CN102153657A - IL-24-TAT PTD fusion protein and construction method and application thereof - Google Patents
IL-24-TAT PTD fusion protein and construction method and application thereof Download PDFInfo
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Abstract
An IL-24-TAT PTD fusion protein is a fusion protein consisting of IL-24 and Tat PTD small peptide, wherein the Tat PTD small peptide consists of 11 amino acid (YGRKKRRQRRR) and is arranged at the carbon end of IL-24. The construction method comprises the steps of construction of fusion protein gene carrying IL-24, prokaryotic expression of IL-24 and Tat PTD fusion protein gene, and preparation of adenoviral vector of IL-24 fusion protein gene. The IL-24-Tat PTD fusion protein is used for preparing drugs for treating cervical carcinoma, lung cancer and malignant melanoma.
Description
The invention belongs to biological technical field, be specifically related to a kind of new fusion rotein and the application in oncotherapy thereof.
Technical background
(Interleukin 24 for interleukin 24, IL-24) be called melanoma differentiation associated gene 7 (Melanomadifferentiation associated gene 7 again, MDA-7), it is subtractive hybridization analytical method such as Jiang, fibroblast Interferon, rabbit and protein kinase c activator mezerein (mezerrein, MEZ) cance high-expression gene that screening is found in the common melanoma cell strain of handling from reorganization.Follow-up discovers, can cause growth of tumour cell retardance and apoptosis after in this gene importing kinds of tumors, reduces tumour cell clone's formation.Prove that thus it is a kind of new cancer suppressor gene.Along with going deep into that MDA-7 is studied, according to structure, chromosomal localization, the amino acid sequence homology of this gene and the feature with cytokine-like, MDA-7 is renamed is IL-24, belongs to IL-10 family.
IL-24 is expressed in monocyte, scavenger cell, T lymphocyte, bone-marrow-derived lymphocyte and the NK cell in immuning tissue's organs such as normal thymus, spleen and peripheral blood.People's peripheral blood lymphocytes (Peripheral blood mononuclear cells, PBMC) in, mainly be activated T lymphocyte and monocytes IL-24; The smooth muscle cell of normal melanocyte, early stage melanoma cell and skin that propagation is active also can detect IL-24 and express, and late in the melanoma cell IL-24 express and reduce or disappearance metastasis melanin tumor cell IL-24 expression deletion.
The inside and outside experimental result of a lot of bodies confirms, IL-24 can suppress the growth of multiple cancer cells such as melanoma, glioblastoma, osteosarcoma and mammary cancer, cervical cancer, colorectal carcinoma, lung cancer, ovarian cancer, prostate cancer and not damage normal cell by apoptosis-induced, and it suppresses the active condition that tumor growth effect does not rely on cancer suppressor gene such as p53, pRB and p21 in such cell.Outside the decapacitation inducing apoptosis of tumour cell, IL-24 also has other biological and learns function such as angiogenesis inhibitor, immunomodulatory, increases radiation and chemotherapy susceptibility etc.But in chronic lymphocytic leukemia B cell, IL-24 presents high expression level, and its mechanism is the phosphorylation that promotes p38 MAPK, protects and promoted the growth of tumour cell.The biological function of IL-24 can play a role by its acceptor.The same with other albumen of IL-10 family, IL-24 albumen can with II cytokines acceptor IL-20RA/IL-20RB and IL-22R/IL-20RB receptors bind, thereby activate JAK/STAT (Janus familykinase/signal transducer and activator of transcription, JAK/STAT) path.High expression level IL-20R and IL-22R can not obviously improve the ability of IL-24 inducing apoptosis of tumour cell, and utilize the specific inhibitor Genistein and the AG18 of Tyrosylprotein kinase, perhaps use the specific inhibitor AG490 of JAK/STAT signal path, do not influence the inducing action of IL-24 apoptosis of tumor cells.Therefore people infer, the mechanism of the apoptosis of tumor cells that IL-24 causes exists difference with the molecular mechanism of the inflammatory cytokine that mainly plays a role by the IL-20/IL-22 acceptor.
