CN100341897C - Tumour new-born blood vessel specific binding polypeptide CRGDC and novel recombinant tumor necrosis factor nrhTNF fusion protein and their preparation method - Google Patents
Tumour new-born blood vessel specific binding polypeptide CRGDC and novel recombinant tumor necrosis factor nrhTNF fusion protein and their preparation method Download PDFInfo
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Abstract
The present invention discloses a tumor new born blood vessel specific binding polypeptide CRGDC and novel recombinant human nrhTNF fusion protein and a preparation method thereof. 1st to 7th position amino acids at the amidogen terminal of a human tumor necrosis factor are removed, 8th to 10th position amino acids are converted into Arg-Lys-Arg(arginine-lysine-arginine), and 157th position amino acids on a carboxyl terminal are converted into Phe (phenylalanine) to construct a novel human tumor necrosis factor nrhTNF with high efficiency and low toxicity. On the basis that cyclic peptide CRGDC which comprises five amino acids, is particularly and effectively combined with alpha v beta 3 integrin and is sieved from a phage display library is used to be merged on the amino terminal of nrhTNF by using a genetic engineering method to prepare the tumor new born blood vessel specific binding polypeptide and the nrhTNF fusion protein. The tumor new born blood vessel specific binding polypeptide and the nrhTNF fusion protein can be concentrated on the new born blood vessels of tumor. Consequently, the treatment specificity of the tumor necrosis factor is enhanced, a whole body dosage is decreased, poison side reaction is reduced, and a quality efficiency ratio is enhanced.
Description
Technical field
The invention belongs to the medical biotechnology field, be specifically related to the inside and outside determination of activity of purifying, tumour new-born blood vessel-specific bonding polypeptide and novel recombinant human tumour necrosis factor (nrhTNF) fusion rotein of the expression in prokaryotic cell prokaryocyte of gene clone, transgenation, foreign gene, target protein matter and the technology such as evaluation of physico-chemical property.Further relate to tumour new-born blood vessel specific binding polypeptide CRGDC and novel recombinant human tumour necrosis factor (nrhTNF) fusion rotein and preparation method thereof, tumour new-born blood vessel specific binding polypeptide CRGDC and novel recombinant human tumour necrosis factor (nrhTNF) fusion rotein is as antitumor drug, can specific effect in tumor tissues new vessel endotheliocyte and tumour cell.
Background technology
2.1 tumour necrosis factor correlative study
Tumour necrosis factor (TNF) is a kind of multi-functional cytokine, and the immune metabolism that participates in body is regulated, and some cancer cells is had propagation suppress and cytotoxicity.Tumour necrosis factor still is a kind of infectious mediated factor, plays an important role in the inflammation of body.
2.1.1 the biological effect of tumour necrosis factor
Antitumor and tumor-inhibiting action: tumour necrosis factor all kills and wounds or restraining effect kinds of tumors, and susceptibility is different because of tumor cell type.The knurl mechanism that presses down of tumour necrosis factor still imperfectly understands, and may comprise 1., tumour necrosis factor is the excitability cytokine, can start immunoinflammatory reaction widely, and mediation natural killer cell, monocyte and scavenger cell are to the lethal effect of cancer cells; 2., tumour necrosis factor vasoactive endotheliocyte but, make the blood vessel distortion of tumour impaired or form thrombus, cause tumour generation hemorrhagic necrosis or nutritive deficiency and disappear; 3., tumour necrosis factor can suppress tumor cell proliferation by inducing some apoptosis of tumor cells.
Antivirus action: tumour necrosis factor is dose-dependently ground and suppresses virus-mediated cytopathic development, and dna virus and RNA viruses are all had restraining effect.Tumour necrosis factor can induce non-infected cells to produce anti-virus ability, also optionally kills and wounds the cell of virus infection.Confirm that now multiple virus etc. also can stimulate the generation of tumour necrosis factor.
Immunoregulation effect: tumour necrosis factor can strengthen the lymphokine of T cell generation based on IL-2, improves the expression level of IL-2, thereby promotes the propagation of T cell; Can also promote propagation, differentiation and the generation antibody of B cell.In addition, tumour necrosis factor can also be induced the precursor cell differentiation of monokaryon-macrophage system, strengthens its phagocytic activity and oxidative metabolism level, improves scavenger cell angtigen presentation ability.And can promote marrow stromal cell and scavenger cell to produce CSF, particularly GM-CSF, can impel marrow to discharge neutrophil leucocyte and scavenger cell.
Tumour necrosis factor can also bring out inflammatory reaction, is a kind of powerful inductor of acute phase reaction.
Effect to reticular tissue: can induce osteoclast to absorb sclerotin, promote the chondrocyte to carry out the cartilage renewal, suppress new bone forming.Tumour necrosis factor can also stimulate inoblast and synovial cell's hyperplasia, causes the fibrosis and the hyperplasia of joint tissue.
2.1.2 the application present situation of tumour necrosis factor
Because the antitumor action and the panimmunity regulating effect of tumour necrosis factor, the clinical study of tumour necrosis factor therapy is carried out in many countries.Experimentation on animals and clinical study all show: tumour necrosis factor has the obvious suppression effect to some tumour; But, limited its clinical application because side effect is bigger.The side effect of tumour necrosis factor comprises: heating, headache, feel sick, vomiting, general lassitude, sore muscle etc.; Can cause during high dosage that shock, renal insufficiency and DIC form etc.Set up rational therapeutic regimen and treatment measure, be expected to reduce consumption, alleviate side effect, reach optimum therapeuticing effect.
Intravenous injection rhTNF can make the part tumour dwindle, but side effect is big, and human body is difficult to tolerance.
Intratumor injection can occur downright bad in the part, and side effect is lighter, and some tumor treatment effect is better than intravenous injection.Effective case of having reported comprises kidney, cancer of the stomach, liver cancer etc., and Metastatic Colorectal Cancer ascites is reduced.
The combined utilization of tumour necrosis factor and other drug: use the TNF consumption big separately, the effect that is not easy to obtain, patient Chang Yin can not tolerate its side effect and end medication.Other had cytokine (as IL-2, IFN etc.) or some antitumor drug (as ADM, MMC, DDP) and TNF combined utilization of tumor inhibition effect, both can reduce various amount of drug, reduce toxic side effect, can improve curative effect again, a kind of feasible method of the oncotherapy of can yet be regarded as.
Heat therapy: obviously the antitumor action of enhance TNF but heating have also strengthened the toxicity of TNF to body in its anti-tumor activity of enhancing to body heating.Therefore normal suggestion with therapeutic dose under the normal temperature 1/10th as the initial dose in the heating therapy, help reduce the TNF consumption so have, the increase curative effect.
Gene therapy:,, it is fed back to make it to produce TNF in the body then as killer cell, tumor infiltrating lymphocyte, cellulotoxic lymphocyte and the scavenger cell of lymphokineactivation with the anticarcinogenic effect cell of tnf gene transfection from body; Perhaps transfection also further is prepared into cancer cell vaccine from the cancer cells of body, thereby reaches therapeutic purpose.Rosenberg once used the TIL of tnf gene transfection melanoma patients, and 50 routine patients with terminal have been accepted the TIL of transfection, had 1 routine tumour to disappear fully among the 5 routine valuable patients.Studies show that TIL has high degree of specificity to tumour cell, is the ideal targeted cells.Make up hTNF retroviral vector and the transfection l cell (NIH3T3) and the melanoma cell (B16, F10) of Induced by Radioactive Ray, thereby carry out intratumor injection tnf gene carrier and use local radiotherapy to lay a good foundation for clinical.Recently obtained the positive colony of gene transfection stomach cancer cell MKN28, BGC823 and l cell NIH3T3, the former reaches about 70% two strain vitro growth of gastric cancer cell inhibiting rates, and the latter can obviously suppress liver cancer cell H22 growth (P＜0.05) and improve mouse survival rate (P＜0.025).With the defective type retrovirus TNF α gene is imported liver cancer cell H22, be prepared into the knurl seedling with the 60Co irradiation then, it is reported that the tumorigenicity of this knurl seedling obviously descends, liver cancer H22 is had good therapeutic action.
