLC purifying of fusion rotein
The target protein sample that ion exchange chromatography is collected carries out the RP-HPLC purifying on AKTA Explore 100.Select 3ml Resource RPC reversed-phase column for use, column length 15cm, diameter 3.5cm.Upper prop behind 5 times of the diluted samples behind the ion exchange chromatography, moving phase is acetonitrile solution, and flow velocity 1.0ml/min, ultraviolet monitoring wavelength are 280nm, through 0%～60% solution gradient wash-out, collect main peak in 10 times of column volumes, and low-temperature freeze drying is standby.
7.Ag85B/IL-2 the SDS-PAGE electrophoresis of fusion rotein is identified and purity check
Separation gel with 11%, 4% concentrated glue carry out electrophoretic analysis to the sample of purifying, and initial voltage 8V/cm treats to increase when sample enters separation gel voltage to 15V/cm, electrophoresis 3h.Electrophoresis is taken gel off and is placed glass dish after finishing, and adds the long-pending stationary liquid room temperature of 3～5 times of colloids fixedly more than the 1h; Outwell stationary liquid, more than Xylene Brilliant Cyanine G R-250 staining fluid dyeing 4h, the sucking-off staining fluid adds an amount of stationary liquid detergent gel once, adds destainer and carries out rinsing, changes destainer therebetween for several times, till protein band is clear.Scan with Bio-Rad gel scanning analysis system.Accompanying drawing 10 shows the about 45KD of Ag85B/IL-2 fusion protein molecule amount size, and purity is greater than 95%.Measure its iso-electric point with isoelectric focusing method and be about 5.31.
Target protein is carried out the purity check result referring to accompanying drawing 11 with RP-HPLC again.The result shows that the purity of Ag85B/IL-2 fusion rotein is greater than 98%.
8.Ag85B/IL-2 the N-terminal determined amino acid sequence of fusion rotein
After the SDS-PAGE electrophoresis of Ag85B/IL-2 fusion rotein finished, electricity was transferred to pvdf membrane, carries out the N-terminal determined amino acid sequence, is finished by Peking University's school of life and health sciences.
Sequencing result: Ag85B/IL-2 fusion rotein N-terminal determined amino acid sequence is in full accord with design.
Embodiment 6
The biological activity test of Ag85B/IL-2 fusion rotein
One, the IL-2 determination of activity of Ag85B/IL-2 fusion rotein: with the IL-2 activity of IL-2 dependent cell strain CTLL-2 raji cell assay Raji Ag85B/IL-2 fusion rotein.
1, method
(1) with RPMI1640 dilution IL-2 standard substance and testing sample, the IL-2 standard substance begin doubling dilution from 100U/ml concentration, and testing sample also is a doubling dilution, and dilution back final volume in 96 orifice plates is 100ul, and each concentration is established 3 repeating holes;
(2) contain 10-15% calf serum (FCS), 200
uThe RPMI-1640 of/ml rIL-2 cultivates CTLL-2 and goes down to posterity after 2-4 time, collect the CTLL-2 cell (immunity teaching and research room of Third Military Medical University provides) of logarithmic phase, with RPMI-1640 cell culture fluid 1500rpm, 10min, 4 ℃ of washed cells 2-3 time, be resuspended in then among the RPMI1640 that 2-5ml contains 10%FCS, make cell suspension, detect cell survival rate (greater than 95%) with 0.5% trypan blue.
(3) adjusting concentration is 1 * 10
5/ ml.In the culture plate of dilute sample, every hole adds the 100ul cell:
(4) Tissue Culture Plate is put 37 ℃, 5%CO
2, cultivate 32-40h (when treating negative hole 90% necrocytosis) in the temperature of saturation;
(5) add MTT (5mg/ml) 20ul/ hole, the 1min that on vibrator, vibrates, 37 ℃, 5%CO
2Cultivate 2-5h;
(6) take out culture plate, with the centrifugal 5min of 2000r/min, inhale and abandon supernatant liquor, every hole adds DMSO120ul, behind the vibration 30s, surveys OD on microplate reader
570Value.
(7) experimental result observation analysis: the extent of dilution with IL-2 testing sample and standard substance is a transverse axis, to detect cell OD value is longitudinal axis mapping, obtain 50% extent of dilution of the highest OD value of standard substance, find out the corresponding 50%IL-2 standard substance of the testing sample extent of dilution of the highest OD.