IL-24 has become in the oncotherapy field one research focus at present, by in IL-24 carried out the nearly more than ten years bodies and in vitro study, and the clinical I phase of thereupon carrying out test, confirmed that all IL-24 has anti-tumor activity widely.Because IL-24 is the apoptosis of inducing tumor cell optionally, and normal cell is not had toxicity, be a kind of ideal antitumor drug therefore.Recombinant protein as IL-24 can enter target cell by acceptor or other approach on target cell surface at present, with the performance antitumor action.But IL-24 awaits further raising to the oncotherapy effect at present.Tat nexin transduction domain (Tat Protein transduction domain, Tat PTD) derives from people HIV Tat albumen.Tat PTD has been widely used in and albumen at present, and siRNA or medicine are crosslinked, enters in the cell to improve these molecules.
Summary of the invention
A technical problem to be solved by this invention is the effective IL-24-Tat PTD fusion rotein of oncotherapy.
Another technical problem to be solved by this invention is to provide a kind of construction process for the IL-24-TatPTD fusion rotein.
To be solved by this invention also have a technical problem to be to provide a kind of new purposes for IL-24-Tat PTD fusion rotein.
Solving the problems of the technologies described above the technical scheme that is adopted is: it is by the fusion rotein that IL-24 and little peptide two portions are formed, and little peptide is positioned at the one of carbon tip of IL-24, it is characterized in that: said little peptide is Tat PTD, and (YGRKKRRQRRR) forms by 11 amino acid.
The construction process of above-mentioned IL-24-Tat PTD fusion rotein is made up of following step:
1, carries the structure of IL-24 antigen-4 fusion protein gene
Adopting the polymerase chain reaction is that template amplification obtains the IL-24 antigen-4 fusion protein gene with human IL-2's 4 genes, is to be connected with pGEM-T easy carrier after 1.0% agarose electrophoresis is returned sheet this gene product of being increased through massfraction, the DH5 α cell of product transformed competence colibacillus will be connected, coat in the LB flat board of the penbritin that contains 100 μ g/ml, picking colony is inoculated in the LB nutrient solution of the penbritin that contains 100 μ g/ml, after 14～16 hours, extract plasmid DNA through the alkaline bleach liquor cleavage method, by the enzyme evaluation positive colony of cutting and check order, obtaining positive colony is the plasmid vector that has the IL-24 antigen-4 fusion protein gene.
2, the prokaryotic expression of IL-24-Tat PTD antigen-4 fusion protein gene
Fragment is cut and reclaimed to the carrier that carries the IL-24 antigen-4 fusion protein gene by ClaI and SpeI enzyme, be connected with the prokaryotic expression carrier pGEX4-3-6His that cuts processing through same enzyme.Connect product transformed competence colibacillus cell, cut through enzyme and identify the acquisition positive colony, be the prokaryotic expression carrier that carries the IL-24 antigen-4 fusion protein gene.
To carry the prokaryotic expression carrier transformed into escherichia coli BL21 of IL-24 antigen-4 fusion protein gene,, behind the Ni column purification, obtain the IL-24 fusion rotein through sec.-propyl-β-D-sulfo-galactopyranoside abduction delivering target protein.
Detect protein content, and carry out the albumen that SDS-PAGE detects purifying.
3, the preparation of IL-24 antigen-4 fusion protein gene adenovirus carrier
The pGEMT/IL-24-Tat PTD carrier that has IL-24-Tat PTD fusion rotein reclaims the purpose fragment behind ClaI and SpeI double digestion, obtain positive colony with transforming after the adenovirus E 1 shuttle vectors pacAd5 CMV K-N pA of SpeI double digestion is connected then with through ClaI, cut evaluation through enzyme and obtain the adenovirus shuttle vector that positive colony is the IL-24 antigen-4 fusion protein gene, the positive colony carrier that obtains behind XhoI and NotI double digestion, obtain fragment, with be connected back transformed competence colibacillus cell with pAd5-E4-PGK-miniCMV-eGFP-Fiber-RGD carrier behind the NotI double digestion through SalI, cut through enzyme and identify that obtaining positive colony is and carries the IL-24 antigen-4 fusion protein gene and be used for the shuttle vectors that adenovirus carrier E4 modifies.
To be used for the shuttle vectors that carries the IL-24 antigen-4 fusion protein gene that adenovirus carrier E4 modifies and be the adenovirus carrier that carries the IL-24 antigen-4 fusion protein gene through obtaining positive colony with pCRAd5-backbone behind the SwaI linearization for enzyme restriction in intestinal bacteria BJ5183 homologous recombination after the XhoI linearizing.