The chemically modified of tumour necrosis factor: the polymkeric substance (DIVEMA) of TNF and divinyl ether and maleic anhydride is connected, and the antitumour activity of the IVEMA-TNF α of generation is higher approximately 100 times than natural TNF.Experimentation on animals shows that this hybrid molecule antitumour activity in vivo is difficult for decay, and the tumour of 5 lotus knurls (Meth-A) mouse of treatment all disappears.Tsutsumi etc. modify TNF to polyoxyethylene glycol (PEG) and have carried out series of studies.Discovery has 56% lysine residue to be attached on the PEG, and the anti-tumor activity of hybrid molecule is bigger 100 times than prototype TNF, and nontoxicity; The size of the molecular weight of PEG and the degree of modification decision PEG-TNF α hybrid molecule; The antitumour activity of the PEG-TNF α of molecular weight 100-110kD is the strongest in vivo; The synergism of the TNF that modifies through PEG has longer plasma half-life and tumour is had higher " affine " effect because of it.
The gene location of tumour necrosis factor sudden change:, displacement, insertion or the deletion of the Nucleotide of TNF come the sequence of directed change gene, thereby reach the purpose that makes the TNF efficacy enhancing and toxicity reducing according to result of study to the TNF structure-function relationship.Obtained a large amount of TNF derivatives with this method.
2.2 tumor vessel inhibitor present Research
The vascular development of Showed Very Brisk is an essential condition of malignant growth.Tumor vessel has 3 key elements to make it become exploitation one of cancer therapy drug target preferably.The first, compare with the normal blood vessels that remains static substantially, tumor vascular endothelial cell is in the height growth conditions, therefore becomes outstanding target.The second, the tumor vessel cell is the normal cell that enters tumour from healthy tissues, and genome is stable, is easy to produce multiple resistance unlike tumour cell.Although the 3rd various tumour cells differ greatly, the difference of its vascular cell is very little, if therefore with a kind of medicine at tumor vessel then may all produce effect to kinds of tumors.
2.2.1 the research strategy of angiogenesis inhibitor
The research of angiogenesis inhibitor mainly contains following 5 kinds of strategies: 1. soluble cell factor blocker; 2. angiogenesis inhibitor; 3. stromatin enzyme inhibitors; 4. α v β 3 puts in order and plain inhibitor; 5. endotheliocyte target inhibitor.
Soluble cell factor blocker: vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) a and b are considered to the important promotor of tumor-blood-vessel growth.The method of the angiogenic action of interference VEGF and bFGF has proved mouse tumor is disappeared that these methods comprise using monoclonal antibody antagonism VEGF and discharge the necessary conjugated protein active release of cellulosa bFGF that prevents by eradicating bFGF.
Angiostatin: the synthetic endothelial cell proliferation inhibitor, as synthetic fumidil derivative, or the endogenous angiogenesis inhibitor, their physiologic function is to make blood vessel maintenance stationary state or cancel physiological vasculogenesis.The endogenous preparation is efficient endothelium antiblastic, and may be more effective than synthesising preparation, remakes because they also may suppress the capillary vessel relevant with the tumor vessel expansion.Angiostatin and endostatin are the most effective current preparations, and these two kinds all have notable antitumor activity.
The stromatin enzyme inhibitors: matrix is also being brought into play important effect in the tumor vessel forming process, tumour cell, endotheliocyte can be secreted some degrading enzymes, the degraded basement membrane, be beneficial to the migration infiltration of tumour and endotheliocyte and the formation of blood vessel, substrate degradation product such as Laminin ELISA, but also stimulating endothelial cell is bred.Anti-Laminin ELISA antibody has blocked the formation of knurl strain capillary structure and the adhesion of endotheliocyte and Laminin ELISA.BB
94Be a kind of small-molecule substance, combine, suppress its activity, the BB of lower concentration with Zn ionic bond site among the MMP
94The active decline 50% of MMP be can make, the infiltration of tumour and the formation of blood vessel, patient's abdominal injection BB clinically in the animal model suppressed
94, obviously reduced advanced ovarian cancer patient's ascites.
α v β 3 integrin inhibitors: based on the clinical preceding data that in animal model, obtain, carried out α v clinical I, the II phase whole and plain antagonist and test in the patient who suffers from terminal cancer, the clinical I phase of having finished that the monoclonal antibody LM609 (vitaxin) of humanized anti-α v β 3 is successful tests.In 14 patients that tried, 8 stable disease and/or tumor regression.After using 22 months continuously, without any side effects.Current clinical experiment is an anti-angiogenic agent of using guidance quality first in human body.Clinical data conforms to data in the animal model.At present, for further estimating the validity of vitaxin life-time service, carrying out II phase clinical experiment.Simultaneously, the clinical I phase of also having carried out the small molecules antagonist curative effect of α v β 3/ α v β 5 in the patient who suffers from terminal cancer tests.
2.2.2 the problem that exists
Though in anti-angiogenic treatment of tumour and tumour necrosis factor clinical application, obtained some achievements, also existed many problems
Genetically engineered endogenous angiogenesis inhibitor: discharge the necessary conjugated protein active release of cellulosa bFGF that prevents as using monoclonal antibody antagonism VEGF and by eradicating bFGF, or use matrix metallo-proteinase inhibitor etc., they only suppress a kind of positive divisor.In fact there is one group of angiogenesis factor, up-regulated in tumor tissues.
The gene therapy that antineoplastic vascular generates: at present still in the primary stage, also need further to keep foreign gene and continue high-caliber expression in vivo, comprise that 1. the carrier of design can enter target cell efficiently; 2. the permanent target cell genome that enters of the gene of Zhuan Yiing; 3. carrier does not cause host's immune response; 4. select effective medicine to suppress the immune response of body to carrier; 5. the design can produce in vivo than the long half-lift bioactive anti-angiogenic proteins gene-code; 6. prevent that promotor from being sealed by host cell.In addition, also to more understand the normal blood vessels phenotype and generate the different of neovascularity with tumour.Some data show: the neovascularity that tumour forms is different with the structure of normal blood vessels, and is unusual as tumor vessel easy to leak and arrangement, becomes fan-shaped or volution.