2, result: the IL-2 specific activity of Ag85B/IL-2 fusion rotein is 2500
u/ mg.
Two, the Ag85B/IL-2 fusion rotein is to the promoter action of human peripheral blood single nucleus cell (PBMC) Th1 cytokines release
1, the separation of human peripheral blood single nucleus cell (PBMC)
The heparin anti-coagulating 5ml of healthy, add 37 ℃ of Hank ' s of equivalent liquid mixing, about 1cm is superimposed on the 5ml lymphocyte layering liquid gently along tube wall on the liquid level apart from separating to get dilute blood 10ml, keeps the boundary between lymphocyte layering liquid and the blood sample clear, 2000rmp, centrifugal 30min.Draw the mononuclearcell layer liquid of middle level tunica albuginea shape, add 37 ℃ of Hank ' s of 8ml liquid washing 2 times, each centrifugal 10min of 1500rmp.Sedimentary cell is made into 1.0 * 10 with the RPMI-1640 nutrient solution that contains 10% calf serum
6/ ml single cell suspension is put in the Tissue Culture Flask, 37 ℃, 5%CO
2Incubator is hatched, and is stand-by.
2, the effector cell's induces
(cell concn is 1 * 10 to the PBMC of adding prepared fresh in 6 well culture plates
6/ ml), every hole 1ml adds 0.8ug/ml Ag85B/IL-2 recombinant protein, Ag85B albumen, 500 respectively
u/ ml rIL-2,0.8ug/ml Ag85B albumen associating 500
u/ ml rIL-2,0.8ug/ml BCG put 37 ℃, cultivate in the 5%CO2 incubator, measured the content of Th1 cytokines in the cells and supernatant in the 3rd day.
3, the mensuration of Th1 cytokines IL-12 and IFN-γ (ELISA method) in the cells and supernatant
(1) takes 96 orifice plates of anti-people IL-12 and IFN-γ monoclonal antibody bag quilt respectively, the PBMC culture supernatant (100ul/ hole) that adds IL-12 and IFN-γ standard substance and the stimulation of different albumen, other establishes the blank hole, every group three multiple holes, seal reacting hole with the shrouding gummed paper, 37 ℃ of incubators of lucifuge were hatched 60 minutes.
(2) get rid of liquid in the most hole, every hole adds washings 350ul/ hole, washes plate 4 times.
(3) except that blank well, add biotinylated antibody working fluid (100ul/ hole), seal reacting hole with the shrouding gummed paper, 37 ℃ of incubators of lucifuge were hatched 60 minutes.
(3) liquid in the most hole, every hole adds washings 350ul/ hole, washes plate 4 times.
(4) except that blank well, add the plain working fluid (100ul/ hole) of affinity of horseradish peroxidase-labeled, seal reacting hole with the shrouding gummed paper, 37 ℃ of incubators of lucifuge were hatched 60 minutes.
(5) get rid of liquid in the most hole, every hole adds washings 350ul/ hole, washes plate 4 times.
(6) add developer (100ul/ hole), 37 ℃ of incubators of lucifuge were hatched 20 minutes.
(7) add stop buffer (100ul/ hole), measure OD in 5 minutes behind the mixing
450Value.
4, result
Table 1: IL-12 in the human peripheral blood mononuclear cell's culture supernatant under the effect of different stimulated thing and IFN-γ content (pg/ml)
The different stimulated thing
Detect index
Ag85B/IL-2 Ag85B IL-2 Ag85B+IL-2 BCG
IL-12 99.072±4.502 37.452±2.131
* 25.362±1212
* 71.521±3.562
▲ 51.855±3.087
*
IFN-γ 100.135±2.837 10.367±4.834
** 81.067±1.804
▲ 98.667±1.001 6.667±4.202
**
Compare with the Ag85B/IL-2 group,
*Expression P＜0.01;
*Expression P＜0.001;
▲Expression P＜0.05
The cylindricality comparison diagram of above-mentioned table is referring to accompanying drawing 12,13.
