CN1566348A - G type non-viral vector and pharmaceutical composition containing same - Google Patents
G type non-viral vector and pharmaceutical composition containing same Download PDFInfo
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- CN1566348A CN1566348A CN 03123941 CN03123941A CN1566348A CN 1566348 A CN1566348 A CN 1566348A CN 03123941 CN03123941 CN 03123941 CN 03123941 A CN03123941 A CN 03123941A CN 1566348 A CN1566348 A CN 1566348A
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Abstract
The invention relates to a non-viral vector and fusion genes encoding it, wherein the non-virus carrier can lead the protein expression type recombinant member with destroying action to cells targetedly into stomach hormone emancipation peptide acceptor positive anoxybiotic tumor cells. The invention also relates to the pharmaceutical compositions containing the non-viral vectors and the expression type recombinant and their use.
Description
Technical field
The invention belongs to the gene therapy medicament field, specifically, the present invention relates to non-virus carrier and its fusion gene of encoding, described non-virus carrier is to pack and to carry the protein expression type recombinant chou target ground importing specific tumors cell that coding has lethal effect, so that this expression type recombinant chou goes out protein at this kind cell inner expression, thereby kill and wound this kind tumour cell and do not injure other cell.The invention still further relates to the pharmaceutical composition and the application thereof that comprise described non-virus carrier and described expression type recombinant chou.
Background technology
Nature exists thousands of kinds to have antitumor and the toxin protein with cytotoxicity antiviral activity, respectively from animal (as snake venom, centipede toxin, scorpion venom, terrestro-lumbrolysin etc.), plant (Snakegourd Root, Phytolacca acinosa, castor-oil plant etc.) and microorganism (pyocyanin, diphtheria toxin, other morbid poisons), especially marine organism (sponge, sea anemone, starfish, Ascidian, king crab, marine alga) is containing abundant anti-tumor virus resource.But nearly more than ten years, the research of using toxin anti-tumour still is trapped in external level.
Because toxin protein can combine with various kinds of cell non-specificly, using the toxitherapy tumour simultaneously toxin protein to be imported separately normally has in the cell of toxoreceptor, produces extremely strong toxic side effect.For fear of the toxic side effect of toxin, method commonly used is that toxin protein and guide molecule amalgamation and expression are formed the guiding fusion toxin over past ten years.Guide molecule commonly used is the antibody of tumor cell surface molecule or the part of its acceptor.Fusion toxin enters cell performance lethal effect by the ligand-receptor mediated endocytosis.Obtained the significant guidance quality fusion toxin of many external anti-knurl effects by this method, TGF α-PE40 for example, CD4 (178)-PE40, IL2-PE40 etc.But these fusion toxins all fail to test by the clinical I phase.Using 2-8 after week,, immune neutralization reaction is taking place, making the very fast inefficacy of this toxin protein because the proteic antibody of toxinicide produces.Use the research of toxitherapy tumour to stagnate Just because of this always.In addition, only the target by guide molecule control is also incomplete, often also have molecule identical or acceptor with tumor cell surface on the normal cell surface, and some overexpression in the acceptor molecule of tumor cell surface simultaneously also at some normal cell surface overexpression, thereby therefore the targeted toxin of this design still can import normal cell and produces uncontrollable toxic side effect.
At using these obstacles that exist in the toxitherapy tumour, there is a need in the art for when avoiding toxin protein immunogenicity and toxic side effect fully, thus the new target therapeutic agent and the related vector of reservation toxin protein activity specific killing tumour cell.
Summary of the invention
In first aspect, the invention provides a kind of non-virus carrier, its essence is a kind of target fusion rotein, described fusion rotein by the receptors ligand polypeptide be rich in positive amino acid whose polypeptide and form.In this another embodiment on the one hand of the present invention, non-virus carrier of the present invention is made up of receptors ligand polypeptide, the polypeptide that is rich in positive amino acid whose polypeptide and concentration of DNA.According to the present invention, described to be rich in positive amino acid whose polypeptide be that the DNA of two placed in-line Protein-tyrosine-phosphatases (PTP) is in conjunction with section or its associated clip; The polypeptide of concentration of DNA is that the proteic DNA of c-myc is in conjunction with section; Described receptors ligand polypeptide be can with gastrin releasing peptide receptor (GRP-R) specificity bonded gastrin release peptide (GRP) or its associated clip, analogue or mutant.Owing to use GRP as guide molecule, so the inventor is called G type non-virus carrier with non-virus carrier of the present invention.
Preferably, obtain fusion gene in vitro recombination, and in prokaryotic cell prokaryocyte or eukaryotic cell, express this fusion gene and obtain non-virus carrier of the present invention (fusion rotein) by gene engineering method.Therefore, on the other hand, the invention provides the fusion gene of coding non-virus carrier of the present invention.
On the other hand, the invention provides the pharmaceutical composition of the mixture that comprises non-virus carrier of the present invention and a kind of expression type DNA recombinant chou, to be used for the gene therapy of target malignant tumour, described expression type recombinant chou comprises expresses the proteinic gene that pair cell has kill capability.
According to the present invention, the protein that described pair cell has kill capability is pseudomonal toxin, diphtheria toxin, Phytolacca acinosa, Ricin, or other all pair cells have the albumen from human body, animals and plants or microorganism of kill capability, or their active function zone, mutant or analogue, and the protein with killer cell activity of synthetic or polypeptide and respective coding gene thereof.Preferably, the protein that wherein said pair cell has kill capability is pseudomonal toxin, or its second and the 3rd structural domain; More preferably, described pair cell has the 3rd structural domain or its mutant that the protein of kill capability is pseudomonal toxin.In a preferred embodiment of the invention, described mutant is a Pseudomonas aeruginosa III functional domain mutant (REDLK of C section is mutated into KDEL), is called PEIIImut.It can produce the stronger toxin protein of lethality, shown in its aminoacid sequence SEQ ID NO.:13.
The present invention changes the mode of toxin protein target transfered cell into toxin protein encoding gene target transfered cell methods of treatment, when avoiding toxin protein immunogenicity and toxic side effect fully, thereby keep toxin protein activity specific killing tumour cell.The present invention should choose the DNA of source protein tyrosine phosphatase in conjunction with section, and the proteic DNA of c-myc in conjunction with section and overexpression in tumor cell surface, only the high-affinity part at the acceptor of limited normal cell trace expression constitutes fusion rotein type non-virus carrier, and carry toxin gene expression type recombinant DNA and form the target medicine composition.In one embodiment of the invention, the toxin gene that the present invention uses is called PEIIImut as Pseudomonas aeruginosa III functional domain mutant (REDLK of C section is mutated into KDEL).It can produce the stronger toxin protein of lethality.The present invention's design is expressed the PEIIImut toxin gene under the regulation and control of the carcinomebryonic antigen CEA of tissue-specific promoter promotor, thereby realizes the controlled expression of PEIIImut.So among the present invention, toxin gene PEIIImut combines section in conjunction with toxin gene with fusion rotein DNA, under the carrying of guide molecule GRP, enters the cell of GRP expression of receptor.But toxin gene can only be at GRP-R under the control of CEA promotor
+And CEA
+Tumour cell in give expression to the PEIIImut toxin protein, thereby kill this kind cell.And at GRP-R
+And CEA
-Normal cell in, because of the PEIIImut gene can't be activated, so that do not have the expression of toxin protein, cell will can not killed and not wounded.Toxin gene can not be imported into GRP-R
-And CEA
+And GRP-R
-And CEA
-Non-tumor cell in.Like this, pharmaceutical composition of the present invention is subjected to guide molecule and promotor " biswitch " dual regulation and control, realized targeting killing effect accurately.This design has been guided out " individuation diagnosis and individualized treatment " necessity, so the indication of this pharmaceutical composition can only be GRP-R
++ CEA
+Tumours such as cancer of the stomach, lung cancer, mammary cancer, carcinoma of the pancreas and colorectal carcinoma.This is a principal character of the present invention.
Description of drawings
Fig. 1 is the overall design drawing of pharmaceutical composition of the present invention.
Fig. 2 is the structure collection of illustrative plates of expression type recombinant chou pGL3-PEIIImut and pGL3-CEA-PEIIImut.
Fig. 3 contains the recombinant chou pET28a-P2G of encoding gene of fusion rotein P2G and the structure collection of illustrative plates of recombinant chou pET28a-P2CG that contains the encoding gene of fusion rotein P2CG.
Fig. 4 cuts qualification result for the enzyme of expression type recombinant chou pGL3-PEIIImut and expression type recombinant chou pGL3-CEA-PEIIImut, and wherein sequence number 1～7 is represented respectively: the 1:HindIII molecular weight marker; The two pGL3 that cut of 2:BamHI-XhoI; The two pGL3CEA-PEIIImut that cut of 3:KpnI-XbaI; The two pGL3 that cut of 4:BamHI-HindIII; The two pGL3-PEIIImut that cut of 5:HindIII-XbaI; 6:CEApromoter PCR reclaims product; The 7:BstNI molecular weight marker.
Fig. 5 cuts qualification result for the enzyme of expression type recombinant chou pET28a-P2G and pET28a-P2CG, and wherein sequence number 1～8 is represented respectively: 1: and λ DNA/HindIII (23130bp, 9416bp, 6551,436Ibp, 2322bp, 2027bp, 564bp, 125bp); 2:pGEX4T-1/BamHI ﹠amp; XhoI (4954bp); 3:P2G-pGEX4T-1/BamHI ﹠amp; XhoI (4954bp, 614bp); 4:P2G-pGEX4T-1/BamHI ﹠amp; XhoI (4954bp, 437bp); 5:P2CG-pET28a/BamhI ﹠amp; XhoI (5328bp, 615bp); 6:P2G-pET28a/BamHI ﹠amp; XhoI (5328bp, 437bp); 7:pET28a/BamHI ﹠amp; XhoI (5328bp); 8:BstNI (1857; 1060; 929; 383; 121).