Use the monoclonal antibody effector agent: occur and sophisticated gradually hybridoma technology the beginning of the eighties, and what make monoclonal antibody mediation transports video picture or medicine to tumor tissues orientation (target), thereby the toxic side effect that reduces systemic administration becomes possibility.About effector agent couplings such as monoclonal antibody and nucleic, chemotherapeutics, toxin, enzyme and somatomedins, and it is of common occurrence to be used for the report of animal tumor model and clinical trial.Yet, still have distance apart from reaching " having enough antibody to carry effector agent arrives tumor locus and continue the anti-knurl effect of performance and healthy tissues is had no side effect " this target up to now, mainly be because the antibody built-in problem, as the immunogenicity of lower targeting specific, low penetration power, weak avidity and antibody etc.Part is because the obstacle that tumour is brought, as matter HT between the polytropy of the heterogeneity of tumour blood confession and tumour antigen and distribution thereof, tumor tissues press, tumour binding site sterically hindered in the solid tumor particularly, promptly antibody is difficult for combining with the oncocyte of tumor tissues inside (amount and tumour size that antibody enters tumour are inversely proportional to).Therefore in actual applications, only there is the minute quantity antibody capable to arrive tumour and combination with it, usually less than 1% of injection total amount.Recently the progress of genetic engineering technique is improved the specificity of genetic engineering antibody of new generation, avidity, the pharmacokinetics of people's antagonist effector agent mixture understands in depth in addition, and the coupling of multiple effector agent, efficient, special, hypotoxicity ground transports treatment or imaging medicament has become possibility to tumor tissues.
2.3 the present Research of specific binding polypeptide
The appearance of phage display technology and develop into the extensive development of people and the screening new type antineoplastic medicine provides strong means.Along with the development of phage-displayed polypeptides storehouse technology, use the trend that little peptide analogue antigen antibody response has become development simultaneously.This be because: (1) contains the small peptide of the Key residues antigenic determinant on can simulated albumin matter; (2) in most cases, several Key residues and the formed non covalent bond of its binding molecule have constituted whole bonded major portions, i.e. interaction between the protein is to realize by the intersegmental interaction of partial oligopeptide.This class guidance quality medicine not only has binding specificity and high efficiency, and it is bigger also to have overcome monoclonal antibody effector agent molecular weight, the shortcoming that penetrativity is weak.At present, development trend based on guide molecule miniaturization in the targeted drug, utilize phage-displayed polypeptides storehouse technology screening go out can with molecules of interest bonded small peptide, nucleotide sequence at external this small peptide of composite coding, merge with the bullet molecular gene, give expression to polypeptide-bullet molecule, by small peptide performance guide effect with guide effect, the bullet molecule can be bacteriotoxin, cytokine, chemotherapeutics etc., and this medicinal design strategy has demonstrated good prospects for application.
1998, Ruoslahti etc. utilize that phage random peptide library screening obtains with integrate plain molecule α v β 3 height bonded peptides, and crosslinked with this peptide and Zorubicin, medicine can be enriched to tumor tissues, acquisition excellent treatment effect in mouse tumor model.Subsequently, but they again the peptide of the peptide that screens with one section cell death inducing is connected, in mouse tumor model, demonstrate extremely knurl effect equally.Thereby prompting: peptide guiding cancer therapy drug has a good application prospect.
Angelo Corti etc. utilizes genetic engineering means, energy specificity that will filter out from phage display peptide library and aminopeptidase (CD13) bonded pentapeptide CNGRC link the aminoterminal of natural human tumour necrosis factor by a glycine, experimental results show that, fusion rotein after the reconstruction has the activity close with natural tumour necrosis factor in the tumor cell in vitro killing experiments, in vivo in the experiment, compare with natural tumour necrosis factor, fusion rotein after the reconstruction has higher amount effect ratio, lower toxic side effects, though there is not direct histology evidence to prove that the fusion rotein after the reconstruction can be in the enrichment of tumor vessel position, but below experimental results show that the relation of active raising of the fusion rotein after the reconstruction and CNGRC: inject the interior antitumor activity of body that free CNGRC Toplink effectively suppresses fusion rotein simultaneously, make it to reach and the close level of the natural bad factor of tumour, and antitumor activity in the body of natural tumour necrosis factor is not had influence; With tryptic digestion CNGRC-TNF and TNF, obtain and separating digesting product TNF
3-156Segment, the digestion product of CNGRC-TNF and TNF have antitumor activity in the identical body.The monoclonal anti physical efficiency of anti-CD13 significantly suppresses the interior antitumor activity of body of CNGRC-TNF, makes it to reach and natural tumour necrosis factor par; And natural tumour necrosis factor is not had obvious restraining effect.After transplantation tumor growth 12 days, significant difference just appears in the two killing activity of CNGRC-TNF and TNF, illustrates that CNGRC-TNF may have higher amount effect ratio by the new vessel that acts on tumour.
People such as Zhou Xue design has also made up two chimeric molecules that merge the RGD sequence at r-hirudin C end.In intestinal bacteria, express and purifying expression product, activation analysis shows that chimera protein keeping the hirudin anticoagulant active while of hemase, also presents platelet aggregation inhibitory activity.Thereby also confirmed reasonableness and the feasibility of specific binding polypeptide as guide molecule.
Summary of the invention
In sum, the objective of the invention is to, a kind of tumour new-born blood vessel-specific bonding polypeptide and novel recombinant human tumour necrosis factor (nrhTNF) fusion rotein and preparation method thereof is provided.
To achieve these goals, the technical solution adopted in the present invention is: tumour new-born blood vessel specific binding polypeptide CRGDC and novel recombinant human tumour necrosis factor (nrhTNF) fusion rotein, it is characterized in that, make 7 aminoacid deletion of wild-type hTNF α N end respectively with the gene location mutating technology, and change 8,9,10 proline(Pro), Serine, aspartic acid into arginine, Methionin, arginine, leucine with 157 at C end is replaced as phenylalanine simultaneously, obtains novel recombinant human tumour necrosis factor nrhTNF α; The applying gene clone technology makes up the fusion gene of tumour new-born blood vessel specific binding polypeptide CRGDC and novel recombinant human tumour necrosis factor (nrhTNF), and obtains to efficiently express in intestinal bacteria.