With the linearizing adenovirus carrier transfection HEK293 cell that carries the IL-24 antigen-4 fusion protein gene of PacI, 7～10 days results virus stock solution useds are through the adenovirus that further increases and the IL-24 antigen-4 fusion protein gene is carried in the acquisition of CsCl purifying.
The purposes of IL-24-Tat PTD fusion rotein in preparation treatment cervical cancer medicine.
The purposes of IL-24-Tat PTD fusion rotein in preparation treatment lung-cancer medicament.
The purposes of IL-24-Tat PTD fusion rotein in preparation treatment malignant melanoma medicine.
Effective constituent IL-24-Tat PTD fusion rotein preparation treatment cervical cancer medicine, preparation treatment lung-cancer medicament, preparation treatment malignant melanoma medicine use with the form of conventional medicinal preparations.Described medicinal conventional formulation contains the IL-24-Tat PTD fusion rotein as activeconstituents, this activeconstituents mixes as organic or inorganic solid or the liquid excipient that is suitable for intravenous administration with pharmaceutically acceptable carrier in preparation, in preparation, the mass content of the IL-24-Tat PTD fusion rotein of activeconstituents is 1%～95%, and preferred mass content is 5%～90%.
The present invention adopts Tat PTD and IL-24 gene fusion, constitutes IL-24-Tat PTD fusion rotein, improves IL-24 and enters in the tumour cell, thereby strengthen its anti-tumor activity.The present invention adds the little peptide of a nexin transduction domain (Tat Protein transduction domain, Tat PTD) in the source HIV Tat albumen by the one of carbon tip at IL-24, and the little peptide of this Tat PTD is made up of 11 amino acid (YGRKKRRQRRR).
Description of drawings
Fig. 1 is the result of IL-24-TatPTD fusion protein prokaryotic expression purifying.
Fig. 2 is that the IL-24-Tat PTD fusion rotein of prokaryotic expression is to cervical cancer tumer line Hela growth-inhibiting result.
Fig. 3 is that the IL-24-Tat PTD fusion rotein of prokaryotic expression is to lung cancer tumour A549 growth-inhibiting result.
Fig. 4 is that the IL-24-Tat PTD fusion rotein of prokaryotic expression is to K-1735 B16F10-G5-luc growth-inhibiting result.
Fig. 5 expresses IL-24-Tat PTD adenovirus carrier to suppress the tumor growth result in vivo.
Specific embodiments
The present invention is described in more detail below in conjunction with drawings and Examples, but the invention is not restricted to these embodiment.
Embodiment 1
The IL-24 fusion rotein of present embodiment, by the fusion rotein that IL-24 and the little peptide two portions of Tat PTD are formed, the little peptide of Tat PTD is positioned at the one of carbon tip of IL-24, and the little peptide of Tat PTD is made up of 11 amino acid, and sequence is YGRKKRRQRRR.
IL-24-Tat PTD fusion rotein full-length gene order is as follows:
ATCGATatgaattttcaacagaggctgcaaagcctgtggactttagccagacccttctgccctcctttgctggcgacagcctctcaaatgc
agatggttgtgctcccttgcctgggttttaccctgcttctctggagccaggtatcaggggcccagggccaagaattccactttgggccctgcc
aagtgaagggggttgttccccagaaactgtgggaagccttctgggctgtgaaagacactatgcaagctcaggataacatcacgagtgcccg
gctgctgcagcaggaggttctgcagaacgtctcggatgctgagagctgttaccttgtccacaccctgctggagttctacttgaaaactgttttc
aaaaactaccacaatagaacagttgaagtcaggactctgaagtcattctctactctggccaacaactttgttctcatcgtgtcacaactgcaacc
cagtcaagaaaatgagatgttttccatcagagacagtgcacacaggcggtttctgctattccggagagcattcaaacagttggacgtagaag
cagctctgaccaaagcccttggggaagtggacattcttctgacctggatgcagaaattctacaagctcGGTGGAGGTTCTTatgg
tcgtaagaaacgacgccaacggcgacgttgaACTAGTA
Its amino acid sequence corresponding is as follows:
MNFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLWSQVSGAQGQEFHFGP
CQVKGVVPQKLWEAFWAVKDTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEF
YLKTVFKNYHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRR
AFKQLDVEAALTKALGEVDILLTWMQKFYKLGGGSYGRKKRRQRRR
The construction process step of above-mentioned IL-24-Tat PTD fusion rotein is as follows:
1, the structure of human IL-2 4-Tat PTD fusion gene
Adopting the polymerase chain reaction is that template amplification obtains IL-24-Tat PTD fusion gene with human IL-2's 4 genes.
