CN1277208A - New angio-auxin derivative protein and its code sequence - Google Patents
New angio-auxin derivative protein and its code sequence Download PDFInfo
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Abstract
The new angio-auxin derivative protein has Aspl 16His mutation, and has, experiment shows, 30 times of blood vessel growth promoting effect. The present invention also discloses polynucleotides encoding the poly peptide and the recombination process to produce the poly peptide. Because of the adopted codon favorable to bacteria, the protein has greatly raised expressed amount.
Description
The invention belongs to biological technical field, specifically, the present invention relates to a kind of new angio-auxin derivative protein, this new angio-auxin derivative protein has the Asp116His sudden change.The invention also discloses a kind of new chemosynthesis, with the gene order of the angio-auxin derivative protein of bacterium preference codon coding with produce the method for this peptide species through recombinant technology.
1985, American scholar Fett JW etc. extract from the human colon adenocarcinoma cell line HT-29 of vitro culture and obtain a kind of active protein of very strong angiogenic growth that has, the about 14.1kD of molecular weight, called after angiogenine (angiogenin abbreviates " ANG " as).
The single chain protein molecule now clear and definite, that ANG is made up of 123 amino-acid residues has strong angiogenic growth activity, and iso-electric point belongs to rnase (ribonuclease, RNase) superfamily greater than 9.5.Except the angiogenic growth activity, ANG also has specificity bace rna (ribonuclic acid, multiple biological activity such as RNA).Studies show that the ANG significant feature is to promote the little angiogenic growth of body, in known angiogenic growth factor, the angiogenic growth activity of ANG is better than other factors of great majority.In chick chorioallantoic membrane angiogenic growth (CAM) test, the ANG of 35 fmol can cause typical spoke sample of blood pipe growth patterns, and the ANG of 3.5pmol can make rabbit corneal new vessel occur.People thereby inference are blocked or are utilized its angiogenic growth effect or to treat various ischemic diseases for the growth of checking noumenal tumour new way is provided.Therefore, ANG is subjected to numerous investigators' extensive concern, and relative basic and applied research is carried out in a large number, and progressively illustrates the basic problem such as structure, functional characteristics, mechanism of action of ANG and derivative thereof, has showed good prospects for application simultaneously.
For a long time, ANG acceptor and mechanism of action thereof etc. are the deep difficult problems of restriction ANG research always, the sluggish to a great extent progress of its applied research.Over the past two years, these problems progressively were familiar with.Studies have shown that in a large number Actin muscle can combine with ANG as one of ANG acceptor high-affinity, is mediating the many vital role in the angiogenic growth process.
Recently, found the ANG acceptor of a kind of molecular weight 170kD again on the human endothelial cell surface.This receptor is only expressed under endotheliocyte distributes sparse, discontinuous situation and is combined specifically with ANG, causes the synthetic and cell proliferative response of the DNA relevant with dosage.Though the research data to this receptor is still few at present, the cell proliferative response of its mediation has caused many scholars' interest, and infers that it plays critical effect in the angiogenic growth process of ANG.After ANG and its receptors bind, can further act on: the information conduction of second messenger mediation and receptor-mediated pinocytosis endocytosis and to nuclear transhipment by two kinds of approach.
In view of the These characteristics of angiogenine, if energy great expression angiogenic growth fibroin will play positive pushing effect to the diagnosis and the treatment of diseases related.Yet human normal tissue medium vessels growth hormone content is lower, and the purification difficulty is very big, and cost is very high, can't be used for large-scale production.From tumor tissues, extract and then have bigger risk.Therefore, carried out the research work of a large amount of angiogenine genetically engineered aspects abroad.The result shows that escherichia coli prokaryotic expression ANG protein content is higher, accounts for the 5-10% of total protein content; The eukaryotic expression cost is higher, and yield is extremely low.And the ANG albumen of prokaryotic expression gained and the albumen of the eukaryotic expression active basically identical aspect the promotion angiogenic growth.
Yet, even the expression amount of 5-10% is still unfavorable for large-scale industrial production.Therefore, this area presses for active higher angio-auxin derivative protein.In addition, the present invention also presses for the method for can high expression level ground producing angio-auxin derivative protein.