The method for preparing aforesaid tumour new-born blood vessel-specific bonding polypeptide and novel recombinant human tumour necrosis factor (nrhTNF) fusion rotein, preparation according to the following steps:
1) 7 aminoacid deletion that with the gene location mutating technology wild-type hTNF α N held respectively, and change 8,9,10 proline(Pro), Serine, aspartic acid into arginine, Methionin, arginine, leucine with 157 at C end is replaced as phenylalanine simultaneously, obtains novel recombinant human tumour necrosis factor nrhTNF α;
2) clone of RGD-nrhTNF antigen-4 fusion protein gene
The pGEM3zf-nrhTNF plasmid is preserved by this center; Its cloning site is EcoRI and PstI, has at first designed a pair of primer the 5 ' EcoRI that holds is sported BamHI;
Primer 1:(5 ' holds primer, 41nt): CGGAATTC ATG CGC AAA CGT AAG CCTGTA GCC CAT GTT GTA
Primer 2: (3 ' end primer, 32nt) CGC TGC AGT CAG AAG GCA ATG ATC CCAAAG TA
PGEM3zf-nrhTNF is carried out pcr amplification;
The preparation of pcr amplification reaction pipe is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive components, 94 ℃ of reaction 5min add the Taq enzyme again, carry out the PCR reaction then, and the loop parameter of pcr amplification is: 94 ℃, the 30s reaction of degeneration; 55 ℃, the 30s annealing reaction; 72 ℃, the 30s amplified reaction carries out 30 circulations, extends 10min in 72 ℃;
After the PCR product is used the BamHI+PstI double digestion, separate, reclaim the purpose fragment, called after m-nrhTNF through 1.5% agarose gel electrophoresis;
Designed and synthesized the nucleic acid fragment of coding CRGDC targeted peptide according to the habitual codon of intestinal bacteria;
Fragment 1 (28nt): AATTC ATG TGT GAT TGT CGT GGC GAT TG
Fragment 2 (50nt): TTT TTG TGG ATC CAC CTC TGG TTC TGG TAA ATCTTC TGA AGG TAA AGG TG
Fragment 3 (40nt): GAT CCA CCT TTA CCT TCA GAA GAT TTA CCA GAACCA GAG G
Fragment 4 (38nt): TGG AGC CAC AAA AAC AAT CGC CAC GAC AAT CACACA TG
With four fragments of synthetic in 65 ℃ of sex change 5min, annealing, be connected with m-nrhTNF and with pGEM3zf (-) plasmid that EcoRI+PstI handled again, connect product transformed competence colibacillus e.colidh5, use blue hickie screening method, picking white clone, overnight incubation in the LB nutrient solution is extracted plasmid DNA, cuts evaluation through enzyme, obtain to insert the correct RGD-nrhTNF fusion gene pronucleus cloning vector of clip size, carry out dna sequence analysis.Correct person's called after pGEM3zf-RT checks order.The gene that wherein contains codes for tumor new-born blood vessel specific binding polypeptide CRGDC and nrhTNF fusion rotein;
3) abduction delivering of the structure of expression vector and recombinant protein
Check order correct pGEM3zf-RT through EcoR I and PstI digestion, reclaim the dna fragmentation of about 500bp, be connected the transformed competence colibacillus e.colidh5 with the pBV220 plasmid of handling through above-mentioned same restrictions restriction endonuclease, the random choose clone identifies, screens with EcoR I and PstI double digestion; Correct clone is named as pBV-RT; The pBV-RT/DH5 α bacterium of correct reorganization, 30 ℃ of activation jolting overnight incubation in LB, morning next day, 30 ℃ were continued to be cultured to OD with 1: 100 ratio inoculation LB (containing Amp 100 μ g/ml) substratum
650nm=0.6 o'clock, rapidly elevated temperature to 42 ℃ was cultivated 4h; The centrifugal 15min of 3000rpm collects thalline;
4) purifying of recombinant protein
Adding 5ml in 1 gram thalline, to split the ratio of bacterium damping fluid resuspended with thalline, with high pressure cell destroyer cracking thalline, 1M MgCl
20.4ml, 4 ℃ stir after, add 1mg/ml DNaseI 20 μ l, 4 ℃ are stirred 10m, 12000rpm, centrifugal 15m collects supernatant, adds solid ammonium sulfate to 50% saturation ratio, 4 ℃ leave standstill 1h, 12000rpm, centrifugal 15m collects supernatant; A liquid with 50 times of volumes: 20mM TrisCl pH8.0,1mM EDTA dialysed overnight, liquid is changed three to four times in the centre; Supernatant after the dialysis abundant equilibrated Q-Sepharose of A liquid Fast Flow chromatography purification, wash-out B liquid is 0.3MNaCl, 20mM TrisCl pH8.0,1mMEDTA, 10 column volumes of continuous gradient wash-out are collected each elution peak, measure active, get the C liquid of active peak to 50 times of volumes: 20mM PB pH6.0 dialysed overnight, liquid is changed three to four times in the centre; The dialysis back supernatant abundant equilibrated SP-sepharoseFast of C liquid Flow chromatography purification, wash-out D liquid is 20mMPB, pH6.0,1M NaCl, 4-5 column volume of continuous gradient wash-out collected elution peak, carries out determination of activity and electrophoresis detection;
5) extracorporeal biology of recombinant protein is active detects
The biological activity assay of tumour new-born blood vessel-specific bonding polypeptide and novel recombinant human tumour necrosis factor fusion protein is undertaken by the L929 cell toxicant method of classics;
6) biologic activity detects in the body of recombinant protein
Set up mouse S180 ascitic tumor transplanted tumor model, for being subjected to the reagent thing, nrhTNF, endoxan, injection physiological saline are control drug with RGD-nrhTNF, and mice-transplanted tumor knurl body weight serves as to detect index when finishing with experiment, calculate tumour inhibiting rate, determine to be subjected to the tumor killing effect of reagent thing; With mouse body weight before and after the experiment serves as to detect index, calculates the body weight change rate, determines to be subjected to the toxic side effects of reagent thing;
Inhibition rate of tumor growth can calculate by following formula:
Calculate simultaneously mouse treatment front and back body weight change as follows, as the index of weighing the medicine toxic side effect;
That the present invention has selected to screen from the phage cycle peptide library, can with plain molecule---the α v β 3 differential high efficient bonded polypeptide-CRGDC-(halfcystine-arginine-glycine-aspartic acid acid-halfcystine) of the integration that the new vessel endothelial cell surface efficiently expresses, the applying gene clone technology, made up the fusion gene of tumour new-born blood vessel-specific bonding polypeptide and novel recombinant human tumour necrosis factor (nrhTNF), and in intestinal bacteria, obtained to efficiently express.Prepare tumour new-born blood vessel-specific bonding polypeptide and novel recombinant human tumour necrosis factor (nrhTNF) fusion rotein, make it both have the advantage of novel recombinant human tumour necrosis factor high-efficiency low-toxicity, again can be in the place's enrichment of tumor tissues new vessel, thereby further reduce the clinical application amount of novel recombinant human tumour necrosis factor, improve amount effect ratio, reduce toxic side effect.
Description of drawings
Fig. 1 is the complete sequence determination figure of tumour new-born blood vessel specific binding polypeptide CRGDC of the present invention and novel recombinant human tumour necrosis factor (nrhTNF) fusion gene;
PBV-CRGDC-nrhTNF：ATG TGC CGT GGT GAT TGC GGATCC CGC AAA CGT AAG CCT GTA GCC CAT GTT GTA GCA AAC CCTCAA GCT GAG GGG CAG CTC CAG TGG CTG AAC CGC CGG GCC AATGCC CTC CTG GCC AAT GGC GTG GAG CTG AGA GAT AAC CAG CTGGTG GTG CCA TCA GAG GGC CTG TAC CTC ATC TAC TCC CAG GTCCTC TTC AAG GGC CAA GGC TGC CCC TCC ACC CAT GTG CTC CTCACC CAC ACC ATC AGC CGC ATC GCC GTC TCC TAC CAG ACC AAGGTC AAC CTC CTC TCT GCC ATC AAG AGC CCC TGC CAG AGG GAGACC CCA GAG GGG GCT GAG GCC AAG CCC TGG TAT GAG CCC ATCTAT CTG GGA GGG GTC TTC CAG CTG GAG AAG GGT GAC CGA CTCAGC GCT GAG ATC AAT CGG CCC GAC TAT CTC GAC TTT GCC GAGTCT GGG CAG GTC TAC TTT GGG ATC ATT GCC TTC TGA
Fig. 2 is the tumor specimen picture in the inhibition test in the recombinant protein body of the present invention.Be followed successively by from top to bottom among Fig. 2: the physiological saline negative control group; The endoxan positive controls; NrhTNF 1,200,000 IU/kg treatment groups; RGD-nrhTNF 1,200,000 IU/kg treatment groups.
Embodiment
The present invention is described in further detail below in conjunction with drawings and Examples.
Tumour new-born blood vessel specific binding polypeptide CRGDC and novel recombinant human tumour necrosis factor (nrhTNF) fusion rotein according to technical scheme preparation of the present invention, make 7 aminoacid deletion of wild-type hTNF α N end respectively with the gene location mutating technology, and change 8,9,10 proline(Pro), Serine, aspartic acid into arginine, Methionin, arginine, leucine with 157 at C end is replaced as phenylalanine simultaneously, obtains novel recombinant human tumour necrosis factor nrhTNF α; The applying gene clone technology has made up the fusion gene of tumour new-born blood vessel-specific bonding polypeptide and novel recombinant human tumour necrosis factor (nrhTNF), and obtains to efficiently express in intestinal bacteria.