Primer sequence is as follows: P1:IL-24 Clal for:AATCGATatgaattttcaacagaggctgcaaag; P2:IL-24 TatPTD SpeI Back:tactagttcaacgtcgccgttggcgtcgtttcttacgaccatAAGAACCTC CACCgagcttgtagaatttctgcatc.
The polymerase chain reaction (PCR) amplification condition is: 94 ℃, 30 seconds, and 98 ℃, 10 seconds, 55 ℃, 15 seconds, 72 ℃, 15 seconds, 28 circulations.The polymerase chain reaction product is to return fragment after 1.0% the agarose electrophoresis promptly to obtain IL-24-Tat PTD fusion gene fragment through massfraction.The polymerase chain reaction is obtained IL-24-Tat PTD fusion gene fragment to be connected with pGEM-T easy carrier.Condition of contact is: 2 μ l enzymes are cut the purifying fragment, 1 μ l, 10 * T4 ligase enzyme damping fluid, 0.5 μ lpGEM-T easy carrier, 0.5 μ l T4 ligase enzyme, 6 μ l tri-distilled waters, 16 ℃ of connections are spent the night, and will connect the DH5 α cell of product transformed competence colibacillus, and coat in the LB flat board of the penbritin that contains 100 μ g/ml.Picking colony is inoculated in the LB nutrient solution of the penbritin that contains 100 μ g/ml, after 14～16 hours, extracts plasmid DNA through the alkaline bleach liquor cleavage method, by the enzyme evaluation positive colony of cutting and check order.Institute is obtained positive colony called after pGEMT/IL-24-Tat PTD respectively.
2, the prokaryotic expression of IL-24-Tat PTD antigen-4 fusion protein gene
PGEMT/IL-24 Tat PTD is cut and reclaim fragment by ClaI and SpeI enzyme, be connected with the prokaryotic expression carrier pGEX4-3-6His carrier of cutting processing through same enzyme.Condition of contact is: 2 μ l enzymes are cut the purifying fragment, 1 μ l, 10 * T4 ligase enzyme damping fluid, and the 1ul enzyme is cut carrier, 0.5 μ l T4 ligase enzyme, 5.5 μ l tri-distilled waters.The connection product adopts with above-mentioned same method and transforms, extracts plasmid DNA, enzyme is cut evaluation and obtained positive colony, called after pGEX4-3-IL-24-Tat PTD-6His.
With the positive recombinant plasmid pGEX4-3-IL-24-Tat PTD-6His transformed into escherichia coli BL21 (DE3) that identifies, 37 ℃ are spent the night.The picking positive colony is inoculated in the LB substratum that contains 100 μ g/mL ammonia benzyl mycins, and 37 ℃ of overnight incubation contain in the fresh LB nutrient solution of 100 μ g/mL ammonia benzyl mycins according to 1: 100 inoculum size access next day, and 37 ℃ are cultured to OD
600The nm value is 0.6 o'clock, add sec.-propyl-β-D-sulfo-galactopyranoside (Isopropyl β-D-1-Thiogalactopyranoside, IPTG) to concentration be 1mmol/L, the proteic expression of testing goal respectively in 6-20 hour of 16 ℃ of 225r/min abduction deliverings is to determine the optimum time point of protein induced expression.
