CN1704425A - Human FL polynucleotide and use of human GM-CSF united gene therewith for treating malignant tumor - Google Patents
Human FL polynucleotide and use of human GM-CSF united gene therewith for treating malignant tumor Download PDFInfo
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Abstract
The invention relates to a polynucleotide that encode ligand (FL) of receptor protein flt3, protein encoded thereby, its preparing process and use, the invention also relates to the combination expression of the human FL and human GM-CSF combination gene, and the use of the combination expression in preparing medicament for treating malignant tumor.
Description
Technical field
The present invention relates to biomedicine field.Particularly, the present invention relates to the to encode polynucleotide, its encoded protein, its production method and uses thereof of part of receptor protein flt3.The invention still further relates to that uniting of people FL and human GM-CSF associating gene expresses and the two is united and expresses the purposes that is used for preparing the medicine for the treatment of malignant tumour.
Background technology
People FL is the part (flt3-ligand) of receptor protein flt3, is called for short hFL, is a kind of hematopoietic cell growth factor of finding recently.Adult form hFL is made up of 156 amino acid, the about 17kD of molecular weight.Its major function is similar to human stem cell factor (SCF), mainly acts on early stage primary ancestral cells, promotes its propagation and differentiation, takes place to impel all hemocytes.It has vital role in hemopoietic system primary disease and secondary lesion disease treatment.But FL is different from SCF again, and its biological action does not have species specificity, and mastocyte is not had effect.Experimentation on animals and clinic trial result show that the toxic side effect of FL is littler than SCF.Particularly it should be noted that FL except hemopoietic stem cell is had the vital role, it can also promote cytotoxic T cell, NK cell proliferation significantly, and dendritic cell (DCs) in a large number particularly can increase.And, comparing with cytokine GM-CSF, IL-4 or the G-CSF of other short DC growth, FL is main and the most strong DCs somatomedin.
DC is the strongest antigen presenting cell of finding at present of function, it also is unique antigen presenting cell that can stimulate T cells propagation, in the inducing of immunne responses such as antineoplastic immune, has unique status: immature DC picked-up in the peripheral tissues, handle tumour antigen, migrate to lymphoid organ and ripe gradually, then tumor antigen peptide is offered to T cells, stimulate its activation and proliferation, start anti-tumor immune response.Malignant tumour is escaped one of reason of immunosurveillance and is, tumour cell suppresses the DC precursor cell by secretion IL-10, TGF-β and VEGF etc. and breaks up to ripe DC, and suppress DC (especially tumor-infiltrated position DC) and express MHC II quasi-molecule and B7, make DC can not effectively offer tumour antigen.In recent years, a large amount of antineoplastic immune research and oncotherapies based on DC have obtained challenging achievement.
Above-mentioned functions makes FL at aspects such as immunotherapy of tumors, hematopoietic stem cell transplantation and preparation immune vaccines important potential using value be arranged.At anti-tumor aspect, experimentation on animals confirms that FL all can partly delay tumor growth to noumenal tumours such as malignant melanoma, lymphoma, fibrosarcoma, liver neoplasm and ovarian cancer or leukemia, and may excite the specific immunity protective reaction.
1993, hFL gene (Lyman SD has cloned in Immunex company, James L, Vanden BT, etal.Molecular cloning of a ligand for the flt3/flk-2 tyrosine kinase receptor:aproliferative factor for primitive hematopoietic cells.Cell, 1993,75 (6): 1157-67), and in eukaryotic cell, to express, it is mainly used in the stem cell transplantation of tumour patient behind melanoma, renal cell carcinoma, prostate cancer and the chemicotherapy.But its output is lower, can not satisfy the needs of large-scale application.
Graddis TJ etc. use two film rapid screening methods, screen the mutant of two amino acid changes (K84E/Q122R) among the hFL of yeast expression.This mutant increases about 3 times than normal hFL to the proliferation activity of BAF/hFlt3R cell.They separate the culture supernatant of this mutant yeast transformant → anti-hFL monoclonal antibody affinity chromatography post separation → concentrated → Superdex 200 separation → reconcentration through filtration → FractogelEMD SO3-650, obtain the pure product of reorganization hFL at last.This technology is complicated, cost height (Graddis TJ, Brasel K, Friend D et al.Structure-function analysis of FLT3 ligand-FLT3 receptorinteractions using a rapid functional screen.J Biol Chem 1998,273 (28): 17626-33).Therefore exist the needs of simplifying hFL preparation technology method.
In addition, aspect the immunogene treatment of tumour a lot of breakthroughs are being arranged both at home and abroad at present.Carry out immunomodulatory with adenovirus mediated cytokine, improve the antineoplastic immune of body, the treatment tumour has obtained numerous achievements.Selecting to have each other the cytokine of synergetic immunity regulating effect and antitumor action, carry out combined gene therapy, is a great development direction of tumor immune gene therapy.Research about various kinds of cell factor combination therapy malignant tumour has a lot, but up to now not about in a carrier, uniting the report of expressing hFL and hGM-CSF treatment malignant tumour.
Summary of the invention
Therefore, based on above-mentioned needs, the invention provides the nucleotide sequence of coding people hFL and use the method for this sequence table intelligent hFL and unite the method for expressing hFL and hGM-CSF and the two is united and expresses the purposes that is used for the treatment of malignant tumour.
The present invention relates to the polynucleotide sequence shown in SEQ ID NO.:1.
The invention still further relates to the carrier that contains described polynucleotide sequence, the particular methanol yeast shuttle vector.
The invention still further relates to the methanol yeast positive recombinant that contains described carrier.
The invention further relates to the hFL that expresses by described methanol yeast positive recombinant, i.e. the aminoacid sequence of SEQ IDNO.:2.
The invention further relates to the method for a kind of hFL of production, it may further comprise the steps:
1) will encode the polynucleotide sequence subclone of hFL to shuttle vectors, screening contains the clone of positive shuttle plasmid;
2) described positive shuttle plasmid is changed in the methanol yeast over to the hFL that abduction delivering is expressed with secreted form;
3) described secretory protein is carried out separation and purification;
The polynucleotide sequence that it is characterized in that described coding hFL is the polynucleotide sequence shown in the SEQ ID NO.:1.
The invention further relates to described hFL and be used for preparing the purposes of the medicine of hemopoietic stem cells hyperplasia
The invention still further relates to described hFL and be used for preparing the purposes of the medicine of anti-malignant tumor.
The invention further relates to the polynucleotide sequence of coding hFL shown in SEQ ID NO.:3.
The invention further relates to the dual-gene transformation adenovirus shuttle plasmid of the polynucleotide sequence that contains described polynucleotide sequence and coding hGM-CSF.
The invention further relates to the recombinant adenovirus that contains described carrier.
The invention further relates to described hFL and hGM-CSF and unite the purposes of expressing the medicine be used to prepare anti-malignant tumor.
The invention further relates to a kind of method of producing recombinant adenovirus, it may further comprise the steps:
1) will encode the polynucleotide sequence subclone of hFL and hGM-CSF to the adenovirus shuttle vector of transforming, screening contains the clone of positive shuttle plasmid;
2) with described positive shuttle plasmid and adenoviral gene group plasmid pAdEasy-1 cotransformation competence bacterium BJ5183, homologous recombination in bacterium produces recombinant adenovirus plasmid
3) change recombinant adenovirus plasmid over to 293 cells with the liposome transfection technology, collecting cell after 7 days, multigelation discharge the 1st generation recombinant adenovirus
4) cultivate 293 cells in a large number, infect 293 cells, collecting cell after 3 days, the recombinant adenovirus of the high titre of release behind the multigelation with the recombinant adenovirus that efficiently expresses.Use GFP analysis or MOI method and measure the recombinant adenovirus titre.
Operable in the present invention cell culture system has a lot, for example prokaryotic cell prokaryocyte, Mammals and insect cell culture systems, and they can be used to express the polynucleotide sequence of the hFL of coding shown in the SEQ ID NO.:2.
The hFL's that the present invention is expressed is active higher, and can work in coordination with to strengthen with the mGM-CSF coupling stimulates mouse bone marrow cells GM-CFU colony to form.
The inventor utilizes the methanol yeast expression system to express secretion hFL, and has obtained desired effects, and it can be obtained in methanol yeast and efficiently express, and adopts Q-FF to separate → concentrate → the SephacrylS-200 separation and purification, and step is simply effective.The measuring of hFL biologic activity, using hFL separately can stimulate the amplification of mouse bone marrow cells karyocyte, and can work in coordination with to strengthen with the mGM-CSF coupling stimulates mouse bone marrow cells GM-CFU colony to form.In the treatment experiment of mouse subcutaneous transplantation EL4 lymphoma model, experimental group is injected hFL after 14 days continuously, and tumour reduces than control group, and tumour inhibiting rate is 24.9%, and the P value is less than 0.001, and difference has remarkable statistical significance.