Fig. 6 cuts qualification result for the enzyme of expression vector pET30a-TrxA-Th-P2G, and wherein sequence number 1～5 is represented respectively: the PCR product (351bp) of 1:TrxA; The 2:DNA molecular weight marker (500,400,300,200,100bp); 3:TrxA-pET30a/BamHI ﹠amp; NdeI (351bp, 5273bp); 4: λ DNA/HindIII ﹠amp; EcoRI (21226,5148,4973,4268,3530,2027,1904,1584,1375,947,831,564,125bp); 5:TrxA-Th-P2G/pET28a (828bp, 5234bp).
Fig. 7 A is that pET28a-P2G and pET28a-P2CG engineering bacteria are induced back protein expression collection of illustrative plates, 1: inductive P2G/pET28a not; 2-4: inductive P2G/pET28a; 5 and 8: inductive P2CG/pET28a; 6: inductive P2CG/pET28a not; 7: molecular weight protein marker (97.4kD, 66.2kD, 43kD, 31kD, 20.1kD, 14.4kD); 9 and 11: inductive P2CG/pET30a; 10: inductive P2CG/pET30a not.Wherein arrow illustrates the P2G and the P2CG of expression.Fig. 7 B is that the pET30a-TrxA-Th-P2G engineering bacteria is induced back protein expression collection of illustrative plates; 1: inductive pET30a-TrxA-Th-P2G engineering bacteria, 2: molecular weight protein marker (97.4kD, 66.2kD, 43kD, 31kD, 20.1kD, 14.4kD), 3: inductive pET30a-TrxA-Th-P2G engineering bacteria.
Fig. 8 is the figure that kills and wounds of MKN45 cell.1: blank; 2:8ug/mlP2G/LIpofectamin 3:8ug/ml CEA-PEIIImut; 4:8ug/ml DYPII; 5:8ug/ml P2G/Lipofectamin/pG13; 6-8 P2G/Lipofectamin/CEA-PEIIImut dosage is respectively: 8,4, and 2ug/ml; 9-11 P2G/Lipofectamin/DYPII dosage is respectively: 8,4, and 2ug/ml.
Fig. 9 is the figure that kills and wounds of MCF-7 cell, 1: blank; 2-4:P2G/Lipofectamin/CEA-PEIIImut pharmaceutical composition dosage is respectively: 8,4, and 2ug/ml; 5-7:P2G/Lipofectamin/DYPII dosage is respectively: 8,4, and 2ug/ml.
Figure 10 is the figure that kills and wounds of MDA-MB-231 cell, 1 blank; 2:8ug/mlP2G/LIpofectamin 3:8ug/ml CEA-PEIIImut; 4:8ug/ml DYPII; 5:8ug/ml P2G/Lipofectamin/pG13; 6-8 P2G/Lipofectamin/CEA-PEIIImut dosage is: 8,4, and 2ug/ml; 9-11 P2G/Lipofectamin/DYPII dosage is: 8,4, and 2ug/ml.
Figure 11 is the figure that kills and wounds of A549 cell, 1: blank; 2:8ug/ml P2G/Lipofectamin3:8ug/ml CEA-PEIIImut; 4:8ug/ml DYPII; 5:8ug/mlP2G/Lipofectamin/pG13; 6-8:P2G/Lipofectamin/CEA-PEIIImut dosage is respectively: 8,4, and 2ug/ml; 9-11:P2G/Lipofectamin/DYPII dosage is respectively: 8,4, and 2ug/ml.
Figure 12 is the figure that kills and wounds of Hela cell, 1: blank; 2:8ug/ml P2G/Lipofectamin3:8ug/ml CEA-PEIIImut; 4:8ug/ml DYPII; 5:8ug/mlP2G/Lipofectamin/pG13; 6-8:P2G/Lipofectamin/CEA-PEIIImut dosage is respectively: 8,4, and 2ug/ml; 9-11:P2G/Lipofectamin/DYPII dosage is respectively: 8,4, and 2ug/ml.
Figure 13 is MCF-7 apoptosis detection figure.
Figure 14 is experiment flow figure of the present invention.
Embodiment
Overall design of the present invention and experiment flow are referring to Fig. 1 and shown in Figure 14.
According to a first aspect of the invention, the invention provides a kind of non-virus carrier, its essence is a kind of target fusion rotein, described fusion rotein by the receptors ligand polypeptide be rich in positive amino acid whose polypeptide and form.In this another embodiment on the one hand of the present invention, non-virus carrier of the present invention is made up of receptors ligand polypeptide, the polypeptide that is rich in positive amino acid whose polypeptide and concentration of DNA.According to the present invention, described to be rich in positive amino acid whose polypeptide be that the DNA of two placed in-line Protein-tyrosine-phosphatases (PTP) is in conjunction with section or its associated clip; The polypeptide of concentration of DNA is that the proteic DNA of c-myc is in conjunction with section; Described receptors ligand polypeptide be can with gastrin releasing peptide receptor (GRP-R) specificity bonded gastrin release peptide (GRP) or its associated clip, analogue or mutant.Because use GRP as guide molecule, so the inventor is called G type non-virus carrier with non-virus carrier of the present invention, non-virus carrier of the present invention is to the human body non-immunogenicity.
Preferably, the encoding gene of above-mentioned receptors ligand polypeptide GRP is the mutant nucleotide sequence that comprises the intestinal bacteria preference codon, as SEQ ID NO:1; Perhaps also can be its wild-type dna sequence dna, as SEQ ID NO:2.
Preferably, in the described non-virus carrier be rich in positive amino acid whose polypeptide be the DNA of two the proteic 332-388 amino acids of placed in-line PTP representatives in conjunction with section, described 332-388 amino acids sequence is: TGLSSKMQDTMEENSESALRKRIRGDRKATTAQKVQQMKQRLNENERKRKRPRLTD T.The dna sequence dna of this section of encoding is the mutant nucleotide sequence that comprises the intestinal bacteria preference codon, the sequence shown in SEQ ID NO:3; Perhaps also can be its wild-type dna sequence dna, shown in SEQ ID NO:4.
Preferably, the polypeptide of the concentration of DNA in the described non-virus carrier be the DNA of c-myc albumen 265-318 amino acids representative in conjunction with section, its aminoacid sequence is: VSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNTAAPPSTRKD.Preferably, the dna sequence dna of this section of encoding is the mutant nucleotide sequence that comprises the intestinal bacteria preference codon, the sequence shown in SEQ ID NO:5; Perhaps also can be its wild-type dna sequence dna, shown in SEQ ID NO:6.
In an especially preferred embodiment, non-virus carrier of the present invention contains 27 amino acid whose gastrin release peptides by the DNA of aforesaid two the proteic 332-388 amino acids of placed in-line PTP representatives in conjunction with section and one from N-terminal to C-terminal and forms, described non-virus carrier called after P2G, its aminoacid sequence is shown in SEQ ID NO:7, it contains 33 basic aminoacidss, is with 24 positive charges.The intestinal bacteria hobby type dna sequence dna of coding P2G and wild-type dna sequence dna are respectively shown in SEQ ID NO:8 and SEQID NO:9.
In another particularly preferred embodiment, non-virus carrier of the present invention is formed in conjunction with section and people's gastrin release peptide in conjunction with the DNA of section, a c-myc albumen 265-318 amino acids representative from N-terminal to C-terminal by the DNA of aforesaid two the proteic 332-388 amino acids of placed in-line PTP representatives, described non-virus carrier called after P2CG, its aminoacid sequence is shown in SEQ ID NO:10, it contains 42 positive amino acid, is with 20 positive charges.The intestinal bacteria hobby type dna sequence dna of coding P2CG and wild-type dna sequence dna are respectively shown in SEQ ID NO:11 and SEQ ID NO:12.
Preferably, obtain fusion gene in vitro recombination, and in prokaryotic cell prokaryocyte or eukaryotic cell, express this fusion gene and obtain non-virus carrier of the present invention (fusion rotein) by gene engineering method.For example in one embodiment of the invention, described fusion gene is recombinated with expression vector pET28a respectively, respectively called after pET28a-P2G and pET28a-P2CG; The P2G fusion gene also can obtain recon pET30a-TrxA-Th-P2G with the reorganization of pET30a-TrxA expression vector in addition.With above-mentioned plasmid transformed into escherichia coli DH5 α respectively, the engineering bacteria that acquisition can expressed fusion protein.
On the other hand, the invention provides the pharmaceutical composition of the mixture that comprises non-virus carrier of the present invention and a kind of expression type DNA recombinant chou, to be used for the gene therapy of target malignant tumour, described expression type recombinant chou comprises expresses the proteinic gene that pair cell has kill capability.
Preferably, the protein that wherein said pair cell has kill capability is pseudomonal toxin, or its second and the 3rd structural domain; More preferably, described pair cell has the 3rd structural domain or its mutant that the protein of kill capability is pseudomonal toxin.
Pseudomonal toxin (PE) is by a kind of toxin of Pseudomonas aeruginosa excretory, and it is a kind of virulent toxin, only can make it dead in 24 hours to intravenous injection 0.3 microgram of mouse.The importing of the toxin of several molecule can make this necrocytosis concerning a cell.Because this particular performances of PE makes it be applied in the genetic treatment of tumor as a kind of up-and-coming toxin and goes.PE has three structural domains, structural domain Ia (AA1-252) is the cell binding domains, domain II I (AA405-613) is ADP glycosylation zone, and domain II and domain II I are collectively referred to as PE40 again, and structural domain Ib (AA365-404) does not have tangible biological function.
That the present invention uses is toxin gene PEIII
MutExpression recombinant.Preferably, described expression type recombinant chou is the expression type recombinant chou pGL3-PEIII that makes up based on pGL3 (Promega)
MutOr the recombinant expressed type recombinant chou of the eucaryon pGL3-CEA-PEIII that controlled by the CEA promotor
MutOr the recombinant expressed type recombinant chou of the eucaryon pGL3-CEA-IntronII-PEIIImut that controlled by CEA promotor and rabbit globin second intron.