5, conclusion
IL-12 concentration in the human peripheral blood single nucleus cell culture supernatant that the Ag85B/IL-2 recombinant protein stimulates is significantly higher than Ag85B, IL-2, Ag85B+IL-2 and BCG stimulating group; IFN-γ concentration in the human peripheral blood single nucleus cell culture supernatant that the Ag85B/IL-2 recombinant protein stimulates is significantly higher than Ag85B, and IL-2 and BCG stimulating group are with Ag85B+IL-2 stimulating group there was no significant difference.
Presentation of results: the Ag85B/IL-2 recombinant protein has the function that stronger promotion human peripheral blood single nucleus cell Th1 cytokines discharges.
Three, Ag85B/IL-2 fusion rotein activated human peripheral blood single nucleus cell (PBMC) is to the killing activity of tumor of bladder cell
1. prepare the effector cell
(adjust concentration is 1 * 10 to the PBMC of adding prepared fresh in 6 well culture plates
6/ ml), every hole 1ml adds Ag85B/IL-2 by the concentration of 0.8ug/ml and induces AIAK effector cell, with the protein induced AAK effector cell of 0.8ug/ml Ag85B, 500
u/ ml rIL-2 inductive LAK cell, 0.8ug/ml Ag85B albumen associating 500
uThe ALAK effector cell of/ml rIL-2 combined induction and 0.8ug/ml BCG inductive BAK effector cell put 37 ℃ in contrast, cultivate in the 5%CO2 incubator, make cell toxicant in the 3rd day harvested cell and measure.
2. cellulotoxic experiment (mtt assay)
On (1) 6 well culture plate to imitate target than 40: 1 adding effector cells earlier, add again the respective target cell (T24, BIU-87, BTT739, EJ), other establishes each target cell control wells, effector cell's control wells sets up 3 repeating holes separately.
(2) in 37 ℃, 5%CO
2Cultivate 24h.
(3) every hole adds MTT (5mg/ml) 20ul, continues to cultivate 4h.
(4) 2000rpm, centrifugal 5min abandons supernatant.
(5) each hole adds DMSO120ul
(6) concussion 10min fully dissolves MTT reduzate first the part between the ribs and the hips.
(7) on microplate reader, survey the 570nm OD of place value.
(8) calculation formula of killing activity:
3. experimental result and conclusion
Table 2: the different effect cell is to the cytotoxic activity (%) (n=3 is imitated target than 40: 1) of tumor of bladder cell
Effector cell BIU-87 T24 EJ BTT739
Fusion rotein inductive 60.4 ± 2.1 94.6 ± 4.3 89.4 ± 3.5 77.7 ± 1.5
The AIAK cell
IL-2 inductive LAK cell 43.0 ± 1.3
▲92.5 ± 2.5
▲62.9 ± 6.1
▲61.1 ± 3.8
▲
Ag85B inductive AAK is thin by 48.8 ± 1.6
▲93.4 ± 4.3
★75.7 ± 2.4
▲71.6 ± 2.5
▲
Born of the same parents
Ag85B+IL-2 inductive 52.6 ± 2.7
▲94.4 ± 1.5 89.1 ± 3.8 75.2 ± 1.5
▲
The ALAK cell
BOG inductive BAK cell 52.0 ± 2.2
▲49.7 ± 2.5
▲73.6 ± 2.5
▲53.9 ± 3.4
▲
Annotate:
▲Expression is compared P＜0.01 with the AIAK groups of cells;
★Expression is compared P＜0.05 with the AIAK groups of cells
The cylindricality comparison diagram of above-mentioned table is referring to accompanying drawing 14.
The result shows: Ag85B/IL-2 fusion rotein inductive AIAK cell is significantly higher than LAK cell, AAK cell, ALAK cell and BAK cell to the cytotoxic activity of BIU-87 and BTT739 tumor of bladder cell; Cytotoxic activity to T24 and EJ tumor of bladder cell is significantly higher than LAK cell, AAK cell and BAK cell.
Conclusion: Ag85B/IL-2 fusion rotein inductive AIAK cell has stronger cytotoxic activity to the tumor of bladder cell, than single cytotoxic activity height with Ag85 or IL-2 inductive killer cell.