Realize the preparation method of tumour new-born blood vessel specific binding polypeptide CRGDC of the present invention and novel recombinant human tumour necrosis factor fusion protein, according to the following steps preparation:
1) 7 aminoacid deletion that with the gene location mutating technology wild-type hTNF α N held respectively, and change 8,9,10 proline(Pro), Serine, aspartic acid into arginine, Methionin, arginine, leucine with 157 at C end is replaced as phenylalanine simultaneously, obtains novel recombinant human tumour necrosis factor nrhTNF α;
The construction process of above-mentioned recombination nrhTNF α is as follows:
1. adopt the method for PCR, (Clontech company) is template with human leukocyte cDNA library, carries out the nucleotide fragments that the PCR reaction obtains encoding wild type hTNF α, simultaneously EcoRI and PstI restriction enzyme site introduced 5 ' end and 3 ' end respectively; PCR reaction product and pGEM-3Zf (-) behind EcoRI and PstI double digestion, are reclaimed big fragment, and carry out ligation.Transformed competence colibacillus cell DH5 α, the picking positive colony is cut evaluation through enzyme, obtains to insert the procaryotic clone carrier of the correct hTNF α gene of clip size, carries out dna sequence analysis.Correct person's called after pGEM-3zf (-)-hTNF α checks order.
2. adopt the gene location mutating technology to make 7 aminoacid deletion of wild-type hTNF α N end, and the employing round pcr changes 8,9,10 and 157 proline(Pro), Serine, aspartic acid and leucine into arginine, Methionin, arginine and phenylalanine.PCR reaction design of primers is as follows: primer 1 is 5 '-CGGAATTCATGCGCAAACGTAAGCCTGTAGCCCAT-3 ', wherein contains an EcoRI restriction enzyme site, is Arg after the initiator codon
8Lys
9Arg
10Codon, the 11st later amino acid coding of natural human TNF alpha molecule thereafter; Primer 2 is 5 '-CGCTGCAGTCAGAAGGCAATGATCCCAAAGTA-3 ', contain a PstI restriction enzyme site and terminator codon, be the Phe codon with TNF α the 157th for the Leu codon mutation, all the other are TNF α gene 3 ' end complementary sequence.
With pGEM-3zf (-)-hTNF α is template, carries out the PCR reaction with above-mentioned primer.The recombinant plasmid that obtains is carried out determined dna sequence, and the correct person's called after pGEM-3zf-nrhTNF that checks order wherein contains the gene of encoding novel recombinant human tumor necrosis factor (nrhTNF);
2) clone of RGD-nrhTNF antigen-4 fusion protein gene
Zhi Bei PGEM-3zf-nrhTNF plasmid is preserved by this center according to the method described above.Its cloning site is EcoRI and PstI, and we have at first designed a pair of primer the 5 ' EcoRI that holds is sported BamHI.
Primer 1:(5 ' holds primer, 41nt): CGGAATTC ATG CGC AAA CGT AAG CCT GTAGCC CAT GTT GTA
Primer 2: (3 ' end primer, 32nt) CGC TGC AGT CAG AAG GCA ATG ATC CCA AAGTA
P3zf-nrhTNF is carried out pcr amplification by following condition:
The composition of pcr amplification reaction pipe:
25 μ l/ reaction tubess
P3zf-nrhTNF (template DNA, 200ng/ml) 2.0
4mM dNTPS 2.0
40mM MgCl
2 1.25
Primer 1 (0.05mg/ml) 2.5
Primer 2 (0.05mg/ml) 2.5
Taq enzyme (1.0U) 1.0
H2O 13.75
Amount to 25.0
The preparation of reaction tubes is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive components, 94 ℃ of reaction 5min add the Taq enzyme again, carry out the PCR reaction then, and the loop parameter of pcr amplification is: 94 ℃, 30s (reaction of degeneration); 55 ℃, 30s (annealing reaction); 72 ℃, 30s (amplified reaction) carries out 30 circulations, extends 10min in 72 ℃.
After the PCR product is used the BamHI+PstI double digestion, separate, reclaim the purpose fragment through 1.5% agarose gel electrophoresis.Called after m-nrhTNF.
Designed and synthesized the nucleic acid fragment of coding CRGDC targeted peptide according to the habitual codon of intestinal bacteria.
Fragment 1 (28nt): AATTC ATG TGT GAT TGT CGT GGC GAT TG
Fragment 2 (50nt): TTT TTG TGG ATC CAC CTC TGG TTC TGG TAA ATC TTCTGA AGG TAA AGG TG
Fragment 3 (40nt): GAT CCA CCT TTA CCT TCA GAA GAT TTA CCA GAA CCA GAGG
Fragment 4 (38nt): TGG AGC CAC AAA AAC AAT CGC CAC GAC AAT CAC ACA TG
With four fragments of synthetic in 65 ℃ of sex change 5min, annealing, be connected with m-nrhTNF and with pGEM3zf (-) plasmid that EcoRI+PstI handled again, connect product transformed competence colibacillus e.colidh5, use blue hickie screening method, picking white clone, overnight incubation in the LB nutrient solution is extracted plasmid DNA, cuts evaluation through enzyme, obtain to insert the correct RGD-nrhTNF fusion gene pronucleus cloning vector of clip size, carry out dna sequence analysis.Correct person's called after pGEM3zf-RT checks order.The gene that wherein contains codes for tumor new-born blood vessel-specific bonding polypeptide and nrhTNF fusion rotein.
3) structure of expression vector and expression of recombinant proteins analysis
Check order correct pGEM3zf-RT through EcoR I and PstI digestion, reclaim the dna fragmentation of about 500bp, be connected the transformed competence colibacillus e.colidh5 with the pBV220 plasmid of handling through above-mentioned same restrictions restriction endonuclease, the random choose clone identifies, screens with EcoR I and PstI double digestion.Correct clone is named as pBV-RT.The pBV-RT/DH5 α bacterium of correct reorganization, 30 ℃ of activation jolting overnight incubation in LB, morning next day, 30 ℃ were continued to be cultured to OD with 1: 100 ratio inoculation LB (containing Amp 100 μ g/ml) substratum
650nm=0.6 o'clock, rapidly elevated temperature to 42 ℃ was cultivated 4h.The centrifugal 15min of 3000rpm collects thalline.Analyze through SDS-PAGE, discovery induces the back at molecular weight about 18, there is a newborn protein band at the 000Dalton place, with mouse-anti-human T NF antibody is one anti-, enzyme mark rabbit anti-mouse igg is two anti-, carry out Western blot and analyze, confirmed and to have combined with anti-TNF antibodies generation specificity by new life's protein band.