Described expression type recombinant chou combines the component combination with the DNA of non-virus carrier of the present invention, and is imported in the cell of GRP acceptor (GRP-R) expression by specificity under the effect of guide molecule GRP, expresses in this kind cell, kills and wounds this kind tumour cell.Be imported into for fear of toxin gene in the cell of normal expression GRP-R, thereby give expression to toxin protein and kill and wound normal cell, cause toxic side effect, as mentioned above toxin gene PEIIImut is connected the downstream of the CEA of tissue-specific promoter promotor (carcinoembryonic antigen promoter), regulates and control its expression.Because only in the tumour cell that CEA expresses, just have some specific trans controlling element, can interact with the cis element in the CEA promotor, promotor is activated, and then starts the expression of PEIIImut toxin gene.And in the cell that CEA does not express, activate the necessary trans factor of promotor owing to lacking, and even the pGL3-CEA-PEIIImut recombinant chou is imported into normal cell, but can not start the expression of downstream gene because of promotor, normal cell can not killed and not wounded yet.Whether GRP-R and CEA express can be detected by trans PCR; In some tumour, CEA can appear in the blood, so also can adopt the expression of the common method detection CEA in the clinical tumor diagnosis.
Therefore, in an embodiment preferred of the present invention, provide the recombinant chou of the PEIIImut expression type under the CEA promoter regulation, the CEA promoter sequence in this recombinant chou is shown in SEQ ID NO.:14.CEA promoter sequence and PEIIImut encoding sequence can be inserted a kind of PEIIImut expression recombinant pGL3-CEA-PEIII of acquisition among the carrier for expression of eukaryon pGL3 commonly used according to technology well known in the art
MutDescribed expression recombinant is used to transform proper host cell such as bacillus coli DH 5 alpha and keeps.
In an embodiment preferred of the present invention, the pharmaceutical composition that is used for the malignant tumour target gene therapy is provided, it comprises the mixture of above-mentioned expression type recombinant chou and non-virus carrier.In this application that is used for the medicine of targeting gene therapy of the present invention, comprise PEIII
MutExpression type recombinant chou and non-virus carrier of the present invention in can be in conjunction with the component of DNA, the fusion sequence that is people PTP DNA land or PTP DNA land and c-myc DNA land is combined closely, guiding component GRP in the fusion rotein by combining with the GRP-R on malignant cell (comprising MKN45 cell, MCF-7 cell etc.) surface with in the mixture transfered cell, PEIII in this cell
MutThe expression type recombinant chou is expressed toxin protein, thereby cell is killed.
Specifically,, in the cell that can express CEA, express, therefore, just do not need the transmembrane transport structural domain of toxin because pharmaceutical composition of the present invention is that toxin gene is imported the malignant cell that GRP-R expresses.Just because of this reason, toxin gene PEIII of the present invention
MutIn only contain the encoding sequence with the active III structural domain of ADP glycosylase of pseudomonal toxin.The albumen that this encoding sequence gives expression in target cell can be used as the toxin that kills target cell.In addition, 4 amino-acid residue KDEL that the inventor holds with diphtheria toxin C have substituted 5 amino-acid residue REDLK of PE toxin C end, have strengthened the cell killing ability of toxin thus greatly.In addition, because toxin protein is to express in cell, this has just thoroughly been avoided the immunogenicity problem of toxin protein.And because the toxic side effect of toxin protein to the cell of normal expression GRP-R avoided in the biswitch design of CEA and GRP-R.
Use in the pharmaceutical composition of the present invention that with GRP-R the part GRP of high-affinity to be arranged be guide molecule, needs for high expression level, based on bacterium consideration to the preferences of genetic codon when the expressing protein, when construction expression type recombinant chou, it is combined section, c-myc DNA be the suitableeest or suitable codon with PTP DNA in conjunction with naturally occurring codon mutation in the section.Adopt gradation PCR and long primer PCR splicing, obtained required target gene fragment.For the ease of the operation of the reorganization between targeting part in the fusion rotein and the DNA bound fraction, in P2G, two PTP DNA connect by the cut of DraI enzyme in conjunction with section in specific implementation process; Two PTP DNA connect, link to each other, are connected by NdeI with GRP by EcoRI in conjunction with section with c-myc DNA by the cut of DraI enzyme in conjunction with section among the P2CG.
Non-virus carrier of the present invention utilizes a part in the conjugated protein molecule of DNA of normal presence in the human body as the functional zone in conjunction with DNA.The PTP fragment that is adopted contains 32 positive amino acid after series connection, this polypeptide fragment is positively charged under certain pH conditions, can combine with electronegative DNA by electrostatic interaction.The proteic DNA of C-myc is not positively charged in conjunction with section, combines with DNA by specific sequence signature, has the effect of concentration of DNA.Like this She Ji two kinds of fusion roteins both can in conjunction with and wrap up dna molecular and guaranteed the reduced immunogenicity of fusion rotein and combination drug and can be simultaneously by the target cell endocytosis.
Further describe the present invention by the following examples.
The structure of embodiment 1 PEIIImut expression type recombinant chou
1, the structure of expression type recombinant chou
Design CEA promotor (Pcea) specific PCR amplimer, from genomic dna, amplify the promoter sequence of 204bp.Primer sequence is as follows:
Upstream (31bps): 5 ' cg ctc gag tgg aga gca tgg gga gac ccg gg 3 '
Downstream (30bps): 5 ' cg cca tgg gtc tct gct gtc tgc tct gtc c 3 '
The PCR reaction conditions is: 94 ℃ of sex change begin circulation after 3 minutes: 94 ℃ of sex change 40 seconds, and 60 ℃ of annealing 40 seconds, 72 ℃ were extended 40 seconds.After 35 circulations, 72 ℃ were extended 10 minutes.The amplification enzyme that uses is the high-fidelity Pfu of Promega company (Madison, Wisconsin, the U.S.).Agarose gel electrophoresis identifies that the purpose clip size of pcr amplification is correct, cuts glue and reclaims small segment, reclaims fragment (Hereinafter the same) with glass milk purification kit purifying.The recovery fragment that takes a morsel, and in system, add dNTP, the Taq enzyme, 10X Taq damping fluid, 70 ℃ of reaction 30min utilize the Taq enzyme after the characteristic that PCR product end adds A adds the A reaction, with the purpose fragment cloning to the T carrier.Connexon transforms DH5 α competent escherichia coli cell.Be coated on the LB agar plate that contains penbritin (100 μ g/ml), utilize blue blank screening positive clone.Picking hickie transformant is according to " molecular cloning experiment guide " (second edition) (J. Sa nurse Brooker, Deng. the chief editor, Jin Dongyan etc. translate, Science Press, 1992) alkaline lysis prepares the plasmid DNA of resistance clone in a small amount, and restriction analysis confirms that plasmid inserts clip size and conforms to goal gene.Cut evaluation with XhoI and HindIII are two, positive transformant T-Pcea should cut out small segment and the big fragment of 3kb of 216bp.Reclaim small segment.Handle pGL3-promoter with identical enzyme, the big fragment and the 219bp small segment that obtain 4791bp reclaim the big fragment of carrier.Pcea is cloned in the big fragment of carrier.XhoI and HindIII are two to be cut and will decide transformant, and positive transformant pGL3-Pcea should cut out the big fragment of small segment and the 4791bp of 216bp.
With EcoRI and the two recon pA-PEIIImut that cut of EcoRv, reclaim the purpose fragment PEIIImut of 649bp.With the two carrier pBSKS that cut of identical enzyme, reclaim the big fragment of carrier.PEIIImut is cloned on this carrier.Connexon transforms DH5 α competent escherichia coli cell.Be coated on the LB agar plate that contains penbritin (100 μ g/ml), utilize blue blank screening positive clone.Picking hickie transformant is according to " molecular cloning experiment guide " (second edition) (chief editor such as J. Sa nurse Brooker, Jin Dongyan etc. translate, Science Press, 1992) alkaline lysis prepares the plasmid DNA of resistance clone in a small amount, restriction analysis confirms that plasmid inserts conform to the goal gene transformant enzyme of picking hickie of clip size and cuts evaluation, can excise the segmental positive transformant pBSKS-PEIIImut of purpose of 649bp.Go up the two 675bp of cutting-out PEIIImut fragments with XbaI and HindIII from pBSKS-PEIIImut, it is cloned on the identical restriction enzyme site of pGL3-Pcea.Connexon transforms DH5 α competent escherichia coli cell.Be coated on the LB agar plate that contains penbritin (100 μ g/ml), utilize blue blank screening positive clone.The picking transformant is according to " molecular cloning experiment guide " (second edition) (J. Sa nurse Brooker, Deng. the chief editor, Jin Dongyan etc. translate, Science Press, 1992) alkaline lysis prepares the plasmid DNA of resistance clone in a small amount, and restriction analysis confirms that plasmid inserts clip size and conforms to goal gene.Cut the fragment that evaluation should obtain 924bp and 3081bp with KpnI and XbaI are two.Positive recombinant called after pGL3-Pcea-PEIIImut.Handle carrier pGL3 with XbaI and two the cutting of HindIII, PEIIImut is inserted wherein.Cut the fragment that evaluation should obtain 3321bp and 687bp with same enzyme is two.Positive recombinant called after pGL3-PEIIImut.Building process and enzyme are cut and are identified collection of illustrative plates such as Fig. 2 and 4.