Embodiment 7
The Ag85B/IL-2 fusion rotein is observed the knurl effect that presses down of bladder cancer tumor-bearing mice
1. material:
BTT739 is a kind of cell strain of setting up the portable transitional cell carcinoma of bladder in T739 mouse body, and Nanjing Railway College of Medicine provides, and these section office are frozen, and the T739 mouse is available from animal housing of the Chinese Academy of Medical Sciences.
2. grouping
40 of the T739 mouse in 5 ages in week are divided Ag85B/IL-2 amalgamation protein vaccine group and blank group, 20 every group
3. experimental technique
(1) tumor of bladder animal model preparation
Property bladder cancer cells BTT739 conventional cultivation the in the RPMI1640 substratum of dividing a word with a hyphen at the end of a line in mouse source promptly with the RPMI-1640 substratum that contains 15% calf serum, added an amount of microbiotic (100 units/ml penicillin and 100 μ g/ml Streptomycin sulphates), puts 37 ℃, 5%CO
2Cultivate in the mixed gas incubator.Cell changes 2-3 days liquid time, goes down to posterity in 2-4 days, and with 0.25% tryptic digestion 2min, adjusting cell concn is 3 * 10 before going down to posterity
6/ ml, stand-by.The mice with tumor model adopts the cell suspension inoculation method: the homogenic mouse T739 of 5 week inbred lines in age, right front oxter subcutaneous vaccination BTT739 tumour cell 100 μ l/ mouse (3 * 10 under the aseptic condition
5Individual cell/only), preparation tumor-bearing mice animal model.
(2) medication:
The blank group: mice with tumor gives PBS 0.1ml intratumor injection; The treatment group: mice with tumor gives Ag85B/IL-2 fusion rotein (200ug/ml) 0.1ml intratumor injection
Two groups respectively at the 21st day dislocation method put to death mouse in the administration of inoculated tumour position in the 0th, 3,7,13,18 day behind the inoculated tumour cell, and it is heavy to take by weighing knurl, calculates the growth of xenografted inhibiting rate according to following formula, with frozen in the tumor tissues liquid nitrogen, standby then.
The result:
Table 3:Ag85B/IL-2 fusion rotein presses down the knurl effect to the bladder cancer mice with tumor
The knurl growth-inhibiting
The average knurl of group mouse number heavy (g)
Rate (%)
PBS organizes 20 9.26 ± 1.15-
Ag85B/IL-2 fusion rotein 20 4.49 ± 1.06
★51.52
Annotate:
★P＜0.01 is compared in expression with control group
Conclusion: the Ag85B/IL-2 fusion rotein can obviously suppress the growth of tumor of bladder mice with tumor in-vivo tumour.
The Ag85B/IL-2 fusion rotein is to the observation of the prolongation effect of the survival time of tumor of bladder tumor-bearing mice
1. the preparation of material and animal model is the same
2. experimental technique:
Control group: mice with tumor gives PBS 0.1ml intratumor injection; The treatment group: mice with tumor gives Ag85B/IL-2 fusion rotein (200ug/ml) 0.1ml intratumor injection
Two groups respectively at behind the inoculated tumour cell the 0th, 3,7,13,18 day in the administration of inoculated tumour position, observe two groups of mice with tumor survival fates.
The result:
Table 4:Ag85B/IL-2 fusion rotein is to the prolongation effect of the lifetime of bladder cancer mice with tumor
Mouse existence prolongs
Group mouse number survival fate (d)
Rate (%)
PBS organizes 20 23.88 ± 3.56-
Ag85B/IL-2 fusion rotein 20 33.00 ± 3.16
★38.19
Annotate:
★P＜0.01 is compared in expression with control group
Conclusion: the survival time of Ag85B/IL-2 recombination fusion protein energy significant prolongation tumor of bladder tumor-bearing mice.
Embodiment 9
The Ag85B/IL-2 fusion rotein is to the tumor treatment method
After when shallow type tumor of bladder and prevention tumor of bladder postoperative recurrence are shown in treatment, can being achieved in that tumor of bladder patient emptying bladder, with Ag85B/IL-2 fusion rotein physiological saline solution, by aseptic catheterization urinary catheter is inserted bladder, slowly Ag85B/IL-2 fusion rotein solution is injected in the bladder cavity.Kept 2 hours, changed 1 position in per 15 minutes, be replaced by prosposition in turn, face upward the position, bow position and left and right sides clinostatism, so that medicine is extensively contacted with The bladder wall.Pour into weekly 1 time, continuous 6 weeks are a course of treatment.But proper extension course of treatment in case of necessity.