The purpose of this invention is to provide a kind of new angio-auxin derivative protein, it has the activity of the angiogenic growth that is higher than natural angiogenine.
Another object of the present invention provides the polynucleotide of this angio-auxin derivative protein of coding.
Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.
In a first aspect of the present invention, a kind of isolating angio-auxin derivative protein is provided, to compare with natural angiogenine, it comprises the Asp116His sudden change.Preferably, this albumen is the polypeptide with SEQ ID NO:2 aminoacid sequence.
In a second aspect of the present invention, a kind of isolating polynucleotide are provided, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQID NO:2.Preferably, these polynucleotide have the sequence shown in the SEQ ID NO:1.
In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.
In a fourth aspect of the present invention, a kind of preparation method of angio-auxin derivative protein is provided, this method comprises:
(a) under the condition that is fit to the expression angio-auxin derivative protein, cultivate above-mentioned host cell;
(b) from culture, isolate angio-auxin derivative protein.
In a fifth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains the described angio-auxin derivative protein of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.Preferably, this albumen is the polypeptide with SEQ ID NO:2 aminoacid sequence.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
Fig. 1 is a chemosynthesis angio-auxin derivative protein encoding sequence and with the electrophorogram behind the pcr amplification.Swimming lane A is an amplified production, and swimming lane Marker is the molecular weight marker thing.
Fig. 2 is the sequencing result of angio-auxin derivative protein encoding sequence of the present invention.
Fig. 3 is the SDS-PAGE picture after angio-auxin derivative protein of the present invention is expressed.Among the figure, the angio-auxin derivative protein for expressing of arrow indication.Be respectively 4,4.5,4.5,5,5,3.5,4,3,8,12 hours protein expression situations of 42 ℃ of inducing culture for swimming lane 1-10 from left to right.
Fig. 4 is the SDS-PAGE picture of angio-auxin derivative protein of the present invention after purified.Among the figure, the angio-auxin derivative protein for expressing of arrow indication.For swimming lane 1-10 be respectively from left to right: the 1-3 swimming lane is a Q-Sepharose Fast Flow anion column purification result; The 4-6 swimming lane is a SP Sepharose Fast Flow cation seperation column purification result; 7,8 swimming lanes are Sephacryl S-100 molecular sieve purification result.
Fig. 5 is the chick chorioallantoic membrane test photo of angio-auxin derivative protein of the present invention.
As used herein, " separation " refers to that material separates (if natural thing from its primal environment Matter, primal environment namely are natural surroundingses). Such as the polynucleotide under the native state in the active somatic cell and polypeptide be Do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials that exist Separately, then for separation and purification.
As used herein, " angio-auxin derivative protein of separation or polypeptide " refers to angio-auxin derivative protein Polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material. The technology of this area Personnel can use the purified technology of protein purifying angio-auxin derivative protein of standard. Basically pure polypeptide is gone back non-Can produce single master tape on the former polyacrylamide gel. Usually, the purity of angio-auxin derivative protein greater than 50%, preferably, greater than 60%, more preferably greater than 70%, best greater than 80% or higher. Among the present invention Polypeptide preferably provides with the form of separating with polynucleotides, more preferably is purified to homogeneous.
As used herein, " Asp116His " or " D116H " refers to corresponding to the 116th of natural angiogenine Asp be replaced by His.