Have obvious synergy between FL and the hematopoietic cytokine GM-CSF, The combined injection mouse, the quantity that causes spleen DC is 75～100 times of control group apparently higher than both single usefulness.So, if use the recombinant adenovirus of carrier FL and two genes of human GM-CSF, express FL and GM-CSF in vivo simultaneously, may obtain better antitumous effect.To the encode polynucleotide sequence of people FL and human GM-CSF of the inventor is recombinated on the transformed adenovirus shuttle plasmid of the inventor, obtains containing dual-gene recombinant adenovirus, and, in the mouse anti tumor experiment, obtain 30% press down knurl efficient.People FL and human GM-CSF that combined utilization is adenovirus mediated, the quantity that increases dendritic cell in vivo is to suppress tumour, and this point is not report in the prior art.And unexpected is to make two goal gene all obtain to efficiently express through the transformed adenovirus shuttle plasmid of the inventor.
Description of drawings
Fig. 1 describes the synthetic synoptic diagram of hFL gene fragment.
Fig. 2 describes PCR product agarose gel electrophoresis and identifies.
Fig. 3 describes the building process of recombinant plasmid pPIC9K-hFL.
Fig. 4 describes positive colony pPIC9K-hFL enzyme and cuts evaluation collection of illustrative plates: M: λ DNA/Hind IIImarker; 1:pPIC9K-hFL/Xho I+EcoR I; 2:pPIC9K/Xho I+EcoR I; 3:pPIC9K-hFL.
Fig. 5 describes in the screening by hybridization high expression level transformant of two pleurodiaphragmatic in terspace positions, and different yeast transformant hFL expression level differences cause the strong and weak different of immunity colour developing, and arrow is depicted as the last one dyeing clone, represents its expression level height.
Fig. 6 describes the hFL in the Q-FF purifying yeast expression supernatant.
Fig. 7 describes RPHPLC (reversed-phase high-performance liquid chromatography) purifying hFL.
The SDS-PAGE that Fig. 8 describes the hFL purifying analyzes (coomassie brilliant blue staining) M:marker; 1. culture supernatant; The 2:Q-FF elution peak; The 3:S-200 elution peak; 4:RP-HPLC elution peak (containing DTT); 5:RP-HPLC elution peak (not containing DTT).
Fig. 9 describes the hFL of purifying and the Western blotting of part de-glycosylation hFL analyzes 1: purifying hFL (p.pastoris); 2: by the hFL (p.pastoris) after the de-glycosylation of EndoH part; 3: purifying hFL (E.coli); 4: negative control.
Figure 10 describes the growth curve of hFL to the bone marrow nucleated cell proliferation function.
Figure 11 describes the medullary cell colony and forms curve.
Figure 12 describes EL4 lymphoma area change curve in time.
Figure 13 describes the restriction enzyme mapping of T carrier-hFL and T carrier-hGM-CSF.M: λ DNA/Hind IIImarker; 1:T carrier-hFL/Sal I+Xba I; 2:T carrier-hGM-CSF/AgeI+AleI.
Figure 14 describes the restriction enzyme mapping of pAdTrack-CMV-hFL, pAdTrack-CMV-hFL-hGM-CSF.M：λDNA/Hind?III?marker；1：pAdTrack-CMV-hFL/Sal?I+Xba?I；
2：pAdTrack-CMV-hFL-hGM-CSF/Age?I+Ale?I。
Figure 15 describes the structure of shuttle plasmid pAdTrack-CMV-hFL-hGM-CSF.
Figure 16 describes pAd-hFL-hGM-CSF PacI restriction enzyme mapping.M：λDNA/Hind?III?marker；1：pAd-hFL-hGM-CSF/PacI；2：pAd-hFL-hGM-CSF。
Figure 17 describes Ad-hFL-hGM-CSF and feels malicious supernatant Western blotting analysis.A: goat-anti hFL one is anti-; B: the anti-hGM-CSF one of rabbit resists.1:Ad-hFL-hGM-CSF feels malicious supernatant 2:Ad-GFP and feels malicious supernatant.
Figure 18 describes the hFL concentration of adenovirus expression in vivo.
Figure 19 describes the hGM-CSF concentration of adenovirus expression in vivo.
Figure 20 describes Ad-hFL-hGM-CSF and feels the proliferation function of malicious supernatant to bone marrow nucleated cell.
Figure 21 describes the proliferation function of hGM-CSF reference substance to TF1.
Figure 22 describes Ad-hFL-hGM-CSF and feels the proliferation function of malicious supernatant to the TF1 cell.
Figure 23 describes CD11c in the spleen
+Cell accounts for the percentage ratio of spleen karyocyte.
Figure 24 describes spleen cell flow cytometer showed figure.
Figure 25 describes EL4 lymphoma area change curve in time.
Preferred implementation
The method that following reference example and experimental example are described polynucleotide of the present invention and the described hFL of preparation of the present invention in detail; its objective is further illustration the present invention; rather than limit its scope; what one of ordinary skill in the art will appreciate that is; under the situation that does not deviate from invention main points of the present invention, can carry out multiple modification to the present invention, and these are revised all within protection scope of the present invention; therefore, the scope of claim of the present invention is determined by appended claim.
1 people FL expression in methanol yeast, purifying, active determination in vitro and mouse anti tumor experiment
1.1 the acquisition of people FL gene
1.1.1 the nucleotide sequence of people FL
Polynucleotide sequence shown in the SEQ ID NO.:1 is in 156 codons of coding people FL gene, has 6 N end codons and replaces with that the methanol yeast preference codon obtains, and they are the 1st, 2,4,9,10,12 bit codons.Its concrete replacement sees Table 1.And the codon AAA of the 84th Methionin of FL is changed into the codon GAG of L-glutamic acid; The codon CAG of the 122nd glutamine changes into arginic codon AGA, to improve the hFL biologic activity.
Table 1 methanol yeast preference codon is revised table
According to SEQ ID NO.:1, design and synthesize 6 pairs of primers (P1-P2, P3-P4, P5-P6, P7-P8, P9-P10, P11-P12) (Fig. 1).Primer P1 contains the XhoI restriction enzyme site, and P12 contains EcoR I restriction enzyme site, overlapping 15～17bp between adjacent two fragments.Below be described PCR primer:
P1(60mers)：CCGCTCGAGA?AGAGAACTCA?AGACTGTTCC?TTCCAACACT?CTCCAATCTC?TTCCGACTTC
P3(55mers)：ACCTGCTTCA?AGATTACCCA?GTCACCGTGG?CCTCCAACCT?GCAGGACGAG?GAGCT
P5(53mers)：GTCCTGGCAC?AGCGCTGGAT?GGAGCGGCTC?AAGACTGTCG?CTGGGTCCAA?GAT
P7(56mers)：AACACGGAGA?TACACTTTGT?CACCGAGTGT?GCCTTTCAGC?CCCCCCCCAG?CTGTCT
P9(55mers)：CCGCCTCCTG?CAGGAGACCT?CCGAGCAGCT?GGTGGCGCTG?AAGCCCTGGA?TCACT
P11(54mers)：GAGCTGCAGT?GTCAGCCCGA?CTCCTCAACC?CTGCCACCCC?CATGGAGTCC?CCGG
P2(59mers)：AATCTTGAAG?CAGGTAGTCA?GACAGCTCAC?GGATTTTGAC?AGCGAAGTCG?GAAGAGATT
P4(55mers)：AGCGCTGTGC?CAGGACCAGC?CGCCAGAGGC?CCCCGCAGAG?CTCCTCGTCC?TGCAG
P6(55mers)：AGTGTATCTC?CGTGTTCACG?CGCTCCAGCA?AGCCTTGCAT?CTTGGACCCA?GCGAC
P8(56mers)：CTCCTGCAGG?AGGCGGGAGA?TGTTGGTCTG?GACGAAGCGA?AGACAGCTGG?GGGGGG
P10(56mers)：GCTGACACTG?CAGCTCCAGG?CACCGGGAGA?AGTTTCTGCG?AGTGATCCAG?GGCTTC
P12(56mers)：GGAATTCTTA?CGGGGCTGTC?GGGGCTGTGG?CCTCCAGGGG?CCGGGGACTC?CATGGG
P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12 are made into 50 μ mol/L concentration.The first round is carried out two fragment assemblies (P1 and P2, P3 and P4, P5 and P6, splicing successively).(P1, P2 are template with P1 and P2, be again primer) respectively get 1 μ l, add in the reaction system of 50 μ l and (include 2.5mmol/L dNTP (Promega company product) 8 μ l, 10x buffer 5 μ l, Taq enzyme 1-2U can add an amount of magnesium ion in case of necessity, supplies volume with the disinfectant redistilled water), the surface adds one deck mineral oil, puts into the PCR instrument.P3 and P4, P5 and P6, P7 and P8, P9 and P10, P11 and P12 also so handle, situation according to template and primer is set response procedures, described response procedures is the known technology of those of ordinary skills, amplifies P1-2, P3-4, P5-6, P7-8, P9-10, P11-12 respectively.It is template that P1-2 and P3-4 are respectively got 1 μ l, adds each 50pmol of P1, P4 more respectively and carries out second and take turns pcr amplification 4 fragment assembly product P 1-4.P5-8, P9-12 also so obtain.It is template that P1-4, P5-8 PCR product are respectively got 1 μ l, and adding each 50pmol of P1, P8 respectively, to carry out third round pcr amplification 8 fragment assembly products be P1-8.P5-12 roughly the same.It is template that P1-8, P5-12PCR product are respectively got 1 μ L, adds each 50pmol of P1, P12 respectively and carries out four-wheel pcr amplification hFL full length gene.Four-wheel PCR product is through 2%agarose electrophoresis result such as Fig. 2.