The acquisition of the mutator gene of embodiment 2 coding GRP
(gastrin releasing peptide GRP) is made up of 27 amino acid the gastrin release peptide.The encoding sequence of its 1-23 amino acids is positioned at first exon of gastrin release peptide gene, is positioned at second exon and remain 4 amino acid.According to this sequence signature and take into account escherichia expression system codon hobby property and design inside and outside two pairs of upstream and downstream primers, can from genomic dna, go out purpose GRP encoding sequence (comprising the long altogether 99bp of terminator codon and restriction enzyme site) by pcr amplification.The upstream inner primer comprises the encoding sequence of 3-12 amino acids, simultaneously the 7th, 8 the original codon GGA of Gly is changed into the GGT of intestinal bacteria hobby.Introduce NdeI restriction enzyme enzyme recognition site in the outer primer of upstream, contain the coding region and 20 Nucleotide identical of 1,2 amino acids with the upstream inner primer.The coding section complementation of downstream inner primer and 15-23 amino acids, and change the original codon GGG of 24 amino acids Gly the GGT of intestinal bacteria hobbies into, also comprise the complementary sequence of 25 amino acids codons.Downstream outer primer and downstream inner primer have 21 Nucleotide identical, simultaneously the original codon TTA of 26 amino acids Leu is changed into the CTG of intestinal bacteria hobby, and introduce two placed in-line terminator codons (TGA TAA) and XhoI restriction enzyme enzyme recognition site.Pcr amplification carries out in two steps, and to be template with the genomic dna amplify the purpose fragment of long 63bp with the upstream and downstream inner primer to the first step, cuts glue and reclaim this fragment of purifying; The PCR fragment of second step with purifying is that template is carried out the PCR reaction with the upstream and downstream outer primer, amplifies the purpose fragment that contains NdeI and BamHI restriction enzyme site and two terminator codons.Final PCR product is cut glue and is reclaimed after agarose gel electrophoresis identifies that size is correct.Cut transition vector pBS-KS with the EcoRV enzyme, cut glue and reclaim the linearizing carrier segments, be connected with the GRP purpose fragment that reclaims.Connexon transforms DH5 α competent escherichia coli cell.Be coated on the LB agar plate that contains penbritin (100 μ g/ml), utilize blue blank screening positive clone.Picking hickie transformant is according to " molecular cloning experiment guide " (second edition) (J. Sa nurse Brooker, Deng. the chief editor, Jin Dongyan etc. translate, Science Press, 1992) alkaline lysis prepares the plasmid DNA of resistance clone in a small amount, and restriction analysis confirms that plasmid inserts clip size and conforms to goal gene.The positive colony called after obtains cloning pBS-KS-GRP.The sequencing result demonstration is inserted fragment except that the codon of revising consistent with the GRP encoding sequence that GenBank provides.
The primer that uses is as follows:
Inner primer:
Upstream (33bps): 5 ' ctg cct gcg ggc ggt ggt acc gtg ctg acc aag 3 '
Downstream (36bps): 5 ' gtg acc cac cgc cca gtg gtt gcc gcg cgg gta cat 3 '
Outer primer:
Upstream (32bps): 5 '
Gtc ccg ctg cct gcg ggc ggt ggt ac3 '
NdeI
Downstream (39bps): 5 '
Tta tca cat cag gtg acc cac cgc cca gtg gtt 3 '
XhoI
The process of inner primer amplification is: 94 ℃ of sex change begin circulation after 3 minutes: 94 ℃ of sex change 40 seconds, and 59 ℃ of annealing 40 seconds, 72 ℃ were extended 30 seconds.After 35 circulations, 72 ℃ were extended 10 minutes.The condition of external primer amplification is that 94 ℃ of sex change begin circulation after 3 minutes: 94 ℃ of sex change 40 seconds, and 56 ℃ of annealing 40 seconds, 72 ℃ were extended 30 seconds.After 35 circulations, 72 ℃ were extended 10 minutes.The amplification enzyme that uses is the high-fidelity Pfu of Promega company (Madison, Wisconsin, the U.S.).
Particularly, the original wild-type sequence of GRP is shown in SEQ ID NO:2, and the dna sequence dna of the GRP after sudden change is shown in SEQ ID NO.:1.
The acquisition of the mutator gene of embodiment 3 coding PTP DNA lands
In order to obtain the molecular encoding sequence of intestinal bacteria hobby property password, PTP DNA is that method by bridging PCR obtains in conjunction with section (PTPc).PTPc total length 171bp (not containing restriction enzyme site), therefore synthetic 4 (two pairs) big primer (length is about 60bp) carries out the PCR splicing altogether, and in addition synthetic two pairs of little primers (containing different restriction enzyme sites, about long 20bp) use in the segmental a large amount of amplifications of purpose.Have between adjacent big primer that 20-25bp's is overlapping.The introductory note thing is held pairing with 5 ' of upstream and downstream primer respectively.Big primer sequence is as follows, and black matrix partly is the overlap:
Upstream (59bps): 5 ' aca ggt ctt tcc tct aaa atg caa gat aca atg
gag?gag?aac?agt?gag?agt?gct?cta?cg?3’(P1)
Upstream middle-end (60bps): 5 ' agc tgt ggt ggc ctt tct gtc ctc tcg aat acg
ttt?gcg?cag?agc?act?ctc?act?gtt?ctc?3’(P2)
Downstream middle-end (60bps): 5 ' gag gac aga aag gcc acc aca gct cag
aag?gtg?cag?cag?atg?aaa?cag?cgt?ctg?aat?gag?3’(P3)
Downstream (60bps): 5 ' ggt gtc agt cag gcg tgg acg ttt tct ttt acg
ttc?att?ctc?att?cag?acg?ctg?ttt?cat?3’(P4)
Splicing comprises three PCR reactions.P1 and P2 are primer template each other again in first reaction, and pcr amplification obtains the fragment (P12) of 96bp, cuts glue and reclaims this fragment.P3 and P4 are primer template each other again in second reaction, can obtain the fragment (P34) of 99bp, cut glue and reclaim this fragment.In the 3rd PCR reaction, take a morsel P12 and P34 splice, and both are primer template and increasing each other again, can obtain the purpose fragment PTPc of long 171bp.The PCR reaction process is 94 ℃ of sex change 40 seconds, anneals 40 seconds for 55 ℃, and 72 ℃ were extended 30 seconds.After 35 circulations, 72 ℃ were extended 10 minutes.The amplification enzyme that uses is the high-fidelity Pfu of Promega company (Madison, Wisconsin, the U.S.).
Particularly, PTP DNA land native sequences is shown in SEQ ID NO:4, and the dna sequence dna of the PTPDNA land after sudden change is shown in SEQ ID NO.:3.The aminoacid sequence of the DNA land of PTP is: TGLSSKMQDTMEENSESALRKRIRGDRKATTAQKVQQMKQRLNENERKRKRPRLTD T.
For the ease of two PTP sequences and with being connected of c-myc, introduce the BamHI site at the 5 ' end of upstream PTP respectively, 3 ' end is introduced the DraI site; PTP 5 ' end in downstream is introduced the DraI site, and 3 ' end is introduced the EcoRI site.
The acquisition of the mutator gene of embodiment 4 coding c-myc DNA lands
Have the extremely low codon of several intestinal bacteria utilization ratios in the c-myc DNA land (c-myc δ), for fear of its influence to protein expression level, this test intended splices the coding section of intestinal bacteria hobby property by the method for bridging PCR.Coding region segment length 162bp needs synthetic three big primers to splice.Codon in every primer is selected according to the utilization ratio that e. coli codon hobby property table shows, the intestinal bacteria in the original series is not had a liking for codon preferably change the high codon of utilization ratio into.Have the base of 20-25bp overlapping between every primer, sequence following (black matrix representative overlap):
Upstream (60bps): 5 ' gtt tct gtg gaa aag cgc cag gct cct ggc aaa cgc tct gag tct ggc
tcc?cct?tct?gct?3’(P1)
Downstream 1 (60bps): 5 ' gac cag tgg gct gtg agg agg ttt gct gtg gcc acc agc aga agg
gga?gcc?aga?ctc?aga?3’(P2)
Downstream 2 (90bps): 5 ' gtc ctt gcg agt gga cgg agg cgc tgc gta gtt gtg ctg atg
ggt?gga?cac?gtg?gca?gcg?ctt?gag?gac?cag?tgg?gct?gtg?agg?agg?ttt?3’(P3)
The process of whole splicing comprises the twice PCR reaction.P1 primer and P2 primer are spliced, and promptly the two is a primer template each other again in the PCR reaction system.First PCR reaction can obtain the PCR product (P12) of long 96bp, and cut glue and reclaim the gained fragment, and the template of P12 as second PCR reaction that take a morsel.P12 in second reaction system with P3 template each other, carry out the splicing second time.Reaction system and first PCR reacting phase seemingly only need to substitute P1 with P12; Reaction process is identical.Splicing promptly can obtain the purpose fragment of long 162bp through twice PCR.The purpose fragment that a pair of little primer is used to increase and splices has been synthesized in this test simultaneously, and sequence is as follows.For the ease of C-myc δ being inserted in the encoding sequence of fusion rotein, introduce restriction enzyme site EcoRI and NdeI (seeing the line sequence) respectively at 5 ' end of 5 ' and 3 ' little primer:
5 ' end primer (27bps): 5 '
Gaa ttcGtt tct gtg gaa aag cgc cag 3 '
EcoRI
3 ' end primer (27bps): 5 '
Cat atgGtc ctt gcg agt gga cgg agg 3 '
NdeI
The final PCR product that obtains is long 174bp, contains the intestinal bacteria hobby property C-myc δ encoding sequence of EcoRI and NdeI restriction enzyme site.Splicing PCR process is 94 ℃ of sex change 40 seconds, anneals 40 seconds for 55 ℃, and 72 ℃ were extended 30 seconds.After 35 circulations, 72 ℃ were extended 10 minutes.Introductory note thing amplification procedure is: 94 ℃ of sex change 3min.94 ℃ of sex change 40 seconds, 55 ℃ of annealing 40 seconds, 72 ℃ were extended 30 seconds, and after 35 circulations, 72 ℃ were extended 10 minutes.The amplification enzyme that uses is the high-fidelity Pfu of Promega company (Madison, Wisconsin, the U.S.).
Shown in the original coding sequence SEQ ID NO:6 of c-myc DNA land, intestinal bacteria hobby type sequence is shown in SEQ ID NO:5.