With 16 ℃ of 225r/min, the IPTG inducing culture was cultivated in 16 hours.7000r/min collected thalline in centrifugal 10 minutes, abandoned supernatant, the sterilized water washing, and 7000r/min collected thalline in centrifugal 10 minutes, abandoned supernatant, repeated twice.With 1: 10 ratio add broken bacterium liquid (the 50mM SODIUM PHOSPHATE, MONOBASIC, 300mM sodium-chlor, the 10mM imidazoles, pH=8.0), adding N,O-Diacetylmuramidase to final concentration simultaneously is 1mg/mL, fully 4 ℃ of cracking 30 minutes are put in piping and druming.With power is 100W, ultrasonic wave 3 seconds, stops 10 seconds, carries out the broken bacterium of ultrasonic wave ice bath in each 5 minutes, repeats 4 times, limpid to solution.4 ℃ of bacterium liquid after ultrasonic, 15, centrifugal 1 hour of 000g abandons precipitation.Getting supernatant, is to add nickel post at 1: 10 by the volume ratio of nickel post and supernatant, places 4 ℃ of rotary shakers in conjunction with 1 hour, carry out purifying in conjunction with liquid with the run by gravity method, add in the gravity post in conjunction with liquid, treat that drips of solution to the greatest extent after, the washings (50mM SODIUM PHOSPHATE, MONOBASIC, 300mM sodium-chlor, the 20mM imidazoles that add 5 times of column volumes, pH=8.0), the abandoned stream fluid adds elutriant (the 50mM SODIUM PHOSPHATE, MONOBASIC of 2 times of column volumes then, 300mM sodium-chlor, the 250mM imidazoles pH=8.0), is collected effluent liquid.With the effluent liquid dialysis tubing of packing into, with dialysis in 500mL, the 0.1M phosphoric acid salt 1 hour, dialyse altogether 4 times, 0.22 μ m filter filtration sterilization, Nanodrop1000 detects protein content, and carries out the albumen of SDS-PAGE detection purifying.Detected result is seen Fig. 1.
3, the preparation of IL-24 antigen-4 fusion protein gene adenovirus carrier
The pGEMT/IL-24-Tat PTD carrier that has IL-24-Tat PTD fusion rotein reclaims the purpose fragment behind ClaI and SpeI double digestion, obtain positive colony with transforming after the adenovirus E 1 shuttle vectors pacAd5CMV K-N pA of SpeI double digestion is connected then with through ClaI, cut evaluation through enzyme and obtain the adenovirus shuttle vector that positive colony is the IL-24 antigen-4 fusion protein gene, the positive colony carrier that obtains behind XhoI and NotI double digestion, obtain fragment, with be connected back transformed competence colibacillus cell with pAd5-E4-PGK-miniCMV-eGFP-Fiber-RGD carrier behind the NotI double digestion through SalI, cut through enzyme and identify that obtaining positive colony is and carries the IL-24 antigen-4 fusion protein gene and be used for the shuttle vectors that adenovirus carrier E4 modifies.
To be used for the shuttle vectors that carries the IL-24 antigen-4 fusion protein gene that adenovirus carrier E4 modifies and be the adenovirus carrier that carries the IL-24 antigen-4 fusion protein gene through obtaining positive colony with pCRAd5-backbone behind the SwaI linearization for enzyme restriction in intestinal bacteria BJ5183 homologous recombination after the XhoI linearizing.
With the linearizing adenovirus carrier transfection HEK293 cell that carries the IL-24 antigen-4 fusion protein gene of PacI, 7～10 days results virus stock solution useds are through the adenovirus that further increases and IL-24-Tat PTD antigen-4 fusion protein gene is carried in the acquisition of CsCl purifying.Purifying obtains virus stock solution used called after CRAd5-RGD.IL-24-Tat PTD, records virus titer (pt/mL) by OD260nm and is divided into 5.3 * 10
12
Embodiment 2
The IL-24 fusion rotein, by the fusion rotein that IL-24 and the little peptide two portions of RAKRRQRRR are formed, the little peptide of RAKRRQRRR is positioned at the one of carbon tip of IL-24.
Its construction process is identical with embodiment 1.
Embodiment 3
The IL-24 fusion rotein, by the fusion rotein that IL-24 and the little peptide two portions of RKARRQRRR are formed, the little peptide of RKARRQRRR is positioned at the carbon art end of IL-24.
Its construction process is identical with embodiment 1.
Embodiment 4
The IL-24 fusion rotein, by the fusion rotein that IL-24 and the little peptide two portions of RRRRRRRRR are formed, the little peptide of RRRRRRRRR is positioned at the one of carbon tip of IL-24.
Its construction process is identical with embodiment 1.
Embodiment 5
The IL-24 fusion rotein, by the fusion rotein that IL-24 and the little peptide two portions of RQIKIWFQNRRMKWKK are formed, the little peptide of RQIKIWFQNRRMKWKK is positioned at the one of carbon tip of IL-24.