1.2 the structure of recombinant expression plasmid pPIC9K-hFL
The PCR product and the expression plasmid pPIC9K (available from Invitrogen, the U.S.) that reclaim are used Xho I and EcoR I double digestion respectively, connect with the T4 dna ligase then, connect product and transform host bacterium DH5 α.Owing to contain the XhoI restriction enzyme site in the kanamycin gene of pPIC9K, pPIC9K with Xho I and EcoR I double digestion after the plasmid skeleton to break at the kanamycin gene place be two big fragments.For guaranteeing correct connection, connect recon (Fig. 3) with the Kana resistance screening.Doubtful positive colony obtains a 501bp district band (Fig. 4) with Xho I and EcoR I double digestion.Utilize invitrogen pPIC9KAOX-5 ' primer and 3 ' primer that positive colony is carried out two-way sequencing, the result is consistent with implementation sequence.
1.3 expression and the purifying of recombinant protein in Pichia pastoris
1.3.1 plasmid transformed yeast host bacterium:
Recombinant expression plasmid pPIC9K-hFL is after the SalI linearizing, getting 5～10 μ g mixes with KM71 competence yeast 80 μ l, change 0.2cm electricity revolving cup over to, behind the ice bath 5min, utilize BioRad electroporation (1500V, 25 μ F, 200 Ω) carrying out electricity changes, then bacterium liquid is coated MD/-His and selected to hatch 2～3 days for 30 ℃ on the flat board, observe the transformant growth.
1.3.2 two pleurodiaphragmatics in terspace position screening by hybridization high expression level hFL transformant
Cellulose acetate film is placed the His of MD/-His flat board
+On the transformant bacterium colony, make cellulose acetate film moistening fully and catch up with most bubble, make all colony lifts to cellulose acetate film with light pressure of spreading rod.On the BMMY plate, put the cellulose nitrate film of a same size, and the cellulose acetate film bacterium colony is upwards placed on the cellulose nitrate film.30 ℃ hatch 18h after, cellulose acetate film is moved on the MD/-His flat board 4 ℃ of preservations.Cellulose nitrate film is taken off, and 37 ℃ of sealings of 6% calf serum 2h, TBS-T wash (each 10min) 3 times.The anti-hFL polyclonal antibody of rabbit (0.1 μ g/ml) incubated at room 2h, TBS-T washes film 3 times.Goat anti-rabbit igg-AP (2u/ml) incubated at room 2h then, TBS-T washes film 3 times.In 15ml BCIP/NBT substrate buffer solution, add 50 μ l BCIP and 99 μ l NBT, behind the room temperature reaction 5min, with the PBS termination reaction that contains 5mmol/L EDTA.
With different cell concn shop MD/-His plates, every plate obtained 100～1000 transformants when electricity changeed.Cellulose nitrate film will be secreted hFL outward through the yeast of cellulose acetate film and catch, identify the height of transformant expression level according to the power of immunity colour developing, each stain is corresponding to the corresponding bacterium colony of cellulose acetate film, and the colored intensity of different bacterium colonies has notable difference (Fig. 5).
1.3.3 the great expression of high expression level transformant and purifying
The transformant that is obtained by KM71 all is Mut
sType.The high expression level transformant that screens is inoculated in the 5mlBMG substratum, 30 ℃ of shaking culture 16～18h, inoculation in 1: 500 is gone in the 400ml BMG substratum, and 30 ℃ of shaking culture are spent the night to OD
600Be 2～6, centrifugal collection thalline is concentrated into OD with 100ml BMM
600Be 10～20 back abduction deliverings, adding methyl alcohol to final concentration every 24h is 2%, coinduction 96h, centrifugal collection yeast culture supernatant.
With culture supernatant usefulness 20mmol/L Tris-Cl (pH8.0) dialysis and behind 0.45-μ m membrane filtration, be splined on anion-exchange column Q-FF, 20mmol/L Tris-Cl (pH8.0) wash-out is not conjugated protein, conjugated protein with linear salt gradient (0～0.8mol/L NaCl, 20mmol/L Tris-Cl, pH8.0) wash-out, collect target protein solution through concentrating, be splined on again with 20mmol/L phosphoric acid buffer (0.15mol/L NaCl, pH7.0) equilibrated S-200 is with same buffer solution elution target protein.More than operation is all carried out at 4 ℃.Target protein is further purified and identifies through RPHPLC (reversed-phase high-performance liquid chromatography) (RP-HPLC).The RP-HPLC post is with solution A (2% acetonitrile, 0.1% trifluoracetic acid TFA) balance, with solution A and solution B (90% acetonitrile, linear gradient elution 0.1%TFA).Respectively go on foot purification effect, gel coomassie brilliant blue staining with the 15%SDS-PAGE detection.
Expression product has an elution peak respectively at 0.2mol/L and 0.5mol/L NaCl gradient place when anion-exchange column Q-FF gradient elution, the former is target protein peak (Fig. 6).Collect this peak, again through molecular sieve S-200 purifying, RP-HPLC purifying and evaluation obtain a symmetric target protein peak after concentrating, and purity reaches (Fig. 7) more than 98%.Each goes on foot SDS-PAGE collection of illustrative plates such as Fig. 8 of purification of samples, the mobility speed of hFL in not containing the irreducibility system of DTT is than fast slightly in the reductibility damping fluid that contains DTT, and its reason may be that protein molecule is tightr than the space structure of all opening when disulfide linkage exists.The main band of hFL is at the 22kD place, and other has sub-fraction hFL owing to the degree of glycosylation difference, and molecular weight is bigger, is positioned at the 34kD place.
1.3.4 the Westem blotting of yeast expression hFL identifies
Using the anti-hFL one anti-Western blotting that carries out of rabbit identifies.The hFL Western blotting result of purifying is positive, illustrates that the yeast expression product can combine with the anti-hFL polyclonal antibody of rabbit, and control group is hybridized be negative (Fig. 9).Think that at first the band at the middle 34kD place of SDS-PAGE collection of illustrative plates (Fig. 8) is a foreign protein, the albumen of finding 34kD and 22kD in each step purge process afterwards always elutes substantially simultaneously, this band develops the color equally in Western blotting, and this may be the hFL of different glycosylation form.Remove the N-sugar chain of hFL through EndoH after, Western blotting shows that 34kD district band disappears, and hFL master's band molecular weight diminishes, but still be higher than the sugar based hFL (Fig. 9) of expression in escherichia coli, not only there is the N-glycosylation among the hFL that the prompting methanol yeast is expressed, also has other forms of glycosylation.
1.4 recombinant protein physical and chemical property determining
To carry out physical and chemical property determining through the hFL of Q-FF, S-200 and RP-HPLC purifying successively.
1.4.1 the hFLN terminal amino acid sequence is analyzed
It is TQDCSFQHSP that the hFL N of yeast expression holds preceding 10 amino acid, and sequencing result is consistent with design.
1.4.2 molecular weight determination
Through Maldi-Tof-MS mass spectroscopy (Voyager-DE PRO) determining molecular weight, the result shows that the recombinant protein molecular weight shows tool glycosylation to a certain degree between 20～22kD.
1.4.3 capillary tube technique is surveyed iso-electric point
Basis institute centralab is 3.12～4.72 with Beckman capillary electrophoresis apparatus (P/ACE System 5500) mensuration hFL iso-electric point, and it is heterogeneity, may be due to the degree of glycosylation difference.
1.5 the external determination of activity of recombinant protein:
1.5.1 hFL is to the proliferation function of bone marrow nucleated cell:
1) the neck dislocation method is put to death mouse, and 75% sterilization in alcohol-pickled 1 minute is dissected and got femur, rejects muscle tissue.
2) cut off femur two ends epiphysis, get physiological saline with the 1ml syringe, marrow is all blown out, pressure-vaccum becomes the individual cells suspension repeatedly.1000rpm * 5 ' abandons supernatant.Add erythrocyte cracked liquid, 1000rpm * 5 ' abandons supernatant.
3) RPMI 1640 re-suspended cells of 20% horse serum, counting, diluting cells is to concentration 2 * 10
5/ ml.
4) in 96 orifice plates, every hole adds 80 μ l hFL, adds 80 μ l cell suspensions again.The hFL final concentration is followed successively by 100ng/ml, 10ng/ml, 1ng/ml, 100pg/ml, 10pg/ml, 0pg/ml.Every concentration is done 4 multiple holes.37 ℃, 5%CO
2Cultivated 9 days.Every day, observation of cell was bred situation therebetween.
5) every hole adds 20 μ l5mg/ml MTT, 37 ℃, 5%CO
2Hatch and add 10% acidifying SDS (containing 10mM HCl) behind the 4hr, dissolving crystallized, 37 ℃, 5%CO
2Overnight incubation is surveyed the OD630 value.