4) purifying of recombinant protein
Adding 5-10ml in 1 gram thalline, to split the ratio of bacterium damping fluid resuspended with thalline, with high pressure cell destroyer cracking thalline, 1M MgCl
20.4ml, 4 ℃ stir after, add 1mg/ml DNaseI 20 μ l, 4 ℃ are stirred 10m, 12000rpm, centrifugal 15m collects supernatant, adds solid ammonium sulfate to 50% saturation ratio, 4 ℃ leave standstill 1h, 12000rpm, centrifugal 15m collects supernatant.A liquid with 50 times of volumes: 20mM TrisCl pH8.0,1mM EDTA dialysed overnight, liquid is changed three to four times in the centre.Supernatant after the dialysis abundant equilibrated Q-Sepharose of A liquid Fast Flow chromatography purification, wash-out B liquid is 0.3M NaCl, 20mM TrisCl pH8.0,1mMEDTA, 10 column volumes of continuous gradient wash-out are collected each elution peak, measure active, get the C liquid of active peak to 50 times of volumes: 20mM PBpH6.0 dialysed overnight, centre are changed liquid three to four times.The dialysis back supernatant abundant equilibrated SP-sepharose of C liquid Fast Flow chromatography purification, wash-out D liquid is 20mMPB, pH6.0,1M NaCl, 4-5 column volume of continuous gradient wash-out collected elution peak, carries out determination of activity and electrophoresis detection.
SDS-PAGE detected through gel electrophoresis and through the gel imaging system analysis, purity is greater than 95%, and protein recovery is 2.54%.
5) extracorporeal biology of recombinant protein is active detects
The biological activity assay of tumour new-born blood vessel-specific bonding polypeptide and novel recombinant human tumour necrosis factor fusion protein is undertaken by the L929 cell toxicant method of classics.Measuring method is as follows:
The L929 cell is with the DMEM culture medium culturing that is added with 10% calf serum, and behind trypsin digestion cell, at the microscopically counting, adjusting cell concn is 1.0 * 10
5Cell/ml, and with cell inoculation in 96 well culture plates, every hole 100 μ l (1.0 * 10
4Cells/well), 37 ℃, 5%CO
2Overnight incubation to cell is sticked wooden partition more than 80%.
Each dosage of TNF standard substance is respectively surveyed two holes, every hole adds 0.1ml, 4 times of gradient dilutions are done with the DMEM nutrient solution that contains 3% calf serum (FBS) in each hole later on, and promptly the standard substance initial concentration is 100IU/ml, is followed successively by 25IU/ml later on, 6.25IU/ml, 1.56IU/ml, 0.39IU/ml, 0.1IU/ml, 0.025IU/ml, 0.00625IU/ml.Sample then is diluted to initial concentration in advance according to practical situation and is about 100IU/ml, does 4 times of gradient dilutions again, and final volume is 100 μ l/ holes.(above operation is all operated in 96 orifice plates).Place 37 ℃, in the 5%CO2 incubator, cultivate 16h.Use violet staining, survey OD
570nmValue, (the diplopore application of sample is surveyed the average of two point values) is X-coordinate with the logarithmic value of extension rate, OD
570nmFor ordinate zou is figure, with the minimum OD of standard substance
570nmThe highest OD
570nmMean value do a straight line that is parallel to X-axis and meet at curve, on X-axis, read the extent of dilution of median effective dose with the corresponding curve of sample and this straight-line intersection, be calculated as follows and tire:
Sample activity (IU/ml)=standard substance are tired * (the pre-extension rate of the pre-extension rate/standard substance of sample) * (standard substance median effective dose extent of dilution/sample is equivalent to the extent of dilution of standard substance median effective dose)
The external activity experiment shows that nrhTNF reaches 1.0 * 10 to L929 cell killing specific activity
9The IU/mgRGD-nrhTNF fusion rotein has tangible killing activity to the L929 cell, and specific activity is 5.6 * 10
8IU/mg.
6) activity in vivo of recombinant protein detects
Detection method: set up mouse S180 ascitic tumor transplanted tumor model, with RGD-nrhTNF for being subjected to the reagent thing, nrhTNF, endoxan, injection physiological saline are control drug, mice-transplanted tumor knurl body weight serves as to detect index when finishing with experiment, calculate tumour inhibiting rate, determine to be subjected to the tumor killing effect of reagent thing; With mouse body weight before and after the experiment serves as to detect index, calculates the body weight change rate, determines to be subjected to the toxic side effects of reagent thing.
Experiment content:
1, being subjected to reagent thing: RGD-nrhTNF is lyophilisate, and it is standby to be stored in-20 ℃ of refrigerators.Face with preceding following to physiological saline solution, dilution according to dosage in aseptic condition.
Control drug:
1., injection novel recombinant human tumour necrosis factor work in-process, be diluted to required dosage during use.
2., endoxan: the Cyclophosphamide for injection of selecting for use Hualian Pharmaceutical Co., Ltd., Shanghai to produce.Be mixed with 40mg/ml, 4 ℃ of refrigerators are standby.
3., injection physiological saline
2, laboratory animal: by the secondary Kunming mouse that The Fourth Military Medical University's Experimental Animal Center is bred, body weight 21 grams-23 grams, totally 40, the male and female dual-purpose is tested under secondary breeding observing condition.
3, experiment knurl strain: the strain of mice-transplanted tumor knurl is the ascitic tumor cell strain S180 that regularly goes down to posterity in this chamber, uses for pressing down the knurl experiment in the animal body.
4, experiment grouping: 40 mouse are divided into 4 groups at random, i.e. guidance quality tumour necrosis factor RGD-nrhTNF1.2 * 10
6The U/kg experimental group, new tumor necrosin nrhTNF 1.2 * 10
6The U/kg group, endoxan 100mg/kg positive controls, physiological saline negative control group, every group of 10 animals.
5, aseptic technique is in the right inguinal region subcutaneous vaccination 1 * 10 of mouse
7The S180 oncocyte of/0.1ml.At random be divided into 4 group with postvaccinal animal next day, weighs before treatment.Treated by above-mentioned each treated animal dosage and medication respectively in the 6th day after the strain of inoculation knurl, all in subcutaneous injection endoxan, guidance quality tumour necrosis factor, novel recombinant human tumour necrosis factor and physiological saline were treated 7 days continuously.After the last administration 24 hours, weigh in once more, put to death animal with the cervical vertebra dislocation method then, isolate tumor tissues rapidly and claim its weight in wet base, calculate inhibition rate of tumor growth according to formula, and carry out statistical study.
Inhibition rate of tumor growth can calculate by following formula:
Calculate simultaneously mouse treatment front and back body weight velocity of variation as follows, as the index of weighing the medicine toxic side effect.
In vivo test shows that RGD-nrhTNF has the obvious suppression effect to the growth of mice-transplanted tumor S180.RGD-nrhTNF organizes inhibitory rate to 84.84%, with control group notable difference (P＜0.01) is arranged.Compare with the nrhTNF (tumor control rate is 59.09%) of same dose, curative effect has notable difference.And toxic side effect has the trend of the nrhTNF that is lower than same dose.
Table 1 is the active detected result of RGD-nrhTNF in purge process, and table 2 is tumor killing effects of recombinant protein.