The structure of embodiment 5 pET28a-P2G, pET28a-P2CG recombinant chou
5.1 the structure of pGEX4T-1-P2G and pGEX4T-1-P2CG
With corresponding restriction enzyme various fragments of GRP, PTPc and C-myc DNA land encoding sequence are scaled off from transition vector, and cut glue and reclaim.Cut carrier pGEX-4T-1 and reclaim the big fragment of carrier with EcoRI and XhoI are two.Simultaneously terminal complementary fragment GRP is connected with the carrier pGEX-4T-1 that handles well with C-myc δ.Connexon transforms DH5 α competent escherichia coli cell.Be coated on the LB agar plate that contains penbritin (100 μ g/ml).The picking transformant is according to " molecular cloning experiment guide " (second edition) (J. Sa nurse Brooker, Deng. the chief editor, Jin Dongyan etc. translate, Science Press, 1992) alkaline lysis prepares the plasmid DNA of resistance clone in a small amount, identify that with EcoRI and two the cutting of XhoI the transformant of selecting, positive transformant should cut out the big fragment of size for the small segment of 267bp and 4963bp.Choose positive transformant pGEX4T-1-CG and carry out next step clone.Cut pGEX4T-1-CG and reclaim big fragment with BamHI and EcoRI are two.Simultaneously BamHI-PTPc-DraI is connected with the pGEX4T-1-CG that handles well with DraI-PTPc-EcoRI.Connexon transforms DH5 α competent escherichia coli cell.Be coated on the LB agar plate that contains penbritin (100 μ g/ml).The picking transformant is according to " molecular cloning experiment guide " (second edition) (J. Sa nurse Brooker, Deng. the chief editor, Jin Dongyan etc. translate, Science Press, 1992) alkaline lysis prepares the plasmid DNA of resistance clone in a small amount, with BamHI and the two transformants of identifying picking of cutting of XhoI, positive transformant should cut out size and be the small segment of 614bp, the big fragment of 4954bp.The clone of P2CG fusion rotein encoding sequence adopts two small segments to be cloned into method on the carrier simultaneously, the ratio of two small segments is 1: 1 in the linked system of using, two small segments add and with the big segmental ratio of carrier be 1: 1-1: 3, linked system is generally 20 μ l, the ligase enzyme consumption is 1-2U, and 16 ℃ of connections are spent the night.
The encoding sequence of P2G fusion rotein is to cut, mend flat method by EcoRI and NdeI to obtain on the basis of P2CG sequence.All form 3 ' recessed end after EcoRI and the NdeI cutting, mend the connection of flat back with the big fragment of Klenow and can not cause the codon displacement, so do not change aminoacid sequence.Transformant is cut evaluation with BamHI and XhoI are two, and positive transformant should cut out the big fragment of small segment and the 4954bp of 437bp.Sequencing result shows that clone's P2G and P2CG encoding sequence is entirely true.
The sequence of P2G fusion rotein is seen SEQ ID NO.:7; The sequence of P2CG fusion rotein is seen SEQ ID NO.:10; The sequencing result of encoding gene is seen respectively: SEQ ID NO.:8 and 11.
5.2 the structure of pET28a-P2G and pET28a-P2CG recon
Cut pGEX4T-1-P2CG with BamHI and XhoI are two, cut glue and reclaim the P2CG small segment.Cut carrier pET28a (5369bp) with identical enzyme is two, cut glue and reclaim big fragment, two fragments are connected.Connexon transforms DH5 α competent escherichia coli cell.Be coated on the LB agar plate that contains penbritin (100 μ g/ml), utilize blue blank screening positive clone.Picking hickie transformant is according to " molecular cloning experiment guide " (second edition) (J. Sa nurse Brooker, Deng. the chief editor, Jin Dongyan etc. translate, Science Press, 1992) alkaline lysis prepares the plasmid DNA of resistance clone in a small amount, cut the evaluation transformant with BamHI and XhoI are two, positive transformant pET28a-P2CG should cut out the big fragment of small segment and the 5328bp of 615bp.Cut pGEX4T-1-P2G with BamHI and XhoI are two, cut glue and reclaim the P2G small segment.Cut carrier pET28a (5369bp) with identical enzyme is two, cut glue and reclaim big fragment, two fragments are connected.Cut the evaluation transformant with BamHI and XhoI are two, positive transformant pET28a-P2G should cut out the big fragment of small segment and the 5328bp of 456bp.Positive transformant pET28a-P2G and pET28a-P2CG are transformed BL21 (DE3) bacterium competence, obtain to express engineering bacteria.The sequence of P2G fusion rotein is shown in SEQ ID NO.:7; The sequence of P2CG fusion rotein is shown in SEQ ID NO.:10; The sequencing result of encoding gene is seen respectively: SEQ ID NO.:8 and 11.
The structure of embodiment 6 pET30a-TrxA-Th-P2G
6.1 PCR obtains the TrxA encoding sequence
With pET32a is template amplification Trx encoding sequence, introduces restriction enzyme site NdeI and BamHI respectively in the upstream and downstream of sequence.At the terminal encoding sequence of introducing the Gly-Ser-Gly-Ser-Gly joint of TrxA sequence, these 5 amino acid can form a kind of flexible joint, are beneficial to the exposure of the Thrombin recognition site of hereinafter mentioning simultaneously.The primer is as follows:
TrxA5 ' primer: CG CAT ATG AGC GAT AAA ATT ATT CAC
TrxA3 ' primer; CG GGA TCC GCC AGA ACC AGA ACC GGC CAG
The PCR reaction conditions is: 94 ℃ of sex change begin circulation after 3 minutes: 94 ℃ of sex change 30 seconds, and 53 ℃ of annealing 40 seconds, 72 ℃ were extended 40 seconds.After 35 circulations, 72 ℃ were extended 5 minutes.The amplification enzyme high-fidelity Pfu that uses.
The purpose clip size that amplifies is 351bp.Reclaim the fragment that electrophoresis identifies that size is correct, be inserted on the linearizing transition vector pBSKS of EcoRv.Connexon transforms DH5 α competent escherichia coli cell.Be coated on the LB agar plate that contains penbritin (100 μ g/ml), utilize blue blank screening positive clone.Picking hickie transformant is according to " molecular cloning experiment guide " (second edition) (J. Sa nurse Brooker, Deng. the chief editor, Jin Dongyan etc. translate, Science Press, 1992) alkaline lysis prepares the plasmid DNA of resistance clone in a small amount, with NdeI and two evaluation, the small segments that recovery positive transformant pBSKS-TrxA downcuts cut of BamHI.Positive transformant identifies that through order-checking the TrxA sequence of pcr amplification is consistent with the sequence that the pET32a carrier provides.With NdeI and the two carrier pET30a that cut of BamHI, reclaim the big fragment of carrier, TrxA is inserted wherein.Cut the evaluation transformant with NdeI and BamHI are two, positive transformant pET30a-TrxA should cut out the big fragment of small segment and the 5273bp of 351bp.The aminoacid sequence of TrxA is:
MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEI
ADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKV
GALSKGQLKEFLDANLAGSGSGGS。
Show that through order-checking the dna encoding sequence of TrxA is shown in SEQ ID NO.:15.
6.2 the structure of pET30a-TrxA-Th-P2G recombinant chou
For the ease of separating of target protein and TrxA, synthetic primer is introduced BamHI site and 7 amino acid whose zymoplasm recognition sites in 5 ' end primer upstream again.With pGEX4T-1-P2CG is that template is carried out pcr amplification.The PCR reaction conditions is: 94 ℃ of sex change begin circulation after 3 minutes: 94 ℃ of sex change 30 seconds, and 56 ℃ of annealing 40 seconds, 72 ℃ were extended 40 seconds.After 35 circulations, 72 ℃ were extended 5 minutes.The amplification enzyme high-fidelity Pfu that uses.The use primer is:
Upstream (49bp): 5 ' ggatccggcgttcgtggtccgcgttctacaggtctttcctctaaaat gc 3 '
Downstream (39bps): 5 ' ctc gag tta tca cat cag gtg acc cac cgc cca gtg gtt 3 '
Electrophoresis is identified the fragment of pcr amplification, and correct size is 477bp.Cut glue and reclaim the purpose fragment, link to each other with the T-carrier after adding A.Connexon transforms DH5 α competent escherichia coli cell.Be coated on the LB agar plate that contains penbritin (100 μ g/ml), utilize blue blank screening positive clone.Picking hickie transformant according to " molecular cloning experiment guide " (second edition) (J. Sa nurse Brooker, etc. chief editor, Jin Dongyan etc. translate, Science Press, 1992) alkaline lysis prepares the plasmid DNA of resistance clone in a small amount, cuts evaluation with BamHI and XhoI are two, should cut out the small segment of 477bp.With BamHI and the two positive transformant T-Th-P2G that cut of XhoI, reclaim the purpose small segment.Small segment is inserted among the pET28a, cut evaluation with BamHI and XhoI are two again, positive transformant should cut out the big fragment of small segment and the 5328bp of 477bp.Positive transformant is transformed BL21 (DE3) bacterium competent cell, obtain to express engineering bacteria.Contain the P2G sequence of zymoplasm recognition site shown in SEQ ID NO.:16 through the order-checking demonstration.
1. the expression of target protein and evaluation
Engineering bacteria is inoculated in 5ml LB substratum respectively, adds the kantlex that final concentration is 50mg/ml simultaneously, 37 ℃ of jolting overnight incubation.Respectively be inoculated in two pipe 5ml LB/Kan+ nutrient solutions by 1% inoculum size next day.Be cultured to OD600 37 ℃ of following joltings and be about 0.4-0.6.Respectively getting a pipe adding final concentration is the IPTG (Takara company product) of 0.6mM, continues at 37 ℃ of shaking culture 3-4h with another pipe; Contrast is not induced in the conduct that does not add IPTG.The centrifugal collection thalline of 4000g adds sample-loading buffer (50mmol/L Tris-HCl, pH6.8,100mmol/L DTT, 2% SDS, 0.1% tetrabromophenol sulfonphthalein, 10% glycerine), and mixing places 100 ℃ of boiling water baths to keep 5min.The centrifugal 1min of 10000g gets supernatant and carries out polyacrylamide gel electrophoresis (SDS-PAGE) analysis.Target protein matter expression amount scans in 626nm wavelength place by Chromscan3 PhastGel scanner, and the calculation expression amount.Detailed method is referring to " modern molecular biology experimental technique " second edition referring to the Lu Shengdong chief editor, 365-387 page or leaf.
2. result:
Analyze demonstration through SDS-PAGE, the expressed target protein of pET28a-P2CG engineering bacteria has tangible protein band to occur at about 30kD place, more bigger than the molecular weight that theoretical aminoacid sequence is calculated.And contrast empty carrier, contain recon but inductive equal this band not on the corresponding position not, tentatively predicate desired target protein.Analyze through the laser scanning of SDS-PAGE gel, target protein accounts for 23% of total protein of cell.Use the same method, engineering bacteria pET28a-P2G and the expressed albumen of pET30a-TrxA-Th-P2G are carried out SDS-PAGE analysis demonstration, there is protein band to occur at about 20KD and 31KD place respectively, but all big than theoretical calculate molecular weight, and obviously do not take out of existing on the empty carrier corresponding position.Analyze through the laser scanning of SDS-PAGE gel, target protein all accounts for 18% of total protein of cell respectively.