Angio-auxin derivative protein of the present invention is characterised in that to have the Asp116His sudden change. Yet, except carrying out Outside the above-mentioned amino acid substitution, those skilled in the art will appreciate that also and can repair the amino acid of other positions Decorations are as replacing, insert or disappearance. For replacement, the amino acid that available character is close is replaced. Preferably, This 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor is to carry out in the same group in following table 1 (representational replacement), and this replacement is best Preferably replace in the following table 1.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | val；leu；ile | val |
| Arg(R) | lys；gln；asn | lys |
| Asn(N) | gln；his；lys；arg | gln |
| Asp(D) | glu | glu |
| Cys(C) | ser | ser |
| Gln(Q) | asn | asn |
| Glu(E) | asp | asp |
| Gly(G) | pro；ala | ala |
| His(H) | asn；gln；lys；arg | arg |
| Ile(I) | Leu; Val; Met; Ala; Phe; Nor-leucine | leu |
| Leu(L) | Nor-leucine; Ile; Val; Met; Ala; Phe | ile |
| Lys(K) | arg；gln；asn | arg |
| Met(M) | leu；phe；ile | leu |
| Phe(F) | leu；val；ile；ala；tyr | leu |
| Pro(P) | ala | ala |
| Ser(S) | thr | thr |
| Thr(T) | ser | ser |
| Trp(W) | tyr；phe | tyr |
| Tyr(Y) | trp；phe；thr；ser | phe |
| Val(V) | Ile; Leu; Met; Phe; Ala; Nor-leucine | leu |
The Summary on technology that DNA is carried out genetic modification is in mutagenesis: a kind of practical approach (Mutagenesis:a Practical Approach), M.J.McPherson, Ed., (IRL Press, Oxford, UK. (1991) for example comprise direct mutagenesis, cassette mutagenesis and polymerase chain reaction (PCR) mutagenesis. The document is at this Quote as a reference.
Can obtain ANG nucleotide sequence natural and/or restructuring by methods such as PCR methods, use then various The method of mutagenesis of knowing in this area carries out sudden change of the present invention. A kind of ANG mutant of the present invention Dna sequence dna is listed in SEQ ID NO:2. But because the degeneracy of genetic code is permitted so can prepare Multiple nucleotides, they all are to instruct synthetic restructuring ANG of the present invention. Therefore, on the function with above-mentioned order Column equivalent dna sequence dna is perhaps recombinated the ANG protein analogue (according to ammonia with instructing synthetic the present invention on the function The base acid sequence produces) sequence sequence of equal value mutually, all be included within the present invention.
For the nucleic acid of encoding novel restructuring ANG of the present invention, by restriction enzyme digestion, connect with the plasmid fragment then Connect, can be cloned in the suitable carrier such as plasmid. Plasmid for selecting has no particular limits, as long as It can copy and express the ANG of restructuring in the host cell of selecting.
Certainly, the coded sequence of this recombinant protein also can be integrated in the genome such as host cell.
Contain the plasmid of the nucleic acid of encoding novel restructuring ANG, generally contain the regulation and control such as suitable promoter, enhancer Element, and suitable Select gene is (such as antibiotics resistance gene, such as ampicillin resistance gene; Or battalion Support defective gene), these all are to know in this area. Can be referring to people such as Sambrook, molecular cloning: real Test chamber handbook (New York:Cold Spring Harbor Laboratory Press, 1989).
Carry the expression vector of restructuring ANG coded sequence of the present invention, available method well known in the art is worn such as electricity Hole, cotransfection method etc. change suitable host cell over to. Host cell can be prokaryotes or eucaryote, as Escherichia coli, hay bacillus, insect cell, Chinese hamster ovary celI etc. But be preferably Escherichia coli.
After transforming, filter out transformant with conventional method. And according to used host cell and expression vector, With the culture medium of the conventional usefulness in this area and the cell that condition of culture is cultivated conversion, thereby the expression restructuring ANG.
After transformed host cells is expressed reorganization ANG of the present invention, if recombinant protein is secreted in the substratum, can be directly from substratum separation and purification go out recombinant protein.If recombinant protein is expressed with the inclusion body form, can from culture, obtain required recombinant protein so by steps such as lysing cell, separation, purifications.These steps and method all are to know in this area, can be referring to people such as for example Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989).
By the recombinant DNA technology of routine, can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce angio-auxin derivative protein polypeptide (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding angio-auxin derivative protein of the present invention, or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, the angio-auxin derivative protein polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains angio-auxin derivative protein DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a LaboratoryManual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage P
LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS or Bowes melanoma cells etc.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Alternative is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
Recombinant polypeptide in the above methods can wrap by in cell, extracellular or on cytolemma, express or be secreted into the extracellular.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The angio-auxin derivative protein or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to directly as pharmacological agent blood vessel infraction property disease, promotion wound healing etc.Angiogenine derivative through scale operation can be used for eliminating foxiness, freckle and skin whitening or other cosmetics production.Antagonism derivative of the present invention or can be used for treatment to tumor tissues.