Its construction process is identical with embodiment 1.
The IL-24 fusion rotein, by the fusion rotein that IL-24 and the little peptide two portions of AGYLLGKINLKALAALAKKIL are formed, the little peptide of AGYLLGKINLKALAALAKKIL is positioned at the one of carbon tip of IL-24.
Its construction process is identical with embodiment 1.
Embodiment 7
The IL-24 fusion rotein, by the fusion rotein that IL-24 and the little peptide two portions of RVIRVWFQNKRCKDKK are formed, the little peptide of RVIRVWFQNKRCKDKK is positioned at the one of carbon tip of IL-24.
Its construction process is identical with embodiment 1.
Effective constituent IL-24-Tat PTD fusion rotein preparation treatment cervical cancer medicine uses with the form of conventional medicinal preparations.Described medicinal conventional formulation contains the IL-24-Tat PTD fusion rotein as activeconstituents, this activeconstituents mixes as organic or inorganic solid or the liquid excipient that is suitable for intravenous administration with pharmaceutically acceptable carrier in preparation, in preparation, the content of the IL-24-Tat PTD fusion rotein of activeconstituents is 1%～95%.
Embodiment 9
Effective constituent IL-24-Tat PTD fusion rotein preparation treatment lung-cancer medicament uses with the form of conventional medicinal preparations.Described medicinal conventional formulation contains the IL-24-TatPTD fusion rotein as activeconstituents, this activeconstituents mixes as organic or inorganic solid or the liquid excipient that is suitable for intravenous administration with pharmaceutically acceptable carrier in preparation, in preparation, the content of the IL-24-Tat PTD fusion rotein of activeconstituents is 1%～95%.
The preparation of effective constituent IL-24-Tat PTD fusion rotein suppresses the malignant melanoma medicine and uses with the form of conventional medicinal preparations.Described medicinal conventional formulation contains the IL-24-Tat PTD fusion rotein as activeconstituents, this activeconstituents mixes as organic or inorganic solid or the liquid excipient that is suitable for intravenous administration with pharmaceutically acceptable carrier in preparation, in preparation, the content of the IL-24-Tat PTD fusion rotein of activeconstituents is 1%～95%.
In order to verify beneficial effect of the present invention, the contriver adopts the embodiment of the invention 1 preparation IL-24-Tat PTD fusion rotein to carry out the test of pesticide effectiveness, and various test situation are as follows:
1, IL-24Tat PTD fusion rotein vitro inhibition cervical cancer tumer line Hela growth
Adopt the influence to cervical cancer tumer line Hela propagation of MTT method detection GST-IL-24 and GST-IL-24-Tat albumen, concrete operations are as follows:
Inoculating cell: the human cervical carcinoma cell in the vegetative period of taking the logarithm (Hela), be inoculated in 96 orifice plates after 0.1% trysinization, every porocyte number is 1 * 10
3
Albumen is handled: with 2 kinds of albumen according to 0.1,0.2,0.4,0.8, the different gradient set handling of 1.6mmol/L group, establish 3 multiple holes for every group.With 1 * PBS treatment group in contrast.Carry out the cell detected downstream after 48 hours.
Colour developing: cultivate the MTT 20uL that adds 5g/L after 48 hours, continue to cultivate 4 hours.
Reading: remove substratum, add dimethyl sulfoxide (DMSO) (DMSO) 150 μ L, place on the horizontal shaking table room temperature effect 10 minutes.Microplate reader 570nm place records light absorption value.Get each hole absorbance value, the gained result is carried out statistical study with the One Way ANOVA of SPSS 13.0 softwares, each group difference relatively adopts variance analysis.Experimental result is seen Fig. 2.As seen from Figure 2, growth has the obvious suppression effect to expressed GST-IL-24-Tat PTD fusion rotein to cervical cancer tumer line Hela.
2, IL-24Tat PTD fusion rotein vitro inhibition human lung carcinoma cell growth
Inoculating cell: the human lung carcinoma cell in the vegetative period of taking the logarithm (A549), be inoculated in 96 orifice plates after 0.1% trysinization, every porocyte number is 1 * 10
3Other experimental procedure is identical with experiment 1.Experimental result is seen Fig. 3.As seen from Figure 3, growth has the obvious suppression effect to expressed GST-IL-24-Tat PTD fusion rotein to human lung carcinoma cell.