The active detected result of table 1:RGD-nrhTNF in purge process
| Active (IU/ml) | Volume (ml) | Protein content (mg/ml) | Than live (IU/mg) | Total protein (mg) | Protein recovery (%) | |
| Split the bacterium supernatant | 5×10 6 | 120 | 0.856 | 5.85×10 6 | 102.72 | |
| Qp1 | 6.4×10 6 | 110 | 0.411 | 1.56×10 7 | 45.21 | 44.01 |
| The Sp peak | 5×10 7 | 30 | 0.088 | 5.6×10 8 | 2.64 | 2.54 |
Table 2 RGD-nrhTNF is to the restraining effect of mice-transplanted tumor S180
| Group tumor control rate % | Dosage | Number of animals (only) | Heavy mg ± the SD of average knurl |
| Experiment contrast group endoxan group 71.21 ** RGD-nrhTNF 84.84 ** nrhTNF 59.09 ** | Physiological saline 0.2ml 100mg/kg 1,200,000 IU/kg 1,200,000 IU/kg | 10 10 10 10 | 132±72 38±21 20±18 54±18 |
*: expression and negative control group significant difference (P＜0.01)
Specific embodiment:
The applicant has selected for use energy specificity and α v β 3 to integrate the cyclic peptide CGRDC that plain bonded contains five amino acid, and the method for applying gene reorganization merges itself and nrhTNF aminoterminal, and obtain expression in intestinal bacteria.On the basis that successfully makes up, expresses fusion gene,, set up purifying process with low cost with reference to the purification process of nrhTNF.In external fragmentation test, this fusion rotein RGD-nrhTNF has lethal effect dose-dependently, tangible to the L929 cell, and specific activity reaches 5.7 * 10
8IU/mg.In animal tumor model, this fusion rotein RGD-nrhTNF has significant inhibitory effect (tumor control rate is 84.84%) to mouse S180 ascitic tumor, apparently higher than the nrhTNF (tumor control rate is 59.09%) of same dose.And toxic side effect is lower than nrhTNF.Though we also do not have direct evidence to confirm that RGD-nrhTNF can be enriched in novel recombinant human tumour necrosis factor at the tumor neogenetic blood vessels position in vivo, proved that tentatively improved RGD-nrhTNF curative effect improves, toxic side effect reduces.Concrete experimental technique and result are as previously mentioned.
The experiment proved that effect of the present invention is as follows:
1) utilize PCR the method success transformation nrhTNF5 ' end restriction enzyme site, and the oligonucleotide fragment of the coding CRGDC of synthetic is connected with nrhTNF5 ' end, made up the cloning vector pGEM3zf-RT of RGD-nrhTNF fusion gene, determined dna sequence confirms entirely true.
2) made up the prokaryotic expression carrier pBV-RT of RGD-nrhTNF fusion gene,, in intestinal bacteria, obtained to efficiently express through temperature-induced.Confirm that through immunoblotting this fusion rotein and anti-TNF alpha antibodies have specific binding capacity.
3) purifying the RGD-nrhTNF fusion rotein.SDS-PAGE detected through gel electrophoresis purity is greater than 95%.Protein recovery is 2.54%.
4) the external activity experiment shows that the RGD-nrhTNF fusion rotein has tangible killing activity to the L929 cell, and specific activity reaches 5.6 * 10
8IU/mg.In vivo test shows that RGD-nrhTNF has the obvious suppression effect to the growth of mice-transplanted tumor S180.The inhibitory rate to 84.84% of RGD-nrhTNF group has notable difference (P＜0.01) with control group.Compare with the nrhTNF (tumor control rate is 59.09%) of same dose, curative effect has notable difference.And toxic side effect has the trend of the nrhTNF that is lower than same dose.
The complete sequence of tumour new-born blood vessel specific binding polypeptide CRGDC and novel recombinant human tumour necrosis factor (nrhTNF) fusion gene:
ATG TGC CGT GGT GAT TGC GGA TCC CGC AAA CGT AAG CCTGTA GCC CAT GTT GTA GCA AAC CCT CAA GCT GAG GGG CAG CTCCAG TGG CTG AAC CGC CGG GCC AAT GCC CTC CTG GCC AAT GGCGTG GAG CTG AGA GAT AAC CAG CTG GTG GTG CCA TCA GAG GGCCTG TAC CTC ATC TAC TCC CAG GTC CTC TTC AAG GGC CAA GGCTGC CCC TCC ACC CAT GTG CTC CTC ACC CAC ACC ATC AGC CGCATC GCC GTC TCC TAC CAG ACC AAG GTC AAC CTC CTC TCT GCCATC AAG AGC CCC TGC CAG AGG GAG ACC CCA GAG GGG GCT GAGGCC AAG CCC TGG TAT GAG CCC ATC TAT CTG GGA GGG GTC TTCCAG CTG GAG AAG GGT GAC CGA CTC AGC GCT GAG ATC AAT CGGCCC GAC TAT CTC GAC TTT GCC GAG TCT GGG CAG GTC TAC TTTGGG ATC ATT GCC TTC TGA
Claims (7)
1. the preparation method of tumour new-born blood vessel specific binding polypeptide CRGDC and novel recombinant human tumour necrosis factor nrhTNF fusion rotein is characterized in that, according to the following steps preparation:
1) 7 aminoacid deletion that with the gene location mutating technology wild-type hTNF α N held respectively, and change 8,9,10 proline(Pro), Serine, aspartic acid into arginine, Methionin, arginine, leucine with 157 at C end is replaced as phenylalanine simultaneously, obtains novel recombinant human tumour necrosis factor nrhTNF α;
2) clone of RGD-nrhTNF antigen-4 fusion protein gene
Get the pGEM3zf-nrhTNF plasmid, its cloning site is EcoRI and PstI, has at first designed a pair of primer the 5 ' EcoRI that holds is sported BamHI;
Primer 1: promptly 5 ' hold primer: CGGAATTC ATG CGC AAA CGT AAG CCT GTAGCC CAT GTT GTA
Primer 2: promptly 3 ' hold primer, CGC TGC AGT CAG AAG GCA ATG ATC CCAAAG TA
P3zf-nrhTNF is carried out pcr amplification;
The preparation of pcr amplification reaction pipe is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive components, 94 ℃ of reaction 5min add the Taq enzyme again, carry out the PCR reaction then, and the loop parameter of pcr amplification is: 94 ℃, the 30s reaction of degeneration; 55 ℃, the 30s annealing reaction; 72 ℃, the 30s amplified reaction carries out 30 circulations, extends 10min in 72 ℃;
After the PCR product is used the BamHI+PstI double digestion, separate, reclaim the purpose fragment, called after m-nrhTNF through 1.5% agarose gel electrophoresis;
Designed and synthesized the nucleic acid fragment of coding CRGDC targeted peptide according to the habitual codon of intestinal bacteria;
Fragment 1:AATTC ATG TGT GAT TGT CGT GGC GAT TG
Fragment 2:TTT TTG TGG ATC CAC CTC TGG TTC TGG TAA ATC TTCTGA AGG TAA AGG TG
Fragment 3:GAT CCA CCT TTA CCT TCA GAA GAT TTA CCA GAA CCAGAG G
Fragment 4:TGG AGC CAC AAA AAC AAT CGC CAC GAC AAT CAC ACATG
With four fragments of synthetic in 65 ℃ of sex change 5min, annealing is connected with m-nrhTNF and with pGEM3zf (-) plasmid that EcoRI+PstI handled again, connects product transformed competence colibacillus e.colidh5, use blue hickie screening method, picking white clone, overnight incubation in the LB nutrient solution is extracted plasmid DNA, cut evaluation through enzyme, obtain to insert the correct RGD-nrhTNF fusion gene pronucleus cloning vector of clip size, carry out dna sequence analysis, correct person's called after pGEM3zf-RT checks order; The gene that wherein contains codes for tumor new-born blood vessel specific binding polypeptide CRGDC and nrhTNF fusion rotein;
3) abduction delivering of the structure of expression vector and recombinant protein