Polypeptide of the present invention, and fragment, derivative, analogue or their cell can be used as antigen to produce antibody.These antibody can be polyclone or monoclonal antibody.Polyclonal antibody can obtain by the method with this polypeptide direct injection animal.The technology of preparation monoclonal antibody comprises hybridoma technology, three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.
Can be with polypeptide of the present invention and antagonist and suitable pharmaceutical carrier combination back use.These carriers can be water, glucose, ethanol, salt, damping fluid, glycerine and their combination.Composition comprises the angio-auxin derivative protein of the present invention or the antagonist of safe and effective amount and carrier and the vehicle that does not influence effect of drugs.These compositions can be used as medicine and are used for disease treatment.
The present invention also provides medicine box or the test kit that contains one or more containers, and one or more medicinal compositions compositions of the present invention are housed in the container.With these containers, can have by the given indicative prompting of government authorities of making, using or selling medicine or biological products, the government authorities that this prompting reflects production, uses or sells permits it to use on human body.In addition, polypeptide of the present invention can be used in combination with other treatment compound.
Pharmaceutical composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the route of administration of intracutaneous.Angio-auxin derivative protein comes administration with the amount that treats and/or prevents concrete indication effectively.The amount and the dosage range that are applied to patient's angio-auxin derivative protein will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.Usually, the consumption of angio-auxin derivative protein is 1 microgram-5 a mg/kg body weight/day, preferably, is 5 micrograms-2.5 mg/kg body weight/day, more preferably is 10 micrograms-1 mg/kg body weight/day.
In an example of the present invention, in order to improve the expression amount of angio-auxin derivative protein in bacterium, the polynucleotide of coding angio-auxin derivative protein are to obtain with the method for chemosynthesis, employing be the codon that bacterium is had a preference for.The polynucleotide sequence total length that it comprises is 378 bases (SEQ ID NO:1), 124 amino acid (SEQ ID NO:2) that its open reading frame has been encoded and comprised Met.Its aminoacid sequence is compared with people's blood vessel growth hormone albumen of having delivered, and except that the 116th amino acids is replaced with the His by Asp, all the other are in full accord.Experiment showed, that through short chick chorioallantoic membrane (CAM) angiogenic growth subliminal dose its angiogenic growth effect has improved 30 times than angiogenine itself.
In addition,, adopt first purifying by improvement to refolding method, the method for back renaturation, not only tens of times of ground have improved the proteic renaturation rate of recovery, and the more original simple renaturation method ratio of the proteic activity of gained is lived high.This reduces production costs to scale operation is very useful.
In addition, by improvement to refolding method, adopt dialysis to combine with dilution and renaturation before the refolding method of adding high density glycerine in the protein solution; not only tens of times of ground have improved the proteic renaturation rate of recovery; and protein-active obtains better protecting, reaches external similar research advanced level.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the chemosynthesis of oligonucleotide fragment
The angiogenine derivative gene of total length 378bp is divided into the oligonucleotide fragment of following 20 28-46mers, sees Appendix, and adopts solid phase phosphorous acid amine method synthetic respectively on ABI381A type dna synthesizer.