3, IL-24Tat PTD fusion rotein vitro inhibition mouse melanin tumor cell growth
Inoculating cell: the murine melanoma lung transitional cell in the vegetative period of taking the logarithm, be inoculated in 96 orifice plates after 0.1% trysinization, every porocyte number is 1 * 10
3Other experimental procedure is identical with experiment 1.Experimental result is seen Fig. 4.As seen from Figure 4, growth has the obvious suppression effect to expressed GST-IL-24-Tat PTD fusion rotein to mouse melanin tumor cell.
4, express the interior therapeutic effect of IL-24-Tat PTD adenovirus (CRAd5-RGD.IL-24-Tat PTD) to malignant melanoma
Get the B16F10-G5-luc melanoma cell that is in logarithmic phase, after 0.2% trysinization, PBS blows and beats suspension cell with PBS washing, and 4 ℃, centrifugal 10 minutes of 2000g abandons supernatant; Use the serum-free medium re-suspended cell, and, be prepared into 6.6 * 10 with the blood counting chamber counting
6The cell suspension of/mL, and be filled in 1.5 milliliters of centrifuge tubes by 1 milliliter of every branch.
Get 49 of the female nude mices of healthy purebred 4-6 week Balb/c in age, raise all laggard action thing experiments in this laboratory adaptability, animal experiment is implemented according to nursing and the instruction explanation of government test animal.Sterilize at its right hind with 75% alcohol earlier, carry out ether inhalation anesthesia, carry out subcutaneous injection after cell suspension shakes up, (total cellular score is 1 * 10 by 150 μ L
6)/only inject.Observe subcutaneous one-tenth knurl situation every day.Subcutaneous vaccination 4 days, nude mice is subcutaneous, and diameter to occur be 5 millimeters black induration, is the growth of plantation knurl.49 nude mices are divided into 7 groups at random, 7 every group.Contrast virus of A d5.eGFP, CRAd5.eGFP, CRAd5-RGD.IL-24 and CRAd5-RGD.IL-24-Tat PTD recombinant adenovirus gene therapy anti-tumor experiment scheme are as follows: each group is all used injection in the knurl body, and every nude mice injecting virus total amount is 2 * 10
9Plaque forming unit (PlaqueForming Units, PFU), the next day, inject 1 time, injects altogether 4 times, and each dosage is 5 * 10
8PFU.Behind the begin treatment, every other day carry out the cubing of transplanted tumor,, by formula calculate knurl body volume (V=a * b2/2), draw gross tumor volume-time changing curve according to major diameter (a), the minor axis (b) of transplanted tumor.Surpass 3 with the nude mice of control group average-volume, 000mm
3The time, as the experiment terminal point, experimental result is seen Fig. 5.As seen from Figure 5, each volume of organizing the subcutaneous plantation knurl of nude mice all has increase, but at same detection time of point relatively (behind the injection tumour cell the 16th day), CRAd5-RGD.IL-24-Tat PTD group is slower than the tumor growth of control group Ad5.eGFP, illustrates that CRAd5-RGD.IL-24-Tat PTD has stronger tumor inhibition effect.
Claims (5)
1. IL-24-Tat PTD fusion rotein, it is by the fusion rotein that IL-24 and little peptide two portions are formed, and little peptide is positioned at the one of carbon tip of IL-24, it is characterized in that: said little peptide is Tat PTD, is made up of 11 amino acid, and sequence is YGRKKRRQRRR.