Mouse bone marrow cells karyocyte vitro culture observed the cell count of adding hFL and begins showed increased, and do not contain the hFL group in the time of 3～4 days, and the most of beginning of cell is dead, and most of cell is dead in 72 hours.In the time of 9 days, use mtt assay, draw the growth curve (Figure 10) of hFL the proliferation function of bone marrow nucleated cell.
1.5.2 medullary cell colony-forming test
1) the neck dislocation method is put to death mouse, and 75% sterilization in alcohol-pickled 1 minute is dissected and got femur, rejects muscle tissue.
2) cut off femur two ends epiphysis, get physiological saline with the 1ml syringe, marrow is all blown out, pressure-vaccum becomes the individual cells suspension repeatedly.1000rpm * 5 ' abandons supernatant.Add erythrocyte cracked liquid, 1000rpm * 5 ' abandons supernatant.
3) 1 * IMDM re-suspended cell, counting, diluting cells is to concentration 2 * 10
5/ ml.
4) in 1ml EP pipe, add 400 μ l, 1 * IMDM successively, cytokine, 100 μ l cell suspensions.Use the hFL group separately, the final concentration that adds hFL is followed successively by 100ng/ml, 10ng/ml, 1ng/ml, 100pg/ml, 10pg/ml, mouse GM-CSF (mGM-CSF) and hFL combined utilization group, add 100ng/ml mGM-CSF, add hFL again and reach final concentration 100ng/ml, 10ng/ml, 1ng/ml, 100pg/ml, 10pg/ml, 0pg/ml.
5) in each 1ml EP pipe, add 1 * IMDM that 500 μ l contain 20% foetal calf serum, 2% methylcellulose gum, put upside down mixing, add in six orifice plates.
6) 37 ℃, 5%CO
2Cultivated 9 days.Counting surpasses clone's number of 50 cells.
The Kong Zhongwu clone of single hFL of adding forms.The hFL+mGM-CSF group increases with hFL concentration, and clone's number increases, and clone's size, and cell number also increases with hFL concentration.Cultivated the 8th day and the 10th day, at 100 * microscopically, counting surpasses 50 cells less than 100 cells respectively, reaches the clone's number greater than 100 cells.See Table 2, table 3, Figure 11.
Compared with prior art, hFL that the present invention is expressed and the synergy of mGM-CSF obviously increase, for example, at the 8th day, 100pg/ml hFL and mGM-CSF coupling group, per 10
5Bone marrow nucleated cell forms 90 clones, and clone's number of organizing than independent application mGM-CSF increases by 9 times; 100ng/ml hFL and mGM-CSF coupling group, per 10
5Bone marrow nucleated cell forms 210 clones, increases by 21 times than clone's number of independent application mGM-CSF.Broxmeyer HE etc. use 200ng/ml hFL and mGM-CSF coupling, per 10
5Bone marrow nucleated cell forms 82 clones, only increase about 1 times than clone's number of using mGM-CSF separately, see: Broxmeyer HE, Lu L, Cooper S, et al.Flt3 ligand stimulates/costimulatesthe growth of myeloid stem/progenitor cells Experimental hematology, 1995,23:1121-1129.
Table 2 hFL+mGM-CSF group is cultivated the 8th day clone's number
| Every hole clone's | 50～100 cell clones | ＞100 cell clones | Add up to | ?10 5Medullary cell forms clone's number |
| ?hFL?100ng/ml+mGM-CSF?100ng/ml ?hFL?10ng/ml+mGM-CSF?100ng/ml ?hFL?1ng/ml+mGM-CSF?100ng/ml ?hFL?100pg/ml+mGM-CSF?100ng/ml ?hFL?10pg/ml+mGM-CSF?100ng/ml ?mGM-CSF?100ng/ml | ?15 ?11 ?12 ?9 ?8 ?2 | ?27 ?16 ?9 ?9 ?7 ?0 | ?42 ?27 ?21 ?18 ?15 ?2 | ?210 ?135 ?105 ?90 ?75 ?10 |
Table 3 hFL+mGM-CSF group is cultivated the 10th day clone's number
| Every hole clone's | 50～100 cell clones | ＞100 cell clones | Add up to | ?10 5Medullary cell forms clone's number |
| ?hFL?100ng/ml+mGM-CSF?100ng/ml ?hFL?10ng/ml+mGM-CSF?100ng/ml ?hFL?1ng/ml+mGM-CSF?100ng/ml ?hFL?100pg/ml+mGM-CSF?100ng/ml ?hFL?10pg/ml+mGM-CSF?100ng/ml ?mGM-CSF?100ng/ml | ?27 ?8 ?11 ?14 ?11 ?5 | ?27 ?24 ?13 ?10 ?7 ?0 | ?54 ?32 ?24 ?24 ?18 ?5 | ?270 ?160 ?120 ?120 ?90 ?25 |
1.6 anti-tumor activity in the recombinant protein body
The treatment experiment of mouse subcutaneous transplantation EL4 lymphoma model: 12 of the male C 57 BL/6 J mouses of 20～22 grams, divide cage according to body weight, be divided into two groups, make every group of 6 mouse body weight even.Collection is in the EL4 lymphoma cell of logarithmic phase, wash once with PBS after, concentration to 2 * 10 are adjusted in cell counting
6/ ml.The above-mentioned cell suspension of the abundant mixing of injection 100 μ l under the right ribbed hide of every mouse, the ophthalmology tweezer clamped for 10 seconds, and anti-non-return leaks.The treatment group is from beginning in the 3rd day, subcutaneous injection every day hFL albumen 30 μ g, continuous 14 days.Control group is injected 100 μ l PBS every day.
The injection 2 * 10 down of the right ribbed hide of mouse
5Behind the EL4 cell, can touch enclosed mass on the 9th day, measure major diameter a of tumour and minor axis b (mm) every other day, draw tumour area S=ab (mm
2) growth curve (Figure 12).Treat that tumour grows to 22 days, put to death mouse, dissect and get tumor tissues, weigh (table 4), calculate tumor control rate:
22 days posterior tuberosities of table 4 mouse inoculation tumour are heavy
The hFL group is compared with the PBS group, and there were significant differences for the tumour size, and tumor control rate is 24.9%, and through the t check, this difference has remarkable statistical significance (P＜0.001).
The hFL of 2 mediated by recombinant adenovirus and the treatment of hGM-CSF anti-malignant tumor
2.1 homologous recombination method is prepared high titre recombinant adenovirus Ad-hFL-hGM-CSF and Ad-GFP in the bacterium
2.1.1 coding hFL and hGM-CSF dna fragmentation two ends primer design and dna fragmentation obtain
As template, design five 5 ' end primers and one 3 ' primer with recombination yeast plasmid pPIC9K-hFL altogether.
Article one, 5 ' end primer adopts Sal I restriction enzyme site GTCGAC, makes that like this 5 ' the UTR length of hFL mRNA is 37bp, is suitable for ribosomal combination to efficiently express.Add KOZAK sequence (gcc gcc accATGg) ,+4 change G into by A, and thus, second amino acid is changed into GGA (gly) by ACA (Thr).The encoding sequence of 26 amino acid: MGVLAPAWSPTTYLLLLLLLSSGLSG of signal peptide of second and third 5 ' end design of primers hFL self, and change the codon of five amino acid ALA into GCC by GCG, can reduce a pairing in the loop-stem structure that primer self forms like this, and be the high expression level codon, to increase the translation efficiency of mRNA; Article fourth, fifth, 5 ' end primer is had a liking for the codon that codon changes the original eukaryotic expression of hFL into yeast.
3 ' primer contains XbaI enzyme cutting site TCTAGA, designs two different terminator codons, and adds purine A (TAA TGA A) thereafter, to guarantee to stop expression.The codon of first and the 4th amino acid Pro of C end changes CCC into by CCG, and C holds the codon of the 3rd amino acid Thr to change ACC into by ACA, is eucaryon preference codon.
The nucleotides sequence of coding hFL is classified the sequence of SEQ ID NO.:3 as.
The recombinant plasmid HTB194 that contains hGM-CSF cDNA sequence that preserves with this chamber design one 5 ' and holds primer and one 3 ' primer as template.5 ' primer contains AgeI restriction enzyme site ACCGGT, KOZAK sequence (ggAG GATG).3 ' primer contains AleI restriction enzyme site CACCAGGGTG, designs two different terminator codons, and adds purine A (TAA TGA A) thereafter, to guarantee to stop expression.