F1：5′-GGGGTACCGTTAACTAAGGAAATACGAATTCATGCAGGACAACTCT-3′
F2：5′-CCCCATGGCAATTGATTCCTTTATGCTTAAGTAC-3′
F3：5′-CGTTACACCCACTTCCTGACCCAGCACTACGACGCTAAACCGCA-3′
F4：5′-GTCCTGTTGAGAGCAATGTGGGTGAAGGACTGGGTCGTGATGCT-3′
F5：5′-GGGTCGTGACGACCGTTACTGCGAATCTATCATGCGTCGTCGT-3′
F6：5′-GCGATTTGGCGTCCCA?GCACTGCTGGCAATGACGCTTAGATAG-3′
F7：5′-GGTCTGACCTCTCCTTGCAAAGACATCAACACCTTCATCCA-3′
F8：5′-TACGCAGCAGCACCAGACTGGAGAGGAACGTTTCTGTAGTTG-3′
F9：5′-CGGCAACAAACGTTCTATCAAAGCTATCTGCGAGAACAAGAAC-3′
F10：5′-TGGAAGTAGGTGCCGTTGTTTGCAAGA?TAGTTTCGATAGACGC-3′
F11：5′-GGTAACCCGCACCGTGAAAACCTGCGTATCTCTAAATCTTCTTT-3′
F12：5′-TCTTGTTCTTGCCATTGGGCGTGGCACTTTTGGACGCATAGAGAT-3′
F13：5′-CCAGGTTACCACCTGCAAACTGCACGGTGGTTCTCCGTGGCCGCC-3′
F14：5′-TTAGAAGAAAGGTCCAATGGTGGACGTTTGACGAGCCACCAAGAGGCACCGGCGG-3′
F15：5′-GTGCCAGTACCGTGCTACCGCTGGTTTCCGTAACGTTGTTGTT-3′
F16：5′-C?ACGGTCATGGCACGATGGCGACCAAAGGCATTGCAACAACAA-3′
F17：5′-GCTTGCGAAAACGGTCTGCCGGTTCACCTGGACCAGTCTATCTTC-3′
F18：5′-CGAACGCTTTTGCCAGACGGCCAAGTGGACCTGGTCAGATAGAA-3′
F19：5′-CGTCGTCCGTGATAAGTTAACGGATCCAG-3′
F20：5′-GGCAGCAGGCACTATTCAATTGCCTAGGTC-3′
Then, the segmental solid phase carrier that hangs with synthetic good DNA is poured in the bottle, added strong aqua to 2/3 volume, the sealing back is separated in 55 ℃ of ammonia and is spent the night to remove blocking group.Next day, the unpacking bottle is rushed behind the ammonia, with the solution for vacuum drying.After heavily being dissolved in saturated urea, last 12% polyacrylamide sex change preparative gel electrophoresis, through the 600V constant voltage, behind the 6-8 hour electrophoresis, take off gel, for setting off, under long wave ultraviolet light, downcut the slowest gel band that contains dna fragmentation of migration with fluorescent plate, add 4-5ml0.5mol/L NaCl after the chopping, 37 ℃ are incubated overnight.
Next day, again this dna solution is crossed the C-18 reversed-phase column to be further purified DNA.Method is: use earlier 100% and 60% washed with methanol C-18 post respectively, with pure water post is cleaned again, then dna solution is crossed post, this moment, DNA was hung on the C-18 post, wash post repeatedly to wash out impurity with pure water, wash DNA with 2ml60% methyl alcohol at last, after ultraviolet detection is also quantitative, packing and vacuum-drying ,-20 ℃ of preservations are standby.After all dna fragmentations prepare, carry out segmental purity detecting.
Embodiment 2: the splicing of gene and pcr amplification:
Except that two fragments of F1, F20, all fragments are respectively got 0.2OD, mix the back and containing 10mM ATP, in the kinase reaction system of 20uKinase 37 ℃ of reaction 90min carry out 5 '-the end phosphorylated.Behind 100 ℃ of 1min deactivation Kinase, add two 5 '-end fragment, and add NaCl to final concentration be 0.1M, then solution is placed 3min so that the DNA sex change at 95 ℃, slowly be annealed to 25 ℃ after, put 65 ℃ of 3min again, for the second time slowly be annealed to 20 ℃, so that the correct renaturation of DNA.Then, use ethanol sedimentation DNA, heavy molten DNA adds the 80u ligase enzyme in 30 microlitre ligase enzyme reaction systems after the vacuum-drying, spends the night in 16 ℃ of connections.Connecting effect detects with 5% polyacrylamide gel electrophoresis.
Though, after connection, obtained required encoding sequence, because the complete genome amount of this method gained is less, thus be template with above-mentioned connection liquid, according to the upstream and downstream sequences Design of full-length gene and synthesize following corresponding primer:
Pp1：5′-ACGAATTCATGCAGGACAACTCTCGTTACACC-3′；
Pm1：5′-CTGGATCCGTTAACTTATCACGGACGACGGAAGATAG-3′；
Use PCR method, the amplification full-length gene.The PCR reaction conditions is 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations.
The electrophorogram of PCR reaction product is seen Fig. 1.
Then isolating pcr amplified fragment after cutting, EcoRI, BamHI enzyme is inserted plasmid vector pUC118 and bacterial infection, obtain the D116H-ANG-pUC118 recombinant plasmid, (Fig. 2) identified in order-checking behind the screening mono-clonal bacterial strain, and the result shows that the encoding sequence of angio-auxin derivative protein is correct (SEQ ID NO:1).