When other tumour of immunotherapy, be achieved in that characteristic according to tumour, behind a certain amount of Ag85B/IL-2 fusion rotein usefulness injection physiological saline solution, by injecting in regular subcutaneous injection, intramuscular injection or the knurl body, activate body immune system and bring into play antitumor action.
Claims (8)
1, a kind of recombination fusion protein Ag85B/IL-2 is characterized in that: it is by mycobacterium tuberculosis Ag 85 B antigen, or the Ag85B antigen in other mycobacterium and human IL-2 are merged the fusion rotein that forms.
2, recombination fusion protein Ag85B/IL-2 according to claim 1 is characterized in that: Ag85B antigen can be the Ag85B antigen in bacille Calmette-Guerin vaccine or the M. smegmatics.
3, recombination fusion protein Ag85B/IL-2 according to claim 1 and 2, it is characterized in that: Ag85B antigen can be positioned at the aminoterminal of fusion rotein, also can be positioned at the carboxyl terminal of fusion rotein.
4, recombination fusion protein Ag85B/IL-2 according to claim 1 and 2 is characterized in that: can be connected by the connexon of flexible structure amino acid coding between Ag85B antigen and the human IL-2, also can not have connexon.
5, recombination fusion protein Ag85B/IL-2 according to claim 4 is characterized in that: its connexon aminoacid sequence can be SEQ ID NO:1, also can be SEQ ID NO:2.
6, recombination fusion protein Ag85B/IL-2 according to claim 1 is characterized in that: it can have shown in SEQ ID NO:3 encoding gene and shown in SEQ ID NO:4 aminoacid sequence.
7, according to the described recombination fusion protein Ag85B/IL-2 of claim 1 to 6, it is characterized in that: can obtain by prokaryotic expression or eukaryotic expression.
8, the Ag85B/IL-2 fusion rotein is used for the medicine of immunotherapy tumour in preparation, especially in the treatment of preparation tumor of bladder with prevent the medicine of its recurrence or the purposes in the protein vaccine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101891825A (en) * | 2010-07-15 | 2010-11-24 | 中国人民解放军军事医学科学院微生物流行病研究所 | Recombinant fusion proteins of hepatitis B core proteins and tuberculosis antigen or antigen fragments and application thereof |
| CN102210860A (en) * | 2011-05-31 | 2011-10-12 | 昆明理工大学 | Mycobacterium tuberculosis TB10.4-F1 fusion protein vaccine and preparation method thereof |
| CN106552259A (en) * | 2015-09-24 | 2017-04-05 | 中国科学院上海巴斯德研究所 | A kind of purposes of fusion protein and its treatment disease |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1342771A (en) * | 2001-09-17 | 2002-04-03 | 复旦大学 | Expression purification of tubercle mycobacterium Ag85B antigen and its application in vaccine |
| CN1339583A (en) * | 2001-09-28 | 2002-03-13 | 靳凤烁 | Recombined smegmatis mycobacterium, its preparation and use in medicine for treating bladder tumor |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101891825A (en) * | 2010-07-15 | 2010-11-24 | 中国人民解放军军事医学科学院微生物流行病研究所 | Recombinant fusion proteins of hepatitis B core proteins and tuberculosis antigen or antigen fragments and application thereof |
| CN101891825B (en) * | 2010-07-15 | 2015-01-07 | 中国人民解放军军事医学科学院生物工程研究所 | Recombinant fusion proteins of hepatitis B core proteins and tuberculosis antigen or antigen fragments and application thereof |
| CN102210860A (en) * | 2011-05-31 | 2011-10-12 | 昆明理工大学 | Mycobacterium tuberculosis TB10.4-F1 fusion protein vaccine and preparation method thereof |
| CN106552259A (en) * | 2015-09-24 | 2017-04-05 | 中国科学院上海巴斯德研究所 | A kind of purposes of fusion protein and its treatment disease |
| CN106552259B (en) * | 2015-09-24 | 2022-03-29 | 中国科学院上海巴斯德研究所 | Fusion protein and application thereof in treating diseases |
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