The nucleotides sequence of coding hGM-CSF is classified as
ACCGGTggAG?GATGTGGCTG?CAGAGCCTGC?TGCTCTTGGG?CACTGTGGCC?TGCAGCATCT
CTGCACCCGC?CCGCTCGCCC?AGCCCCAGCA?CGCAGCCCTG?GGAGCATGTG?AATGCCATCC
AGGAGGCCCG?GCGTCTCCTG?AACCTGAGTA?GAGACACTGC?TGCTGAGATG?AATGAAACAG
TAGAAGTCAT?CTCAGAAATG?TTTGACCTCC?AGGAGCCGAC?CTGCCTACAG?ACCCGCCTGG
AGCTGTACAA?GCAGGGCCTG?CGGGGCAGCC?TCACCAAGCT?CAAGGGCCCC?TTGACCATGA
TGGCCAGCCA?CTACAAGCAG?CACTGCCCTC?CAACCCCGGA?AACTTCCTGT?GCAACCCAGA
TTATCACCTT?TGAAAGTTTC?AAAGAGAACC?TGAAGGACTT?TCTGCTTGTC?ATCCCCTTTG
ACTGCTGGGA?GCCAGTCCAG?GAGTGATAAG?CACCCTGGTG
Below be described PCR primer:
5 ' the primer of hFL
P1(25mers)：GGTCGACGCC?GCCACCATGG?GAGTG
P2(59mers)：GAAGATCTAT?GACAGTGCTG?GCGCCAGCCT?GGAGCCCAAC?AACCTATCTC?CTCCTGCTG
P3(59mers)：CCTATCTCCT?CCTGCTGCTG?CTGCTGAGCT?CGGGACTCAG?TGGGACCCAG?GACTGCTCC
P4(35mers)：CGGGATCCCT?TGTTCCAAGG?ACCCAGGACT?GCTCC
P5(56mers)：ATTTTGACAG?CGAAGTCGGA?GGAGATGGGG?CTGTGTTGGA?AGGAGCAGTC?CTGGGT
3 ' the primer of hFL
P6(33mers)：GCTCTAGATT?CATTAGGGGG?CGGTGGGGGC?TGT
5 ' the primer of hGM-CSF and 3 ' primer
P5(24mers)：GCGACCGGT?GGAG?GATGTGGCTG?C
P6(31mers)：GCACCAGGGTG?CTTATCACTC?CTGGACTGGC
Go up the gene hFL that PCR goes out to contain self signal peptide from expression plasmid of yeast pPIC9K-hFL, be connected to the T carrier, doubtful positive colony obtains a 574bp district band (Figure 13) with Sal I and Xba I double digestion, by the order-checking of TaKaRa company correctly.
Go up the gene hGM-CSF that PCR goes out to contain signal peptide from recombinant plasmid HTB194, be connected to the T carrier, doubtful positive colony obtains a 460bp district band (Figure 13) with AgeI and AleI double digestion, and is correct by the order-checking of TaKaRa company.
2.1.2 the structure of shuttle plasmid pAdTrack-CMV-hFL and pAdTrack-CMV-hFL-hGM-CSF
Shuttle plasmid pAdTrack-CMV Sal I and Xba I double digestion, big fragment is reclaimed in 0.8% gel electrophoresis.T carrier-hFL is used Sal I and Xba I double digestion equally, and the hFL dna fragmentation is reclaimed in 1.5% gel electrophoresis.Both recovery products connect with the T4 dna ligase, in 13 ℃ of incubated overnight, make hFL gene directed cloning to pAdTrack-CMV.To connect product transformed competence colibacillus DH5 α, the positive colony of hFL DNA is inserted in screening, and extracting plasmid cuts evaluation (Figure 14) through Sal I and Xba I enzyme in a small amount.
In order to obtain containing dual-gene shuttle plasmid pAdTrack-CMV-hFL-hGM-CSF, will identify correct pAdTrack-CMV-hFL AgeI and AleI double digestion, big fragment is reclaimed in 0.8% gel electrophoresis.T carrier-hGM-CSF is used AgeI and AleI double digestion equally, and hGM-CSF DNA is reclaimed in 1.5% gel electrophoresis.Both recovery products connect with the T4 dna ligase, and 13 ℃ of incubated overnight make hGM-CSF gene directed cloning to pAdTrack-CMV-hFL (Figure 15).To connect product transformed competence colibacillus DH5 α, the positive colony of hGM-CSF DNA is inserted in screening, extracts plasmid in a small amount and identifies (Figure 14) through AgeI and AleI double digestion.
2.1.3 homologous recombination produces recombinant adenovirus plasmid pAd-hFL-hGM-CSF and pAd-GFP in the bacterium
Get 0.5 μ g shuttle plasmid pAdTrack-CMV-hFL-hGM-CSF and pAdTrack-CMV, cut with Pme I enzyme respectively, make it linearizing, electrophoresis reclaims then, is dissolved in 6 μ l H respectively
2O.Add 1 μ l pAdEasy-1 (about 0.1 μ g) respectively, mixing adds 40 μ l electroreception attitude BJ5183 bacterium, mixing then, be transferred to the electric revolving cup of ice bath, under 2.5kv/200 Ω/25 μ F conditions, carry out electricity and transform, add 500 μ l LB substratum immediately, mixing, transfer to 1.5ml Ep pipe, in 37 ℃ of insulations 15～20 minutes, to get 50～200 μ l and be applied to LB/Kan (50 μ g/mL) flat board, 37 ℃ of incubators spend the night (16～20hrs).The colony inoculation of 10～20 minimums of picking contains among the LB of kan50 μ g/ml in 3mL, and 37 ℃ of shaking tables are cultivated 12～15hrs.Extract plasmid in a small amount by the alkali cracking method, the plasmid electrophoresis that takes a morsel earlier, the probable positive plasmid is cut with the PacI enzyme, and the plasmid of correct reorganization a big fragment (about 35kb) and a little fragment (3.0kb or 4.5kb) can occur (Figure 16).Get and identify that correct pAd-hFL-hGM-CSF and each 1～2 μ l of pAd-GFP plasmid carry out electricity and transform in DH10B, carry out plasmid then and increase in a large number, use EndoFree
TMPlasmid Maxi Kit plasmid purification is standby.
2.1.4 the packing of recombinant adenovirus Ad-hFL-hGM-CSF and Ad-GFP
Preceding 1 day of transfection inoculates 2 * 10 in the T-25 culturing bottle
6293 cells, cell is about 50%～70% degree of converging during to transfection.Get each 4 μ g of plasmid pAd-hFL-hGM-CSF and pAd-GFP and cut with Pac I enzyme respectively, phenol, phenol/chloroform, each extracting of chloroform 1 time, ethanol sedimentation, 70% washing with alcohol twice, the super clean bench room temperature dries up, and is dissolved in 20 μ l ddH
2O adds 20 μ l Lipofectamine2000 again and is mixed together in 500 μ l OPTI-MEM
I, incubated at room 10～30 minutes.Bide one's time waiting, 293 cell culture mediums in the T-25 culturing bottle are gone, use 4ml OPTI-MEM
I washes 1 time, adds 2.5ml OPTI-MEM again
I places 37 ℃ of CO again
2About 10 minutes of incubator.Above-mentioned Lipofectamine2000-DNA mixture is added culturing bottle, put back to 37 ℃ of CO
2Incubator is hatched 4hrs.Discard the nutrient solution supernatant that contains the Lipofectamine2000-DNA mixture, add 6mlDMEM 5%, continue to cultivate 7～10 days, changed 6mL DMEM 5%1 times in per 2～3 days in 5 days after the transfection, no longer changed substratum in 5 days later on.1～2 day pAd-GFP can see green fluorescent protein in the cell (GFP) expression after the transfection under fluorescent microscope, increases gradually later on, reaches the peak in 7～10 days, and left and right sides cell partly came off in 8 days; Centrifugal collecting cell after 7～10 days is abandoned supernatant after the transfection, and cell precipitation is suspended in 2ml DPBS, and 37 ℃/-70 ℃ multigelations 4 times are simply centrifugal, stay supernatant, are stored in-70 ℃, be for the 1st generation the adenopathy venom, titre can reach 10
7Pfu/ml.Get 293 cells that 2 bottles of T-25 50%～70% of 1.0ml supernatant transfection converge, can see CPE after about 2～3 days, under fluorescent microscope, observe, can see that a large amount of GFP express in the cell, after the transfection 3～5 days, when about 1/3～1/2 293 cell detachments, centrifugal collecting cell, collect virus as stated above, be for the 2nd generation adenovirus, titre can reach 10
9More than the pfu/ml.
Owing to do not contain the GFP gene,, still can be observed CPE among the pAd-hFL-hGM-CSF so under fluorescent microscope, can't see green fluorescence, cytopathy, swelling becomes circle, agglomerating floating.All the other steps are the same.
2.1.5 the evaluation of recombinant adenovirus Ad-hFL-hGM-CSF and Ad-GFP
The Ad-GFP recombinant adenovirus does not need further evaluation, if the 1st generation Adenovirus Transfection 293 cells in can see fluorescent substance.Western blotting method is adopted in the evaluation of Ad-hFL-hGM-CSF.
The culture supernatant of pAd-GFP the 3rd generation adenovirus infection 293 cells is as negative control, and the culture supernatant of pAd-hFL-hGM-CSF the 4th generation adenovirus infection 293 cells is a sample to be measured.
It is anti-to use goat-anti hFL one respectively, the anti-hGM-CSF one of rabbit resists, and is the Western blotting that pAd-hFL-hGM-CSF feels malicious supernatant, and the result is all positive, and molecular weight is higher than the molecular weight of prokaryotic expression protein, illustrates that hFL and the hGM-CSF in the culture supernatant all has to a certain degree glycosylation.Control group is hybridized be negative (Figure 17).