Embodiment 3: the vivoexpression of recombinant vascular growth hormone derivative, separate and purifying
Designed a pair of primer at the initiator codon place and the terminator codon place of angiogenine derivative gene respectively, its 5 ' end has the KpnI restriction enzyme site, 3 ' end has the BamHI restriction enzyme site, simultaneously for improving expression efficiency, in one section SD sequence of ATG upstream design.
Pp2：5′-GGTACGTTAACTAAGGAAATACGAA?TTCATGCAGGACAAC-3′；
Pm2：5′-CTGGATCCGTTAACTTATCACGGACGACGGAAGATAG-3′；
With D116H-ANG-pUC118 is that template is carried out pcr amplification acquisition D116H-ANG gene coding region.Amplified fragments is cut insertion expression vector pBV220 (available from Pharmacia Biotech) through enzyme, and Transformed E .coliJM103, containing on the LB flat board of penbritin and X-gal and IPTG, recombinant conversion of screening white carries out dna sequence analysis, and the genes encoding region sequence among result and the D116H-ANG-pUC118 is identical.The gained recombinant clone can be expressed D116H-ANG albumen.
The above-mentioned pBV220/ANG-D116H/JM103 engineering bacteria list bacterium colony that successfully constructs is taken in the 3ml LB nutrient solution that contains penbritin 100mg/ml, and 30 ℃ of constant temperature shaking table shaking culture are spent the night, and rotating speed is 250rpm.Connect this and spend the night bacterium 0.5ml in the fresh LB+Amp nutrient solution of 50ml next day, and 30 ℃ are cultured to bacterial concentration and reach OD
590Value is about at 0.8 o'clock, rapidly shaking table is warming up to 42 ℃ and continues to cultivate, and the 1ml bacterium liquid of sampling per half an hour is induced after 5 hours and received bacterium.Collected bacterium liquid is centrifugal with 5000rpm, add the resuspended thalline of 60 microliters of water behind the bacterium liquid precipitate, add 60 microlitres, 2 * SDS albumen sample-loading buffer, boil do after 15 minutes of short duration centrifugal.Then, get supernatant and be splined on 15%SDS-PAGE, 30mA constant current electrophoresis is after 4 hours, and Coomassie brilliant blue G-250 dyeing is 3 hours under the normal temperature, after 3 hours, can observe protein band with the decolouring of gel destainer again.The SDS-PAGE electrophoresis showed has angiogenine derivative protein product at about 14.4kDa place, and about the 5th hour expressing quantity the highest (swimming lane 3), expressing protein accounted for 30% of total bacterial protein.(see figure 3).
Expression product is successively through Q-Sepharose Fast Flow anion chromatography exchange column, SP Sepharose FastFlow positively charged ion chromatography exchange column and Sephacryl S-100 sieve chromatography column purification.Elutriant is through the SDS-PAGE electrophoresis showed, and most foreign proteins have obtained effective removing (see figure 4).
Embodiment 4: protein renaturation
Adopt dialysis and dilution bonded method to remove urea, recover ANG albumen native conformation.Concrete grammar is: the D116H-ANG protein concentration behind the purifying is adjusted to be not higher than 10mg/ml, add isopyknic aseptic glycerine, the two is mixed at 1: 1, move in the dialysis tubing of molecular weight cut-off 6000-8000, in 4 ℃ of refrigerators, protein renaturation dialyzate to 100 times of volumes was slowly dialysed 24 hours, and the dialysis system is opened in the air.Next day, the protein renaturation diluent to 10-15 times of volume under 4 ℃ was slowly dialysed 24 hours, and the dialysis system is opened in the air.Dialysis finishes, and with Amicon-Stir Cell ultra-fine filter it is concentrated into the volume that needs.
The protein renaturation dialyzate: with 50mmol/LTris-HCl (pH8.5), 100mmol/L NaCl solution and aseptic glycerine do 1: 3 miscible.