2. the construction process of the IL-24-Tat PTD fusion rotein of a claim 1 is characterized in that being made up of following step:
(1) carries the structure of IL-24 antigen-4 fusion protein gene
Adopting the polymerase chain reaction is that template amplification obtains the IL-24 antigen-4 fusion protein gene with human IL-2's 4 genes, is to be connected with pGEM-T easy carrier after 1.0% agarose electrophoresis is returned sheet this gene product of being increased through massfraction, the DH5 α cell of product transformed competence colibacillus will be connected, coat in the LB flat board of the penbritin that contains 100 μ g/ml, picking colony is inoculated in the LB nutrient solution of the penbritin that contains 100 μ g/ml, after 14～16 hours, extract plasmid DNA through the alkaline bleach liquor cleavage method, by the enzyme evaluation positive colony of cutting and check order, obtaining positive colony is the plasmid vector that has the IL-24 antigen-4 fusion protein gene;
(2) prokaryotic expression of IL-24-Tat PTD antigen-4 fusion protein gene
Fragment is cut and reclaimed to the carrier that carries the IL-24 antigen-4 fusion protein gene by ClaI and SpeI enzyme, be connected with the prokaryotic expression carrier pGEX4-3-6His that cuts processing through same enzyme, connect product transformed competence colibacillus cell, cut evaluation through enzyme and obtain positive colony, be the prokaryotic expression carrier that carries the IL-24 antigen-4 fusion protein gene;
To carry the prokaryotic expression carrier transformed into escherichia coli BL21 of IL-24 antigen-4 fusion protein gene,, behind the Ni column purification, obtain the IL-24 fusion rotein through sec.-propyl-β-D-sulfo-galactopyranoside abduction delivering target protein;
Detect protein content, and carry out the albumen that SDS-PAGE detects purifying;
(3) preparation of IL-24 antigen-4 fusion protein gene adenovirus carrier
The pGEMT/IL-24-Tat PTD carrier that has IL-24-Tat PTD fusion rotein reclaims the purpose fragment behind ClaI and SpeI double digestion, obtain positive colony with transforming after the adenovirus E 1 shuttle vectors pacAd5 CMV K-N pA of SpeI double digestion is connected then with through ClaI, cut evaluation through enzyme and obtain the adenovirus shuttle vector that positive colony is the IL-24 antigen-4 fusion protein gene, the positive colony carrier that obtains behind XhoI and NotI double digestion, obtain fragment, with be connected back transformed competence colibacillus cell with pAd5-E4-PGK-miniCMV-eGFP-Fiber-RGD carrier behind the NotI double digestion through SalI, cut through enzyme and identify that obtaining positive colony is and carries the IL-24 antigen-4 fusion protein gene and be used for the shuttle vectors that adenovirus carrier E4 modifies;
To be used for the shuttle vectors that carries the IL-24 antigen-4 fusion protein gene that adenovirus carrier E4 modifies and be the adenovirus carrier that carries the IL-24 antigen-4 fusion protein gene through obtaining positive colony with pCRAd5-backbone behind the SwaI linearization for enzyme restriction in intestinal bacteria BJ5183 homologous recombination after the XhoI linearizing;
With the linearizing adenovirus carrier transfection HEK293 cell that carries the IL-24 antigen-4 fusion protein gene of PacI, 7～10 days results virus stock solution useds are through the adenovirus that further increases and the IL-24 antigen-4 fusion protein gene is carried in the acquisition of CsCl purifying.
3. claim 1IL-24-Tat PTD fusion rotein is preparing the purposes for the treatment of in the cervical cancer medicine.
4. claim 1IL-24-Tat PTD fusion rotein is preparing the purposes for the treatment of in the lung-cancer medicament.
5. claim 1IL-24-Tat PTD fusion rotein is preparing the purposes for the treatment of in the malignant melanoma medicine.
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| CN106581643B (en) * | 2016-12-07 | 2020-05-12 | 黄钟 | Application of interleukin 37 as medicine in treating osteoarthritis and gout |
| CN107022004A (en) * | 2017-03-29 | 2017-08-08 | 华中科技大学同济医学院附属协和医院 | A kind of polypeptide of targets neoplastic cells and its application |
| CN107082799A (en) * | 2017-03-29 | 2017-08-22 | 华中科技大学同济医学院附属协和医院 | A kind of antineoplastic polypeptide HUSP 48 and its application |
| CN107022004B (en) * | 2017-03-29 | 2020-06-05 | 华中科技大学同济医学院附属协和医院 | Polypeptide targeting tumor cells and application thereof |
| CN113234745A (en) * | 2021-06-11 | 2021-08-10 | 山东祥维斯生物科技股份有限公司 | Method for realizing transmembrane transduction of marine microorganism low-temperature lipase gene by PTD-Tat |
| CN113234745B (en) * | 2021-06-11 | 2021-11-02 | 山东祥维斯生物科技股份有限公司 | Method for realizing transmembrane transduction of marine microorganism low-temperature lipase gene by PTD-Tat |
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