2.1.6 a large amount of amplifications of adenovirus pAd-hFL-hGM-CSF and pAd-GFP, preservation
Be accredited as the male recombinant adenovirus, in 293 cells, breed.After infecting 48 hours with MOI=10～20, when complete pathology appears in cell, collecting cell, the centrifugal substratum that discards is with a small amount of DPBS re-suspended cell.Through freeze thawing three times, the centrifugal 10min of 3000rpm resets and add 10% glycerine in the absorption, uses the membrane filtration degerming of 0.2 μ m then, after the packing in-70 ℃ of preservations.
2.1.7 titer determination
1) green fluorescent protein counting process:
Measure preceding 1 day, a peek Φ 60mm culture dish, every ware inoculation 2 * 10
6Individual 293 cells were about 70%～90% to the 2nd day and converge.Press limiting dilution assay, get different dilution viral supernatant 1ml and infect 293 cells, 37 ℃ of CO
2Incubator was hatched 90 minutes, added DMEM5%4ml again, and after transfection 18 hours, counting was expressed the cell count of GFP under fluorescent microscope, by formula: virus titer=viral dilution multiple * GFP positive cell number (pfu/ml).The virus titer of measuring with this method is the complete adenovirus titre (pfu-plaque forming unit, plaque forming unit) with transfection ability.
2) MOI method
With 12 35mm plates (or 26 orifice plates), by 8 * 10
5Cell/plate (hole) amount inoculation 293 cells, overnight incubation.Getting viral liquid 0.15ml adding to be measured is equipped with in the Eppendorf pipe of 1.35ml PBS, make 10 * doubling dilution to the, 5 pipes behind the mixing successively, get each dilution viral liquid and infect 293 cells by 0.5ml/ plate (hole) amount, cultivate after 36～48 hours, the observation of cell pathology viral dilution degree of pathology occurs with nearly 100% cell, and the following formula of substitution calculates virus titer, 10 viruses of each cell infection of supposition can cause cytopathy in the formula in 36～48 hours.
Prepare adenovirus Ad-GFP and Ad-hFL-hGM-CSF according to a conventional method in a large number, concentrating back its titre of mensuration is 5 * 10
10Pfu/ml.
2.2 Expression in Vivo and in Vitro flow measurement
2.2.1 vivoexpression flow measurement
With 1 * 10
6Individual 293 cell inoculations in 6 orifice plates, 37 ℃, 5%CO
2Hatch 4hr and make cell attachment.According to MOI=30, infect 293 cells respectively with adenovirus Ad-GFP, Ad-hFL-hGM-CSF.Collect the 24hr supernatant.Dilute with certain multiple, use R ﹠amp; D hFL and hGM-CSF immunity detection reagent are measured hFL and hGM-CSF content.As table 5, table 6.
The hFL concentration of table 5 adenovirus vivoexpression
| ??293 | |
| ??Ad-GFP | Be lower than 15.6pg/ml * |
| ??Ad-hFL-hGM-CSF | ??96ng/ml |
15.6pg/ml
*Detection Schwellenwert for this test kit
The hGM-CSF concentration of table 6 adenovirus vivoexpression
| ??293 | |
| ??Ad-GFP | Be lower than 7.8pg/ml * |
| ??Ad-hFL-hGM-CSF | ??306ng/ml |
7.8pg/ml
*Detection Schwellenwert for this test kit
2.2.2 expression in vivo flow measurement
Mouse is subcutaneous injection adenovirus Ad-GFP, Ad-hFL-hGM-CSF 5 * 10 respectively
9Pfu gets 4hr, 8hr, 24hr, 48hr, 96hr, 8 days, 11 days mouse tail blood 50 μ l, and room temperature is placed 1hr, and 3000rpm * 15 ' is centrifugal, gets serum, dilutes with certain multiple, uses R﹠amp; D hFL and hGM-CSF immunity detection reagent are measured hFL and hGM-CSF content.
Injection Ad-hFL-hGM-CSF group, hFL reached climax 25ng/ml in the serum at 24 hours, hGM-CSF reached climax 3.3ng/ml at 8 hours, and continued 8～11 days, still can detect expression (Figure 18, Figure 19).Injection Ad-GFP group does not detect the expression of hFL and hGM-CSF all the time in the serum.
2.3 active determination in vitro
2.3.1 recombinant adenovirus infects the proliferation function of 293 hFL that express to bone marrow nucleated cell
With 1 * 10
6Individual 293 cell inoculations in 6 orifice plates, 37 ℃, 5%CO
2Hatch 4hr and make cell attachment.According to MOI=30, infect 293 cells with adenovirus Ad-GFP, Ad-hFL-hGM-CSF.Collect the 24hr supernatant ,-20 ℃ frozen standby.
1) the neck dislocation method is put to death mouse, and 75% sterilization in alcohol-pickled 1 minute is dissected and got femur, rejects muscle tissue.
2) cut off femur two ends epiphysis, get physiological saline with the 1ml syringe, marrow is all blown out, pressure-vaccum becomes the individual cells suspension repeatedly.1000rpm * 5 ' abandons supernatant.Add erythrocyte cracked liquid, 1000rpm * 5 ' abandons supernatant.
3) RPMI 1640 re-suspended cells of 20% horse serum, counting, diluting cells is to concentration 2 * 10
5/ ml.
4) in 96 orifice plates, Ad-GFP, the Ad-hFL-hGM-CSF of every hole 20 μ l feel malicious supernatant, add the RPMI 1,640 20 μ l of 20% horse serum, and each sample is established 4 parallel holes.The cell suspension that adds 80 μ l again.37 ℃, 5%CO
2Cultivated 9 days.Every day, observation of cell was bred situation therebetween.
5) every hole adds 20 μ l5mg/ml MTT, 37 ℃, 5%CO
2Hatch and add 10% acidifying SDS (containing 10mM HCl) behind the 4hr, dissolving crystallized, 37 ℃, 5%CO
2Overnight incubation is surveyed the OD630 value.
The mouse bone marrow cells karyocyte is in vitro culture, in the time of 3～4 days, observe and add Ad-hFL-hGM-CSF and feel malicious supernatant group cell count and begin showed increased, feel malicious supernatant group and blank group and add Ad-GFP, the most of beginning of cell is dead, cell count reduces, and most of cell is dead in 72 hours.In the time of 9 days, use mtt assay and measure cel l proliferation (Figure 20).
2.3.2 Ad-hFL-hGM-CSF infects the effect that 293 hGM-CSF that express stimulate TF1 cell proliferation
With 1 * 10
6Individual 293 cell inoculations in 6 orifice plates, 37 ℃, 5%CO
2Hatch 4hr and make cell attachment.According to MOI=30, infect 293 cells with adenovirus Ad-hFL-hGM-CSF.Collect the 24hr supernatant ,-20 ℃ frozen standby.
After will being in TF1 cell in the logarithmic phase and washing twice, the concentration of cell suspension is adjusted into 2 * 10 with the substratum that does not contain the factor with the RPMI 1640 training bases that do not contain the factor
6/ ml.In 96 orifice plates, every hole adds the sense poison supernatant of four times of gradient dilutions of 100 μ l, inoculate 100 μ l TF1 cell suspensions, the hGM-CSF final concentration is followed successively by 0.009ng/ml, 0.037ng/ml, 0.15ng/ml, 0.59ng/ml, 2.34ng/ml, 9.38ng/ml.The every hole of positive control adds the hGM-CSF standard substance of four times of gradient dilutions of 100 μ l, inoculate 100 μ l TF1 cell suspensions, the hGM-CSF final concentration is followed successively by 0.024ng/ml, 0.097ng/ml, 0.39ng/ml, 1.5625ng/ml, 6.25ng/ml, 25ng/ml.Every concentration is done 3 multiple holes.37 ℃, 5%CO
2Cultivated 4 days.Every day, observation of cell was bred situation therebetween.Every hole adds 20 μ l 5mg/ml MTT, 37 ℃, 5%CO
2Hatch and add 10% acidifying SDS (containing 10mM HCl) behind the 4hr, dissolving crystallized, 37 ℃, the 5%CO2 overnight incubation is surveyed the OD630 value.
The TF1 cell is in the substratum that does not contain hGM-CSF, and is dead gradually from beginning in second day.Add Ad-hFL-hGM-CSF and feel malicious supernatant group cell count showed increased, after 4 days, use mtt assay, draw hGM-CSF TF1 cel l proliferation curve, coincide substantially with the hGM-CSF reference substance (Figure 21, Figure 22).
2.4 DC in adenovirus mediated people hFL and the hGM-CSF associating gene amplification mouse body
1) C57BL/6J mouse subcutaneous injection 5 * 10 respectively
9Pfu Ad-GFP, Ad-hFL-hGM-CSF.
2) the 8th day, the neck dislocation method was put to death mouse, and dissection is got spleen and weighed.On 100 eye mesh screens, grind spleen with the glass syringe core, use PBS ' (PBS ' preparation: PBS+1～2% deactivation FCS+0.09% sodium azide) preparation single cell suspension, centrifugal 1000rpm * 5 ' abandons supernatant.Add erythrocyte cracked liquid, 1000rpm * 5 ' abandons supernatant.