Protein renaturation diluent: 50mmol/LTris-HCl (pH8.5), 100mmol/L NaCl
Aforesaid method is simple and easy to do, and compares with the abroad simple dialysis method or the dilution method of report, has reduced the generation of flocculent precipitate in the renaturation process effectively, and tens of times of ground have improved proteic renaturation and reclaimed yield.The renaturation rate of recovery of the inventive method is 75～85%.
In addition, owing to take the protein solution before the renaturation and glycerine high density glycerine to be dialysed with 1: 1 mixed, albumen precipitation does not almost observe again.Glycerine is to preserve protein protective material commonly used, helps safeguarding the hydrophobicity of protein molecular, has the active ability of protected protein matter.Simultaneously, can significantly the slow down movement velocity of protein molecular of high density glycerine solution helps the formation of intramolecular disulfide bond.Therefore, when improving yield, reduced the forfeiture of protein-active in this process, further improved proteic effectively than living.In shown in the following chick chorioallantoic membrane test, the minimum positive significant quantity of ANG is 1ng/ piece, and the minimum positive significant quantity of D116H-ANG is 0.03ng/ piece.More external similar research is compared and is in tangible leading position.
Embodiment 5:D116H-ANG and ANG angiogenic growth specific activity are
At present, detecting the method that ANG angiogenic growth activity adopted abroad is the chick chorioallantoic membrane test.The methylcellulose gum film that is about to contain different concns ANG places the chorioallantoic membrane of chicken embryo, to observe the change of angiogenic growth situation.
As mentioned above, the inventor adopts dialysis to carry out protein renaturation with the method that dilution combines.This method is when improving the protein renaturation rate of recovery, and protein-active also increases, and gained natural A NG is more consistent with external report than living, and minimum positive significant quantity is 1.0ng/ piece.The condition milder helps the protection to protein-active, and gained ANG angiogenic growth activity is higher, and it is 1.0ng/ piece that chick chorioallantoic membrane is tested minimum positive significant quantity, reaches external similar research advanced level.
And with the resulting D116H-ANG of the present invention of identical refolding method, its minimum positive significant quantity reaches 0.03ng/ piece of (see figure 5).Protein quantification all adopts mass spectrograph to detect.This shows that the angiogenine derivative D116H-ANG that expresses gained through genetic modification improves a lot than ANG itself in the activity aspect the promotion angiogenic growth, and both specific activitys are about 30.
Rabbit acute myocardial infarction model trial
Prick left anterior descending coronary artery (LAD) 2 roads with 5/0 polyester thread at left auricle of heart lower edge horizontal joint, observe the variation of left chamber cardiac muscle color and motion, trace electrocardiogram(ECG.Cardiac muscle is congested garnet when left chamber, antetheca hypomotility, and the obvious back of a bow of ST section appears in electrocardiogram(ECG to be raised or forces down, and thinks that promptly modelling is successful.Prop up blood supply district intersection along left chamber cardiac muscle transformation region edge part, left anterior descending coronary artery and left chamber, the D116H-ANG solution 50 μ l/ points that upper, middle and lower 3 dessert intramuscularlys are dissolved in the PBS damping fluid are got in stringer, contain D116H-ANG 5 μ g.The dirty capable pathology detection of coring after 8 days sees under the high power lens that the myocardial cell does not have obvious granular sex change, and little blood vessel is a bunch shape, accidental endotheliocyte bar rope sample traveling between cardiac muscle fibre, and capillary vessel is counted than the control group showed increased.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
(1) general information:
(ii) denomination of invention: new angio-auxin derivative protein and encoding sequence thereof
(iii) sequence number: 2
(2) information of SEQ ID NO:1:
(i) sequence signature:
(A) length: 378bp