Check order correct pGEM3zf-RT through EcoRI and PstI digestion, reclaim the dna fragmentation of about 500bp, be connected the transformed competence colibacillus e.colidh5 with the pBV220 plasmid of handling through above-mentioned same restrictions restriction endonuclease, the random choose clone identifies, screens with EcoRI and PstI double digestion; Correct clone is named as pBV-RT; The pBV-RT/DH5 α bacterium of correct reorganization, 30 ℃ of activation jolting overnight incubation in LB, be inoculated in the LB substratum that contain 100 μ g/ml Amps with 1: 100 ratio morning next day, and 30 ℃ are continued to be cultured to OD
650nm=0.6 o'clock, rapidly elevated temperature to 42 ℃ was cultivated 4h; The centrifugal 15min of 3000rpm collects thalline;
4) purifying of recombinant protein
Adding 5ml in 1 gram thalline, to split the ratio of bacterium damping fluid resuspended with thalline, with high pressure cell destroyer cracking thalline, 1M MgCl
20.4ml, 4 ℃ stir after, add 1mg/ml DNaseI 20 μ l, 4 ℃ are stirred 10m, 12000rpm, centrifugal 15m collects supernatant, adds solid ammonium sulfate to 50% saturation ratio, 4 ℃ leave standstill 1h, 12000rpm, centrifugal 15m collects supernatant; A liquid with 50 times of volumes: 20mM TrisCl pH8.0,1mM EDTA dialysed overnight, liquid is changed three to four times in the centre; Supernatant after the dialysis abundant equilibrated Q-Sepharose of A liquid Fast Flow chromatography purification, wash-out B liquid is 0.3MNaCl, 20mM TrisCl pH8.0,1mMEDTA, 10 column volumes of continuous gradient wash-out are collected each elution peak, measure active, get the C liquid of active peak to 50 times of volumes: 20mM PB pH6.0 dialysed overnight, liquid is changed three to four times in the centre; The dialysis back supernatant abundant equilibrated SP-sepharoseFast of C liquid Flow chromatography purification, wash-out D liquid is 20mMPB, pH6.0,1M NaCl, 4-5 column volume of continuous gradient wash-out collected elution peak, carries out determination of activity and electrophoresis detection;
5) extracorporeal biology of recombinant protein is active detects
The biological activity assay of tumour new-born blood vessel specific binding polypeptide CRGDC and novel recombinant human tumour necrosis factor fusion protein is undertaken by the L929 cell toxicant method of classics;
6) biologic activity detects in the body of recombinant protein
Set up mouse S180 ascitic tumor transplanted tumor model, for being subjected to the reagent thing, nrhTNF, endoxan, injection physiological saline are control drug with RGD-nrhTNF, and mice-transplanted tumor knurl body weight serves as to detect index when finishing with experiment, calculate tumour inhibiting rate, determine to be subjected to the tumor killing effect of reagent thing; With mouse body weight before and after the experiment serves as to detect index, calculates the body weight change rate, determines to be subjected to the toxic side effects of reagent thing;
Inhibition rate of tumor growth calculates by following formula:
Calculate mouse treatment front and back body weight change simultaneously, as the index of weighing the medicine toxic side effect;
2. method according to claim 1 is characterized in that, contains the gene of codes for tumor new-born blood vessel specific binding polypeptide CRGDC and tumour necrosis factor nrhTNF fusion rotein among the described pGEM3zf-RT.
3. method according to claim 1, it is characterized in that, the gene that contains codes for tumor new-born blood vessel specific binding polypeptide CRGDC and novel recombinant human tumour necrosis factor nrhTNF fusion rotein among the described prokaryotic expression carrier pBV-RT, the intestinal bacteria of carrying this carrier are after 30 ℃ of shaking culture are spent the night, activated, ℃ induce through elevated temperature to 42, can efficiently express tumour new-born blood vessel specific binding polypeptide CRGDC and tumour necrosis factor nrhTNF fusion rotein.
4. method according to claim 1, it is characterized in that, described engineering bacteria e.colidh5, genotype is supE44 Δ lacU169 hsdR17 recA1 endA1 gyrA96 thi-1 relA1, wherein contains the Prokaryotic Expression carrier pBV-RT of codes for tumor new-born blood vessel specific binding polypeptide CRGDC and tumour necrosis factor nrhTNF fusion rotein.
5. method according to claim 1 is characterized in that, the consisting of of described pcr amplification reaction pipe:
25 μ l/ reaction tubess
The p3zf-nrhTNF template DNA, 200ng/ml 2.0
4mM dNTPS 2.0
40mM MgCl
2 1.25
Primer 1 0.05mg/ml 2.5
Primer 2 0.05mg/ml 2.5
Taq enzyme 1.0U 1.0
H2O 13.75
Amount to 25.0.
6. method according to claim 1 is characterized in that, described L929 cell toxicant method measuring method is as follows:
The L929 cell is with the DMEM culture medium culturing that is added with 10% calf serum, and behind trypsin digestion cell, at the microscopically counting, adjusting cell concn is 1.0 * 10
5Cell/ml, and with cell inoculation in 96 well culture plates, every hole 100 μ l, promptly 1.0 * 10
4Cells/well, 37 ℃, 5%CO
2Overnight incubation to cell is sticked wooden partition more than 80%;
Each dosage of TNF standard substance is respectively surveyed two holes, every hole adds 0.1ml, 4 times of gradient dilutions are done with the DMEM nutrient solution that contains 3% foetal calf serum (FBS) in each hole later on, and promptly the standard substance initial concentration is 100IU/ml, is followed successively by 25IU/ml later on, 6.25IU/ml, 1.56IU/ml, 0.39IU/ml, 0.1IU/ml, 0.025IU/ml, 0.00625IU/ml; Sample then is diluted to initial concentration in advance according to practical situation and is about 100IU/ml, does 4 times of gradient dilutions again, and final volume is 100 μ l/ holes, and more than operation is all operated in 96 orifice plates; Place 37 ℃, in the 5%CO2 incubator, cultivate 16h; With the dyeing of Viola crystallina method, survey OD
570nmValue.
7. method according to claim 1 is characterized in that, the concrete steps of determination of activity are in the described recombinant protein body:
1) with 40 secondary body weight between the Kunming mouse between the 18-22 gram under aseptic technique, in right inguinal region subcutaneous vaccination 1 * 10
7The S180 oncocyte of/0.1ml;
2) next day postvaccinal animal is divided into 4 groups at random, i.e. RGD-nrhTNF1.2 * 10
6The U/kg experimental group, nrhTNF 1.2 * 10
6The U/kg group, endoxan 100mg/kg positive controls, physiological saline negative control group, every group of 10 animals;
3) before treatment, weigh; Treated by above-mentioned each treated animal dosage and medication respectively in the 6th day after the strain of inoculation knurl, all in subcutaneous injection endoxan, RGD-nrhTNF, nrhTNF and physiological saline were treated 7 days continuously;
4) after the last administration 24 hours, weigh in once more, push away dislocation method with neck then and put to death animal, isolate tumor tissues rapidly and claim its weight in wet base, calculate inhibition rate of tumor growth according to formula, and carry out statistical study.
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| 3中人肿瘤坏死因子衍生物的研制 和晓龙 等，生物化学与生物物理学报，第27卷第1期 1995 * |
| RGD特异性结合多肽与重组改构人肿瘤坏死因子融合基因的克隆表达极其抑瘤活性 王慧 等，中国生物制品学杂志，第18卷第3期 2005 * |
| 新型重组人肿瘤坏死因子-α的研究进展 黄洁等，中国生物制品学杂志，第23卷第3期 2002 * |
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