3) PBS ' re-suspended cell, counting, adjusting cell concn is 10
7/ ml.
4) get 100 μ l cells in pipe, add the anti-mouse CD11c antibody 5 μ l of PE mark and the anti-mouse CD11b antibody 0.5 μ l of FITC mark, mixing lucifuge, ice bath 30～45min.
5) add the cold PBS ' of 100～200 μ l, 250g/1500rpm * 5 ', 2ml PBS ' washes once again.0.5ml cold PBS ' is resuspended, flow cytometer detects.
The injection Ad-hFL-hGM-CSF mouse spleen enlarge markedly, total spleen cell number be increased to normal value～2 times, the injection Ad-GFP mouse spleen then do not have considerable change.The Ad-hFL-hGM-CSF group, CD11c in the spleen
+Cell (consistent) percentage ratio average out to 14.4%, and the spleen DC less than 0.5% of normal mouse with the surface marker of DC, CD11c
+The DC number significantly increase to normal value～50 times (Figure 23, Figure 24).
2.5 adenovirus mediated people FL and human GM-CSF combined gene therapy mouse malignant tumour
18 of the male C 57 BL/6 J mouses of 20～22 grams divide cage according to body weight, make every group of 6 mouse body weight even.Collection is in the EL4 lymphoma cell of logarithmic phase, wash once with PBS after, concentration to 2 * 10 are adjusted in cell counting
6/ ml.The above-mentioned cell suspension of the abundant mixing of injection 100 μ l under the right ribbed hide of every mouse, the ophthalmology tweezer clamped for 10 seconds, and anti-non-return leaks.At injection tumour first three sky, subcutaneous injection Ad-GFP, Ad-hFL-hGM-CSF 5 * 10 every other day
9Pfu injects four times altogether.Control group is injected 100 μ l PBS every day.After treating that tumour grows, use major diameter a of kind of calliper tumour and minor axis b (mm) every other day, calculate tumour area S=ab (mm
2), draw tumour Area Growth curve (Figure 25).Treat that tumour grows to 21 days, put to death mouse, dissect and get tumor tissues, weigh (table 7) calculates tumor control rate.
21 days posterior tuberosities of table 7 mouse inoculation tumour are heavy
*Test unexpected death in the 5th day, irrelevant with sample.
The Ad-hFL-hGM-CSF group is compared with PBS group, GFP, and the tumour size has notable difference, and tumor control rate is 30%, and through the t check, this difference has statistical significance (P＜0.05).
Sequence table
＜110〉Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
＜120〉a kind of people FL polynucleotide and with the purposes of human GM-CSF combined gene therapy malignant tumour
<130>CGCNC41289
<160>5
<170>PatentIn?version?3.1
<210>1
<211>468
<212>DNA
＜213〉yeast
<220>
<221>CDS
<222>(1)..(468)
<223>
<400>1
act?caa?gac?tgt?tcc?ttc?caa?cac?tct?cca?atc?tct?tcc?gac?ttc?gct??????48
Thr?Gln?Asp?Cys?Ser?Phe?Gln?His?Ser?Pro?Ile?Ser?Ser?Asp?Phe?Ala
1???????????????5???????????????????10??????????????????15
gtc?aaa?atc?cgt?gag?ctg?tct?gac?tac?ctg?ctt?caa?gat?tac?cca?gtc??????96
Val?Lys?Ile?Arg?Glu?Leu?Ser?Asp?Tyr?Leu?Leu?Gln?Asp?Tyr?Pro?Val
20??????????????????25??????????????????30
acc?gtg?gcc?tcc?aac?ctg?cag?gac?gag?gag?ctc?tgc?ggg?ggc?ctc?tgg??????144
Thr?Val?Ala?Ser?Asn?Leu?Gln?Asp?Glu?Glu?Leu?Cys?Gly?Gly?Leu?Trp
35??????????????????40??????????????????45
cgg?ctg?gtc?ctg?gca?cag?cgc?tgg?atg?gag?cgg?ctc?aag?act?gtc?gct??????192
Arg?Leu?Val?Leu?Ala?Gln?Arg?Trp?Met?Glu?Arg?Leu?Lys?Thr?Val?Ala
50??????????????????55??????????????????60
ggg?tcc?aag?atg?caa?ggc?ttg?ctg?gag?cgc?gtg?aac?acg?gag?ata?cac??????240
Gly?Ser?Lys?Met?Gln?Gly?Leu?Leu?Glu?Arg?Val?Asn?Thr?Glu?Ile?His
65??????????????????70??????????????????75??????????????????80
ttt?gtc?acc?gag?tgt?gcc?ttt?cag?ccc?ccc?ccc?agc?tgt?ctt?cgc?ttc??????288
Phe?Val?Thr?Glu?Cys?Ala?Phe?Gln?Pro?Pro?Pro?Ser?Cys?Leu?Arg?Phe
85??????????????????90??????????????????95
gtc?cag?acc?aac?atc?tcc?cgc?ctc?ctg?cag?gag?acc?tcc?gag?cag?ctg??????336
Val?Gln?Thr?Asn?Ile?Ser?Arg?Leu?Leu?Gln?Glu?Thr?Ser?Glu?Gln?Leu
100?????????????????105?????????????????110
gtg?gcg?ctg?aag?ccc?tgg?atc?act?cgc?aga?aac?ttc?tcc?cgg?tgc?ctg??????384
Val?Ala?Leu?Lys?Pro?Trp?Ile?Thr?Arg?Arg?Asn?Phe?Ser?Arg?Cys?Leu
115?????????????????120?????????????????125
gag?ctg?cag?tgt?cag?ccc?gac?tcc?tca?acc?ctg?cca?ccc?cca?tgg?agt??????432
Glu?Leu?Gln?Cys?Gln?Pro?Asp?Ser?Ser?Thr?Leu?Pro?Pro?Pro?Trp?Ser
130?????????????????135?????????????????140
ccc?cgg?ccc?ctg?gag?gcc?aca?gcc?ccg?aca?gcc?ccg????????????????468
Pro?Arg?Pro?Leu?Glu?Ala?Thr?Ala?Pro?Thr?Ala?Pro
145?????????????????150?????????????????155
<210>2
<211>156
<212>PRT
＜213〉yeast
<400>2
Thr?Gln?Asp?Cys?Ser?Phe?Gln?His?Ser?Pro?Ile?Ser?Ser?Asp?Phe?Ala
1???????????????5???????????????????10??????????????????15
Val?Lys?Ile?Arg?Glu?Leu?Ser?Asp?Tyr?Leu?Leu?Gln?Asp?Tyr?Pro?Val
20??????????????????25??????????????????30
Thr?Val?Ala?Ser?Asn?Leu?Gln?Asp?Glu?Glu?Leu?Cys?Gly?Gly?Leu?Trp
35??????????????????40??????????????????45
Arg?Leu?Val?Leu?Ala?Gln?Arg?Trp?Met?Glu?Arg?Leu?Lys?Thr?Val?Ala
50??????????????????55??????????????????60
Gly?Ser?Lys?Met?Gln?Gly?Leu?Leu?Glu?Arg?Val?Asn?Thr?Glu?Ile?His
65??????????????????70??????????????????75??????????????????80
Phe?Val?Thr?Glu?Cys?Ala?Phe?Gln?Pro?Pro?Pro?Ser?Cys?Leu?Arg?Phe
85??????????????????90??????????????????95
Val?Gln?Thr?Asn?Ile?Ser?Arg?Leu?Leu?Gln?Glu?Thr?Ser?Glu?Gln?Leu
100?????????????????105?????????????????110
Val?Ala?Leu?Lys?Pro?Trp?Ile?Thr?Arg?Arg?Asn?Phe?Ser?Arg?Cys?Leu
115?????????????????120?????????????????125
Glu?Leu?Gln?Cys?Gln?Pro?Asp?Ser?Ser?Thr?Leu?Pro?Pro?Pro?Trp?Ser
130?????????????????135?????????????????140
Pro?Arg?Pro?Leu?Glu?Ala?Thr?Ala?Pro?Thr?Ala?Pro
145?????????????????150?????????????????155
<210>3
<211>574
<212>DNA
＜213〉people
<220>
<221>CDS
<222>(16)..(561)
<223>
<400>3
gtcgacgccg?ccacc?atg?gga?gtg?ctg?gcc?cca?gcc?tgg?agc?cca?aca?acc?????51
Met?Gly?Val?Leu?Ala?Pro?Ala?Trp?Ser?Pro?Thr?Thr
1???????????????5???????????????????10
tat?ctc?ctc?ctg?ctg?ctg?ctg?ctg?agc?tcg?gga?ctc?agt?ggg?acc?cag??????99
Tyr?Leu?Leu?Leu?Leu?Leu?Leu?Leu?Ser?Ser?Gly?Leu?Ser?Gly?Thr?Gln
15??????????????????20??????????????????25
gac?tgc?tcc?ttc?caa?cac?agc?ccc?atc?tcc?tcc?gac?ttc?gct?gtc?aaa????147
Asp?Cys?Ser?Phe?Gln?His?Ser?Pro?Ile?Ser?Ser?Asp?Phe?Ala?Val?Lys
30??????????????????35??????????????????40
atc?cgt?gag?ctg?tct?gac?tac?ctg?ctt?caa?gat?tac?cca?gtc?acc?gtg????195
Ile?Arg?Glu?Leu?Ser?Asp?Tyr?Leu?Leu?Gln?Asp?Tyr?Pro?Val?Thr?Val
45??????????????????50??????????????????55??????????????????60
gcc?tcc?aac?ctg?cag?gac?gag?gag?ctc?tgc?ggg?ggc?ctc?tgg?cgg?ctg????243
Ala?Ser?Asn?Leu?Gln?Asp?Glu?Glu?Leu?Cys?Gly?Gly?Leu?Trp?Arg?Leu
65??????????????????70??????????????????75
gtc?ctg?gca?cag?cgc?tgg?atg?gag?cgg?ctc?aag?act?gtc?gct?ggg?tcc????291
Val?Leu?Ala?Gln?Arg?Trp?Met?Glu?Arg?Leu?Lys?Thr?Val?Ala?Gly?Ser
80??????????????????85??????????????????90
aag?atg?caa?ggc?ttg?ctg?gag?cgc?gtg?aac?acg?gag?ata?cac?ttt?gtc????339
Lys?Met?Gln?Gly?Leu?Leu?Glu?Arg?Val?Asn?Thr?Glu?Ile?His?Phe?Val
95??????????????????100?????????????????105