The a large amount of preparations and the purifying of embodiment 8 fusion roteins
1. large volume is cultivated:
Engineering bacteria pET28a-P2G, pET28a-P2CG and pET28a-TrxA-Th-P2G plant respectively and are inoculated in 50ml LB substratum, add the kantlex that final concentration is 50mg/ml simultaneously, 37 ℃ of jolting overnight incubation.Be re-seeded in the 2L triangular flask that contains 500ml LB/Kan+ nutrient solution by 1% inoculum size.Be cultured to OD600 37 ℃ of following joltings and be about 0.4-0.6.The IPTG that adds final concentration 0.6mM then begins to induce, and culture continues to induce 3-4h 37 ℃ of joltings cultivations.4000g is centrifugal, the collection thalline, places-20 ℃ of multigelations.
The frozen thalline that takes a morsel is resuspended in the pH8.0 20mM Tris-HCL damping fluid, has children outside the state plan thalline on ice, and ultrasonic frequency is unsuitable big, and every 10sec intermittently once surpasses three times altogether.The centrifugal 10min of 12000g gets supernatant, the evaluation of precipitation row SDS-PAGE electrophoresis.Find that pET28a-P2G and pET28a-P2CG are inclusion body and express; And pET30a-TrxA-Th-P2G is the supernatant expression.
2. the preparation of inclusion body
2.1 washed cell:
6000g * 15 minute centrifugal collection bacterium, the wet thallus of weighing.With 3%Triton X-100 washing thalline, stirred evenly on the agitator 30 minutes, 6000g * 15 a minute centrifugal collection consumption is 10ml-50ml damping fluid/1g wet thallus (comprising following various lavation buffer solution).
2.2 broken cell
With cell suspension, damping fluid is prepared: 50mmol/LTris.Cl (pH8.5-9.0), 2mmol/L EDTA, 100mmol/L NaCl, 0.5% Triton X-100, N,O-Diacetylmuramidase 1mg/ml with damping fluid).At room temperature stir with agitator, make the effect thickness of suspension because of N,O-Diacetylmuramidase.
2.3 supersound process
Each super making a call to 10 seconds, interval 15 seconds, 20-50 secondary action (deciding on cell concentration) is to thalline no longer till the thickness.Centrifugal then collection, 6000g * 15 minute.Aforesaid operations should carry out in frozen water, and for preventing charing, ultrasonic power is unsuitable excessive, carries out in glass beaker better, and ultrasonic probe gos deep into liquid level greater than 3 centimetres, and is good apart from cup 2 centimetres of positions, the end.
2.4 5%Triton X-100 washing
With the damping fluid precipitation that thoroughly suspends, stir and ultrasonication 30 times, referring to above-mentioned steps 2.3.Damping fluid is: 50mmol/L Tris.Cl (pH8.0), 2mmol/L EDTA, 5%TritonX-100.Centrifugal collecting precipitation, 6000g * 15 minute.
2.5 the preservation of inclusion body and dissolving:
2.5.1 dissolve inclusion body gradually with a small amount of 8M urea, to get 20 μ l solution and add the 2X sample-loading buffer, mixing boiled in the boiling water 3-5 minute.Get 20ul and carry out the SDS-PAGE electrophoretic analysis.All the other inclusion bodies can be frozen in-70 ℃.
If 2.5.2 cross column purification, should operate by following step: with containing body lysate (50mmol/LTris.Cl, pH8.0,2mmol/L EDTA, 10mMDTT, 7M Guanidinium hydrochloride) dissolving inclusion body, inclusion body concentration is about 10mg/ml.With dissolved inclusion body 12000g centrifugal after, get supernatant, obtain can upper prop sample solution.
2.5.3 go up albuminised preparation
Damping fluid 50mM Tris-HCl, the 10mMEDTA, the 100mM NaCl that add 1/20 volume in the frozen again thalline, pH8.5, resuspended thalline.Behind the complete mixing of thalline, place and carry out carrying out ultrasonic bacteria breaking on ice, method is the same.After bacterium liquid was limpid, in 12000g, 4 ℃ centrifugal 15 minutes.Collect supernatant liquor, in 4 ℃ to the initial damping fluid of cation seperation column (0.03M Na
2HPO
3, pH6.5) dialysis is crossed column purification in order to next step.
3. the purifying of target protein
3.1 inclusion body protein cut glue purification
Get inclusion body solution prepared in 2.3 steps, add in the equal-volume 2x sample-loading buffer, carry out 15% SDS-PAGE electrophoresis.
A. gel cutting: downcut the adhesive tape and the small part sample adhesive tape at albumen Marker swimming lane place, put it into the rapid dye liquor middling speed and dyed 15 minutes, the residue gel is wrapped with preservative film and is placed 4 ℃ of refrigerators.After band is showed in the adhesive tape decolouring that speed is dyed,, downcut the gel strips that contains the purpose band portion with adhesive tape and the contrast of remaining gel.
B. electroelution: pack into the gel strips of downcutting in the dialysis tubing and place the horizontal strip electrophoresis groove, add the protein electrophoresis damping fluid and carry out electrophoresis.Counterelectrophoresis is 5 minutes behind electrophoresis 2～4h.Damping fluid in the dialysis tubing is poured in the 50ml centrifuge tube, and changed over to another dialysis tubing deionized water dialysis 1 day.Solution after the dialysis changes in the clean 50ml centrifuge tube of another sterilization
C. the removal of trace SDS: add 5 times of freezing acid acetone-methyl alcohol albumen precipitation liquid more than the volume in centrifuge tube ,-20 ℃ of precipitations are spent the night.10, centrifugal 10 minutes of 000r/m removes the supernatant collecting precipitation.The precipitation of collecting is washed 1 time with albumen precipitation liquid.
D. the preservation of albumen precipitation: dry up albumen precipitation with nitrogen.Precipitation dry powder is put-20 ℃ of refrigerators and can be preserved 1 month.
3.2 go up albuminised chromatography purification
3.2.1 SP Sepherose cationic exchange coloum
After the supernatant protein sample that dialysis is good is crossed 0.22 μ m filter membrane, with cationic exchange coloum (Hitrap
TM1ml SP Sepherose
TMFF Pharmacia) carries out the first step purifying.Earlier with initial damping fluid (0.03M Na
2HPO
3, pH6.5) a balance pillar 5-10 column volume is gone up sample then.Each sample 35ml that goes up.Still wash pillar (2-5 column volume) before the wash-out and wash pillar with initial damping fluid.Rise gradient 0-25%, 2540%, 40-100% and carry out wash-out, wash-out 3-5min under each gradient.Elution buffer is 0.03M Na
2HPO
3/ 1M NaCl.Individual elution peak is identified through the SDS-PAGE electrophoresis.Target protein is at second gradient 25-40%, and electricity is led the complete wash-out into 29.4-43%ms/cm.
3.2.2 the cutting of zymoplasm
To pH8.0,50mM Tris-HCl, 75mM NaCl damping fluid dialyse to salt ionic concentration and pH and cushion liquid phase together with the first step purified product.In protein sample, add the ratio of 2U zymoplasm, add an amount of zymoplasm, 28 ℃ of cutting 2-4hr in 1mg albumen.The cutting system enters the second step purifying immediately.
3.2.3 Resource S cationic exchange coloum
After egg white mixture after zymoplasm (thrombin) cutting crossed 0.22 μ m filter membrane, the 50mM Tris-HCl damping fluid dilution that adds pH8.0 got final product Resouce more than one times
TMThe S cationic exchange coloum carries out purifying (Pharmacia).Pillar more than the column volume, can be gone up sample with 5 of same damping fluid balances.Elution buffer is 50mM Tris-HCl/1MNaCl.Rise gradient 0-40%-100% and carry out wash-out.Purpose P2G albumen under the gradient of 40-100% by wash-out.
3.2.4 reversed-phase column
Dilute more than one times with the purpose elution peak of reversed-phase column A damping fluid (10% acetonitrile, 0.05% trifluoroacetic acid), and, can go up sample with behind 5 column volumes of A liquid balance pillar with second post.Pillar (source 15, self-chambering) is through A damping fluid balance.Elution buffer is 90% acetonitrile, 0.1% trifluoroacetic acid.Rise gradient 0-40%, 40-100% and carry out wash-out.Electrophoresis is identified each elution peak.Target protein is at second elution peak wash-out.The freeze-drying target protein is removed acetonitrile and trifluoroacetic acid.Albumen dry powder is frozen stand-by in-80 ℃.
Embodiment 9 target gene medicine P2G or P2CG and pGL3-PEIII
MutOr the preparation of the mixture of pGL3-CEA-PEIIImut or DYPII and to the lethal effect of tumour cell
The preparation of 1 combination drug
Frozen target protein is dissolved in 2M NaCl solution, and 4 ℃ to normal saline dialysis, to the salt ionic concentration balance.Use the BCA kit measurement protein content of Pierce company.Equal-volume adds an amount of various toxin gene expression recombinant, in room temperature reaction 15-30min, combination drug is formed.DNA in the mixture in agarose gel electrophoresis, can occur and be stranded in situation in the well.Wherein the mass ratio of DNA and P2CG is 1: 4; With the ratio of cutting the P2G that glue reclaims be 1: 3; With the ratio of the P2G of chromatography purification be 1: 5.
For strengthen albumen and DNA combination drug tightness, adding quality in 1: 5 DNA/P2G system is the Lipofectamin (Gibcal) of DNA 1/2.
Various combination drugs are crossed 0.22 μ m filter membrane degerming with the cell culture medium dilution that does not contain serum before the transfectional cell.