(B) type: nucleic acid
(C) chain: two strands
(D) topological framework: linearity
(ii) molecule type: cDNA
(xi) sequence description: SEQ ID NO:1:1 ATG CAG GAC AAC TCT CGT TAC ACC CAC TTC CTG ACC CAG CAC TAC GAC GCT AAA CCG CAG61 GGT CGT GAC GAC CGT TAC TGC GAA TCT ATC ATG CGT CGT CGT GGT CTG ACC TCT CCT TGC121 AAA GAC ATC AAC ACC TTC ATC CAC GGC AAC AAA CGT TCT ATC AAA GCT ATC TGC GAG AAC181 AAG AAC GGT AAC CCG CAC CGT GAA AAC CTG CGT ATC TCT AAA TCT TCT TTC CAG GTT ACC241 ACC TGC AAA CTG CAC GGT GGT TCT CCG TGG CCG CCG TGC CAG TAC CGT GCT ACC GCT GGT301 TTC CGT AAC GTT GTT GTT GCT TGC GAA AAC GGT CTG CCG GTT CAC CTG GAC CAG TCT ATC361 TTC CGT CGT CCG TGA TAA
(2) information of SEQ ID NO:2:
(i) sequence signature:
(A) length: 124 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ii) molecule type: polypeptide
(xi) sequence description: SEQ ID NO:2:1 Met Gln Asp Asn Ser Arg Tyr Thr His Phe Leu Thr Gln His Tyr16 Asp Ala Lys Pro Gln Gly Arg Asp Asp Arg Tyr Cys Glu Ser Ile31 Met Arg Arg Arg Gly Leu Thr Ser Pro Cys Lys Asp Ile Asn Thr46 Phe Ile His Gly Asn Lys Arg Ser Ile Lys Ala Ile Cys Glu Asn61 Lys Asn Gly Asn Pro His Arg Glu Asn Leu Arg Ile Ser Lys Ser76 Ser Phe Gln Val Thr Thr Cys Lys Leu His Gly Gly Ser Pro Trp91 Pro Pro Cys Gln Tyr Arg Ala Thr Ala Gly Phe Arg Asn Val Val106 Val Ala Cys Glu Asn Gly Leu Pro Val His Leu His Gln Ser Ile121 Phe Arg Arg Pro
Claims (10)
1. an isolating angio-auxin derivative protein is characterized in that, it comprises the Asp116His sudden change.
2. albumen as claimed in claim 1 is characterized in that, this albumen is the polypeptide with SEQ ID NO:2 aminoacid sequence.
3. isolating polynucleotide is characterized in that, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.
4. polynucleotide as claimed in claim 3 is characterized in that, these polynucleotide have the sequence shown in the SEQ ID NO:1.
5. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
6. a genetically engineered host cell is characterized in that, it is a kind of host cell that is selected from down group:
(a) host cell that transforms or transduce with the described carrier of claim 5;
(b) host cell that transforms or transduce with the described polynucleotide of claim 3.
7. the preparation method of an angio-auxin derivative protein is characterized in that, this method comprises:
(a) being fit to express under the condition of angio-auxin derivative protein, cultivate the described host cell of claim 6;
(b) from culture, isolate angio-auxin derivative protein.
8. method as claimed in claim 7 is characterized in that, this method also comprises following renaturation step:
Isolating angio-auxin derivative protein concentration adjusted to be not higher than 10mg/ml, add isopyknic aseptic glycerine, mix, move in the dialysis tubing of molecular weight cut-off 6000-8000, slowly dialysed 24 hours at 4 ℃ of protein renaturation dialyzates to 100 times of volumes, protein renaturation diluent to 10-15 times of volume was slowly dialysed 24 hours under 4 ℃ again, concentrate then, wherein the protein renaturation dialyzate is 50mmol/LTris-HCl, pH8.5,1: 3 mixed solution of 100mmol/LNaCl solution and aseptic glycerine, and the protein renaturation diluent contains 50mmol/LTris-HCl, pH8.5,100mmol/L NaCl.
9, a kind of pharmaceutical composition is characterized in that, it contains the described angio-auxin derivative protein of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.
10. pharmaceutical composition as claimed in claim 8 is characterized in that, this albumen is the polypeptide with SEQ ID NO:2 aminoacid sequence.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN00115679A CN1277208A (en) | 2000-05-15 | 2000-05-15 | New angio-auxin derivative protein and its code sequence |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN00115679A CN1277208A (en) | 2000-05-15 | 2000-05-15 | New angio-auxin derivative protein and its code sequence |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1277208A true CN1277208A (en) | 2000-12-20 |
Family
ID=4585123
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN00115679A Pending CN1277208A (en) | 2000-05-15 | 2000-05-15 | New angio-auxin derivative protein and its code sequence |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1277208A (en) |
-
2000
- 2000-05-15 CN CN00115679A patent/CN1277208A/en active Pending
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