acc?gag?tgt?gcc?ttt?cag?ccc?ccc?ccc?agc?tgt?ctt?cgc?ttc?gtc?cag????387
Thr?Glu?Cys?Ala?Phe?Gln?Pro?Pro?Pro?Ser?Cys?Leu?Arg?Phe?Val?Gln
110?????????????????115?????????????????120
acc?aac?atc?tcc?cgc?ctc?ctg?cag?gag?acc?tcc?gag?cag?ctg?gtg?gcg????435
Thr?Asn?lle?Ser?Arg?Leu?Leu?Gln?Glu?Thr?Ser?Glu?Gln?Leu?Val?Ala
125?????????????????130?????????????????135?????????????????140
ctg?aag?ccc?tgg?atc?act?cgc?aga?aac?ttc?tcc?cgg?tgc?ctg?gag?ctg????483
Leu?Lys?Pro?Trp?Ile?Thr?Arg?Arg?Asn?Phe?Ser?Arg?Cys?Leu?Glu?Leu
145?????????????????150?????????????????155
cag?tgt?cag?ccc?gac?tcc?tca?acc?ctg?cca?ccc?cca?tgg?agt?ccc?cgg????531
Gln?Cys?Gln?Pro?Asp?Ser?Ser?Thr?Leu?Pro?Pro?Pro?Trp?Ser?Pro?Arg
160?????????????????165?????????????????170
ccc?ctg?gag?gcc?aca?gcc?ccc?acc?gcc?ccc?taatgaatct?aga?????????????574
Pro?Leu?Glu?Ala?Thr?Ala?Pro?Thr?Ala?Pro
175?????????????????180
<210>4
<211>182
<212>PRT
＜213〉people
<400>4
Met?Gly?Val?Leu?Ala?Pro?Ala?Trp?Ser?Pro?Thr?Thr?Tyr?Leu?Leu?Leu
1???????????????5???????????????????10??????????????????15
Leu?Leu?Leu?Leu?Ser?Ser?Gly?Leu?Ser?Gly?Thr?Gln?Asp?Cys?Ser?Phe
20??????????????????25??????????????????30
Gln?His?Ser?Pro?Ile?Ser?Ser?Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu
35??????????????????40??????????????????45
Ser?Asp?Tyr?Leu?Leu?Gln?Asp?Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu
50??????????????????55??????????????????60
Gln?Asp?Glu?Glu?Leu?Cys?Gly?Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln
65??????????????????70??????????????????75??????????????????80
Arg?Trp?Met?Glu?Arg?Leu?Lys?Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly
85??????????????????90??????????????????95
Leu?Leu?Glu?Arg?Val?Asn?Thr?Glu?Ile?His?Phe?Val?Thr?Glu?Cys?Ala
100?????????????????105?????????????????110
Phe?Gln?Pro?Pro?Pro?Ser?Cys?Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser
115?????????????????120?????????????????125
Arg?Leu?Leu?Gln?Glu?Thr?Ser?Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp
130?????????????????135?????????????????140
Ile?Thr?Arg?Arg?Asn?Phe?Ser?Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro
145?????????????????150?????????????????155?????????????????160
Asp?Ser?Ser?Thr?Leu?Pro?Pro?Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala
165?????????????????170?????????????????175
Thr?Ala?Pro?Thr?Ala?Pro
180
<210>5
<211>460
<212>DNA
＜213〉people
<400>5
accggtggag?gatgtggctg?cagagcctgc?tgctcttggg?cactgtggcc?tgcagcatct????60
ctgcacccgc?ccgctcgccc?agccccagca?cgcagccctg?ggagcatgtg?aatgccatcc????120
aggaggcccg?gcgtctcctg?aacctgagta?gagacactgc?tgctgagatg?aatgaaacag????180
tagaagtcat?ctcagaaatg?tttgacctcc?aggagccgac?ctgcctacag?acccgcctgg????240
agctgtacaa?gcagggcctg?cggggcagcc?tcaccaagct?caagggcccc?ttgaccatga????300
tggccagcca?ctacaagcag?cactgccctc?caaccccgga?aacttcctgt?gcaacccaga????360
ttatcacctt?tgaaagtttc?aaagagaacc?tgaaggactt?tctgcttgtc?atcccctttg????420
actgctggga?gccagtccag?gagtgataag?caccctggtg??????????????????????????460
Claims (10)
1. a polynucleotide sequence is selected from the polynucleotide sequence shown in SEQ ID NO.:1 or the SEQ ID NO.:3.
2. the shuttle vectors that contains the described polynucleotide sequence of claim 1, the particular methanol yeast vector.
3. the methanol yeast positive recombinant that contains the described carrier of claim 2.
4. the aminoacid sequence shown in SEQ ID NO.:2 or SEQ ID NO.4.
5. method of producing people FL, it may further comprise the steps:
1) will encode the polynucleotide sequence subclone of hFL to shuttle vectors, screening contains the clone of positive shuttle plasmid;
2) described positive shuttle plasmid is changed in the methanol yeast over to the hFL that abduction delivering is expressed with secreted form;
3) described secretory protein is carried out separation and purification;
The polynucleotide sequence that it is characterized in that described coding hFL is the polynucleotide sequence shown in the SEQ ID NO.:1.
6. aminoacid sequence according to claim 4 is used for preparing the medicine of hemopoietic stem/progenitor cells propagation or the purposes of anti-malignant tumor medicine.
7. the transformation adenovirus shuttle plasmid that contains the polynucleotide of described polynucleotide sequence of claim 1 and coding hGM-CSF.
8. the recombinant adenovirus that contains the described plasmid of claim 7.
9.hFL and hGM-CSF unites the purposes that expression is used for preparing anti-malignant tumor medicine.
10. produce the method for uniting the recombinant adenovirus of expressing hFL and hGM-CSF for one kind, it may further comprise the steps:
1) will encode the polynucleotide sequence subclone of hFL and hGM-CSF to the adenovirus shuttle vector of transforming, screening contains the clone of the positive shuttle plasmid of hFL and hGM-CSF gene;
2) with described positive shuttle plasmid and adenoviral gene group plasmid pAdEasy-1 cotransformation competence bacterium BJ5183, homologous recombination in bacterium produces recombinant adenovirus plasmid;
3) change recombinant adenovirus plasmid over to 293 cells with the liposome transfection technology, collecting cell after 7 days, multigelation discharge the 1st generation recombinant adenovirus;
4) cultivate 293 cells in a large number, infect 293 cells with the recombinant adenovirus that efficiently expresses, collecting cell after 3 days, the recombinant adenovirus of the high titre of release is measured the recombinant adenovirus titre behind the multigelation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100383984A CN100404683C (en) | 2004-05-27 | 2004-05-27 | Human FL polynucleotide and use of human GM-CSF united gene therewith for treating malignant tumor |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100383984A CN100404683C (en) | 2004-05-27 | 2004-05-27 | Human FL polynucleotide and use of human GM-CSF united gene therewith for treating malignant tumor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1704425A true CN1704425A (en) | 2005-12-07 |
| CN100404683C CN100404683C (en) | 2008-07-23 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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| CN (1) | CN100404683C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105343874A (en) * | 2015-11-11 | 2016-02-24 | 固安鼎泰海规生物科技有限公司 | Prostate cancer nucleic acid vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5554512A (en) * | 1993-05-24 | 1996-09-10 | Immunex Corporation | Ligands for flt3 receptors |
| CN1234855C (en) * | 2001-11-23 | 2006-01-04 | 上海中信国健药业有限公司 | Recombinant Flt3 ligand gene and its fusion gene and product |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105343874A (en) * | 2015-11-11 | 2016-02-24 | 固安鼎泰海规生物科技有限公司 | Prostate cancer nucleic acid vaccine |
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| CN100404683C (en) | 2008-07-23 |
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