The killing experiments of 2 combination drugs
Select GRP-R for use
+With CEA
+Stomach cancer cell line MKN45, mammary cancer MCF-7, GRP-R
+With CEA
-Breast carcinoma cell strain MDA-MB-231, select GRP-R for use
-With CEA
+Lung cancer cell line A549, and GRP-R
-With CEA
-Cervical cancer cell strain Hela carry out transfection experiment.MKN45, MCF-7, A549 and Hela use the DMEM substratum that contains 10% foetal calf serum, and the MDA-MB-231 cell strain uses the L15 substratum that contains 10% foetal calf serum, at 37 ℃, cultivates in the incubator of CO2 dividing potential drop 5%.Serum is the product of folium ilicis chinensis company, and substratum is a Gibcal company product.Before the transfection with various cells with 10
4Concentration be inoculated in 96 orifice plates (Costar, Copenhagen, Denmark), be cultured to 80% degree of converging.Discard old substratum, and wash cell 2 times, add various combination drugs with the corresponding substratum that does not contain serum, and independent DNA, independent albumen, the mixture that albumen and pGL3 empty carrier form carries out target and extremely upward tests.The combination drug that contains toxin gene has: cut glue and reclaim the combination drug that P2G forms with pGL3-PEIIImut, pGL3-CEA-PEIIImut respectively; The combination drug that P2CG forms with these two kinds of recombinant chous respectively, the test dose of these four kinds of mixtures is respectively 0.5,1,4 μ g/ml; Cross column purification P2G and form combination drug with pGL3-CEA-PEIIImut, DYPII and an amount of Lipofectamin respectively, using dosage is respectively 2,4,8 μ g/ml.What various control groups used is and the corresponding maximum dose level of combination drug dosage.
Behind the transfection 6-10hr, discard the nutrient solution that contains various medicines, wash the cell both sides, add the substratum that contains 10% foetal calf serum and continue to cultivate 24-32hr with the substratum that does not contain serum.Mtt assay detects viable count, and the T check analysis shows that the combination drug of high dosage is to GRP-R
+With CEA
+Stomach cancer cell line MKN45, mammary cancer MCF-7 cell has intensive killing area effect (P＜0.05), and all the other 3 kinds of cells are not had lethal effect (P＞0.05); Various control groups do not have lethal effect (P＞0.05) yet.Result such as table 1 are shown in the 2-6.
When the combination drug using dosage is 1ug/ml and 4ug/ml, the P2G/pGL3-CEA-PEIIImut combination drug to the MKN45 cell kill rate be: 20.2% and 29.8%; P2CG/pGL3-CEA-PEIIImut to the kill rate of MKN45 is: 14.1% and 26.2%; P2G/pGL3-PEIIImut is respectively 28.4% and 50.5% to the kill rate of MKN45; P2CG/pGL3-PEIIImut is 27.1% and 45.3%.There was no significant difference between P2G mixture and the P2CG mixture kill rate.4 kinds of combination drugs all do not have and kill and wound when 0.5ug/ml.
When the combination drug using dosage was 2ug/ml, 4ug/ml and 8ug/ml respectively, two kinds of combination drug P2G/Lipofectamin/pGL3-Pcea-PEIIImut and P2G/Lipofectamin/DYPII were respectively 27.9%, 39.4%, 48.4% and 30.4%, 38%, 44.4% to the kill rate of MKN45; Kill rate to MCF-7 is 21.1%, 35.8%, 44.1% and 23.5%, 34.9%, 43.7%.The combination drug of various dosage is all to MDA-MB-231, and A549 and Hela cell do not have lethal effect (P＞0.05).Pharmaceutical composition is because toxin protein is expressed due to the trigger cell apoptosis apoptosis such as Figure 13 in born of the same parents to the lethal effect of MCF-7 and MKN45 cell.
The killer cell of table 1 P2G, P2CG and recombinant plasmid, albumen/DNA mixture
Test
| Protein | Clone | Group | Dosage μ g/ ml | ???????????????????????????OD490 | The P value | Lethal effect | |||
| ?1 | ?2 | ?3 | Mean value ± SD | ||||||
| P2G | 2BS | Blank | ?/ | ?0.338 | ?0.312 | ?0.303 | ?0.318±0.02 | ?/ | ?/ |
| pGL3SV40- PEIIImut | ?4 | ?0.325 | ?0.300 | ?0.353 | ?0.326±0.03 | ?0.34 | ?/ | ||
| pGL3CEA- PEIIImut | ?4 | ?0.345 | ?0.321 | ?0.309 | ?0.325±0.02 | ?0.32 | ?/ | ||
| P2G | ?12 | ?0.311 | ?0.327 | ?0.298 | ?0.312±0.01 | ?0.35 | ?/ | ||
| P2G/ pGL3SV40- PEIIImut | ?4 | ?0.324 | ?0.301 | ?0.348 | ?0.324±0.02 | ?0.36 | ?/ | ||
| MK N 45 | Blank | ?/ | ?0.457 | ?0.402 | ?0.439 | ?0.433±0.03 | ?/ | ?/ | |
| pGL3SV40- PEIIImut | ?4 | ?0.439 | ?0.461 | ?0.402 | ?0.434±0.03 | ?0.21 | ?/ | ||
| pGL3CEA- PEHImut | ?4 | ?0.406 | ?0.473 | ?0.418 | ?0.432±0.03 | ?0.50 | ?/ | ||
| P2G | ?12 | ?0.435 | ?0.444 | ?0.416 | ?0.432±0.01 | ?0.49 | ?/ | ||
| P2G/ pGL3SV40- PEIIImut | ?0.5 | ?0.392 | ?0.432 | ?0.414 | ?0.413±0.02 | ?0.19 | ?/ | ||
| ?1 | ?0.332 | ?0.307 | ?0.29 | ?0.310±0.02 | ?0.002 | ?28.4％ | |||
| ?4 | ?0.216 | ?0.234 | ?0.192 | ?0.214±0.02 | ?0.002 | ?50.5％ | |||
| P 2G/ pGL3CEA- PEIIImut | ?0.5 | ?0.407 | ?0.421 | ?0.448 | ?0.425±0.02 | ?0.37 | ?/ | ||
| ?1 | ?0.368 | ?0.347 | ?0.321 | ?0.345±0.02 | ?0.008 | ?20.2％ | |||
| ?4 | ?0.322 | ?0.305 | ?0.284 | ?0.304±0.02 | ?0.0028 | ?29.8％ | |||
| 2BS | blank | ?/ | ?0.321 | ?0.313 | ?0.34 | ?0.325±0.02 | ?/ | ?/ | |
| pGL3SV40- PEIIImut | ?4 | ?0.335 | ?0.3 | ?0.33 | ?0.322±0.02 | ?0.42 | ?/ | ||
| pGL3CEA- PEIIImut | ?4 | ?0.325 | ?0.314 | ?0.319 | ?0.319±0.006 | ?0.30 | ?/ | ||
| ?P2CG | P 2CG | ??12 | ??0.318 | ??0.307 | ??0.3 | ??0.308±0.01 | ??0.09 | ??/ | |
| P2CG/ pGL3SV40- PEIII | ??4 | ??0.328 | ??0.324 | ??0.315 | ??0.322±0.01 | ??0.41 | ??/ | ||
| P2CG/ pGL3CEA- PEIIImut | ??4 | ??0.297 | ??0.312 | ??0.316 | ??0.308±0.01 | ??0.09 | ??/ | ||
| ?MK ?N45 | Blank | ??/ | ??0.448 | ??0.489 | ??0.428 | ??0.455±0.03 | ??/ | ??/ | |
| SV40- PEIIImut | ??4 | ??0.43 | ??0.479 | ??0.408 | ??0.439±0.04 | ??0.30 | ??/ | ||
| CEA- PEIIImut | ??4 | ??0.452 | ??0.438 | ??0.476 | ??0.455±0.02 | ??0.49 | ??/ | ||
| P2CG | ??16 | ??0.442 | ??0.466 | ??0.455 | ??0.454±0.01 | ??0.49 | ??/ | ||
| P2CG/ pGL3SV40- PEIIImut | ??0.5 | ??0.416 | ??0.409 | ??0.421 | ??0.415±0.06 | ??0.077 | ??/ | ||
| ??1 | ??0.342 | ??0.319 | ??0.334 | ??0.332±0.01 | ??0.006 | ??27.1％ | |||
| ??4 | ??0.239 | ??0.254 | ??0.253 | ??0.249±0.008 | ??0.002 | ??45.3％ | |||
| P 2CG/ pGL3CEA- PEIIImut | ??0.5 | ??0.443 | ??0.405 | ??0.471 | ??0.44±0.03 | ??0.30 | ??/ | ||
| ??1 | ??0.385 | ??0.391 | ??0.397 | ??0.391±0.006 | ??0.03 | ??14.1％ | |||
| ??4 | ??0.335 | ??0.329 | ??0.343 | ??0.336±0.007 | ??0.009 | ??26.2％ |
Dosage is meant the concentration of DNA in the mixture.
The cell killing test of table 2 P2G, recombinant plasmid and mixture transfection MKN45
| Clone | Group | Dosage (ug/ml) | ????????????????????OD490 | The P value | Lethal effect % | |||
| ?1 | ?2 | ?3 | Mean value ± SD | |||||
| MKN45 | Blank | / | ?0.803 | ?0.769 | ?0.793 | ?0.79±0.02 | ||
| P2G/ LIpofectamin | 40/8 | ?0.807 | ?0.786 | ?0.824 | ?0.81±0.02 | ?0.31 ?p＞0.05 | ?/ | |
| CEA-PEIIImu t | 8 | ?0.815 | ?0.767 | ?0.763 | ?0.78±0.02 | ?0.75 ?P＞0.05 | ?/ | |
| DYPII | 8 | ?0.8 | ?0.759 | ?0.784 | ?0.78±0.02 | ?0.66 ?P＞0.05 | ?/ | |
| P2G/ Lipofectamin/ pG13 | 40/4/8 | ?0.758 | ?0.787 | ?0.743 | ?0.76±0.02 | ?0.19 ?P＞0.05 | ?/ | |
| P2G/ Lipofectamin/ CEA-PEIIImut | 40/4/8 | ?0.422 | ?0.385 | ?0.403 | ?0.40±0.02 | ?0.00001 ?P＜0.01 | ?48.4 | |
| 20/2/4 | ?0.462 | ?0.491 | ?0.477 | ?0.48±0.01 | ?0.00002 ?P＜0.01 | ?39.4 | ||
| 10/1/2 | ?0.582 | ?0.568 | ?0.561 | ?0.57±0.01 | ?0.0002 ?P＜0.01 | ?27.9 | ||
| P2G/ Lipofectamin/ DYPII | 40/4/8 | ?0.423 | ?0.432 | ?0.408 | ?0.42±0.01 | ?0.00001 ?P＜0.01 | ?44.4 | |
| 20/2/4 | ?0.501 | ?0.497 | ?0.482 | ?0.49±0.01 | ?0.00009 ?P＜0.01 | ?38.7 | ||
| 10/1/2 | ?0.554 | ?0.564 | ?0.56 | ?0.55±0.005 | ?0.001 ?P＜0.01 | ?30.4 | ||
Dosage is meant the concentration of albumen in the mixture, DNA, Lipofectamin, and corresponding order is described in group (group).
The cell killing test of table 3 P2G, recombinant plasmid and mixture transfection MCF7
| Clone | Group | Dosage (ug/ml) | ????????????????????????OD490 | The P value | Lethal effect (%) | |||
| ??1 | ??2 | ??3 | Mean value ± SD | |||||
| MCF7 | Blank | ??/ | ??0.726 | ??0.694 | ??0.707 | ??0.71±0.02 | ||
| ??Lipofectamin/ ??P2G/ ??CEA-PEIIImut | ??4/40/8 | ??0.379 | ??0.41 | ??0.399 | ??0.40±0.02 | ??0.001 ??(P＜0.01) | ??44.1 | |
| ??2/20/4 | ??0.453 | ??0.432 | ??0.482 | ??0.46±0.03 | ??0.001 ??(P＜0.01) | ??35.8 | ||
| ??1/10/2 | ??0.576 | ??0.612 | ??0.496 | ??0.56±0.06 | ??0.03 ??(P＜0.05) | ??21.1 | ||
| ??Lipofectamin/ ??P2G/DYPII | ??4/40/8 | ??0.42 | ??0.386 | ??0.404 | ??0.4±0.02 | ??0.001 ??(P＜0.01) | ??43.7 | |
| ??2/20/4 | ??0.442 | ??0.477 | ??0.452 | ??0.46±0.02 | ??0.002 ??(P＜0.01) | ??34.9 | ||
| ??1/10/2 | ??0.545 | ??0.579 | ??0.502 | ??0.54±0.04 | ??0.005 ??(P＜0.01) | ??23.5 | ||
Dosage is meant the concentration of albumen in the mixture, DNA, Lipofectamin, and corresponding order is described in group.
The cell killing test of table 4 P2G, recombinant plasmid and mixture transfection MDA-MB-231
| Clone | Group | Dosage (ug/ml) | ????????????????????OD490 | The P value | Lethal effect | |||
| ?1 | ?2 | ?3 | ?Mean±SD | |||||
| MDA-MB -231 | Blank | ?0.765 | ?0.732 | ?0.711 | ?0.74±0.03 | |||
| P2G /lipofectamin | ?40/4 | ?0.698 | ?0.667 | ?0.702 | ?0.69±0.01 | ?0.07 ?(P＞0.05) | / | |
| CEA-PEIII | ?4 | ?0.698 | ?0.706 | ?0.755 | ?0.72±0.03 | ?0.53 ?(P＞0.05) | / | |
| DYPII | ?4 | ?0.735 | ?0.687 | ?0.662 | ?0.69±0.04 | ?0.20 ?(P＞0.05) | / | |
| P2G/ Lipofectamin /pGL3 | ?40/4/8 | ?0.759 | ?0.714 | ?0.679 | ?0.72±0.04 | ?0.55(P＞0.05) | / | |
| P2G /Lipofectamin/ CEA-PEIII | ?40/4/8 | ?0.664 | ?0.702 | ?0.767 | ?0.71±0.05 | ?0.51 ?(P＞0.05) | / | |
| ?20/2/4 | ?0.743 | ?0.713 | ?0.772 | ?0.74±0.03 | ?0.79(P＞0.05) | / | ||
| ?10/1/2 | ?0.745 | ?0.712 | ?0.729 | ?0.73±0.03 | ?0.71 ?(P＞0.05) | / | ||
| P2G /Lipofectamin/ DYPII | ?40/4/8 | ?0.687 | ?0.722 | ?0.756 | ?0.72±0.03 | ?0.60 ?(P＞0.05) | / | |
| ?20/2/4 | ?0.743 | ?0.732 | ?0.716 | ?0.73±0.01 | ?0.77(P＞0.05) | / | ||
| ?10/1/2 | ?0.757 | ?0.714 | ?0.736 | ?0.74±0.02 | ?0.98 ?(P＞0.05) | / | ||
Dosage is meant the concentration of albumen in the mixture, DNA, Lipofectamin, and corresponding order is described in group.
The cell killing test of table 5 P2G, recombinant plasmid and mixture transfection A549
| Clone | Group | Dosage (ug/ml) | ??????????????????????OD490 | The P value | Lethal effect | |||
| ?1 | ?2 | ?3 | ?Mean±SD | |||||
| A549 | Blank | ?0.842 | ?0.788 | ?0.826 | ?0.82±0.03 | |||
| P2G /lipofectamin | ?40/4 | ?0.805 | ?0.821 | ?0.822 | ?0.82±0.01 | ?0.89 ?(P＞0.05) | / | |
| CEA-PEIII | ?4 | ?0.792 | ?0.764 | ?0.815 | ?0.80±0.03 | ?0.35 ?(P＞0.05) | / | |
| DYPII | ?4 | ?0.831 | ?0.849 | ?0.825 | ?0.84±0.01 | ?0.43 ?(P＞0.05) | / | |
| P2G/ Lipofectamin /pGL3 | ?40/4/8 | ?0.773 | ?0.786 | ?0.821 | ?0.80±0.02 | ?0.30 ?(P＞0.05) | / | |
| P2G /Lipofectamin/ CEA-PEIII | ?40/4/8 | ?0.768 | ?0.792 | ?0.784 | ?0.78±0.01 | ?0.13 ?(P＞0.05) | / | |
| ?20/2/4 | ?0.836 | ?0.821 | ?0.81 | ?0.82±0.01 | ?0.85 ?(P＞0.05) | / | ||
| ?10/1/2 | ?0.795 | ?0.836 | ?0.745 | ?0.79±0.05 | ?0.44 ?(P＞0.05) | / | ||
| P2G /Lipofectamin/ DYPII | ?40/4/8 | ?0.806 | ?0.824 | ?0.843 | ?0.82±0.02 | ?0.79 ?(P＞0.05) | / | |
| ?20/2/4 | ?0.811 | ?0.843 | ?0.826 | ?0.83±0.02 | ?0.69 ?(P＞0.05) | / | ||
| ?10/1/2 | ?0.775 | ?0.787 | ?0.813 | ?0.79±0.02 | ?0.25 ?(P＞0.05) | / | ||
Dosage is meant the concentration of albumen in the mixture, DNA, Lipofectamin, and corresponding order is described in group.
The cell killing test of table 6 P2G, recombinant plasmid and mixture transfection Hela
| Clone | Group | Dosage (ug/ml) | ??????????????????????OD490 | The P value | Lethal effect | |||
| ?1 | ?2 | ?3 | ?Mean±SD | |||||
| Hela | Blank | ?0.58 | ?0.652 | ?0.662 | ?0.63±0,04 | |||
| P2G /lipofectamin | ?40/4 | ?0.63 | ?0.68 | ?0.597 | ?0.64±0.04 | ?0.91 ?P＞0.05 | ?/ | |
| CEA-PEIII | ?8 | ?0.608 | ?0.574 | ?0.558 | ?0.58±0.02 | ?0.18 ?P＞0.05 | ?/ | |
| DYPII | ?8 | ?0.661 | ?0.589 | ?0.606 | ?0.62±0.03 | ?0.73 ?P＞0.05 | ?/ | |
| P2G/ Lipofectamin /pGL3 | ?40/4/8 | ?0.694 | ?0.616 | ?0.606 | ?0.64±0.05 | ?0.86 ?P＞0.05 | ?/ | |
| P2G /Lipofectamin/ CEA-PEIII | ?40/4/8 | ?0.662 | ?0.656 | ?0.637 | ?0.651±0.01 | ?0.52 ?P＞0.05 | ?/ | |
| ?20/2/4 | ?0.575 | ?0.526 | ?0.542 | ?0.55±0.02 | ?0.06 ?P＞0.05 | ?/ | ||
| ?10/1/2 | ?0.582 | ?0.555 | ?0.54 | ?0.56±0.02 | ?0.09 ?P＞0.05 | ?/ | ||
| P2G /Lipofectamin/ DYPII | ?40/4/8 | ?0.531 | ?0.579 | ?0.552 | ?0.55±0.02 | ?0.07 ?P＞0.05 | ?/ | |
| ?20/2/4 | ?0.522 | ?0.575 | ?0.621 | ?0.57±0.05 | ?0.2P＞0. ?05 | ?/ | ||
| ?10/1/2 | ?0.641 | ?0.572 | ?0.528 | ?0.58±0.06 | ?0.3P＞0. ?05 | ?/ | ||
Dosage is meant the concentration of albumen in the mixture, DNA, Lipofectamin, and corresponding order is described in group.
Sequence table
＜110〉Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
＜120〉G type non-virus carrier and the pharmaceutical composition that comprises it
<130>I20031303CB
<160>16
<170>PatentIn?version?3.1
<210>1
<211>99
<212>DNA
＜213〉artificial sequence
<400>1
catatggtcc?cgctgcctgc?gggcggtggt?accgtgctga?ccaagatgta?cccgcgcggc????60
aaccactggg?cggtgggtca?cctgatgtga?taactcgag???????????????????????????99
<210>2
<211>81
<212>DNA
＜213〉people
<400>2
gtcccgctgc?ctgcgggcgg?agggaccgtg?ctgaccaaga?tgtacccgcg?cggcaaccac????60
tgggcggtgg?ggcacttaat?g??????????????????????????????????????????????81
<210>3
<211>171
<212>DNA
＜213〉artificial sequence
<400>3
acaggtcttt?cctctaaaat?gcaagataca?atggaggaga?acagtgagag?tgctctgcgc?????60
aaacgtattc?gagaggacag?aaaggccacc?acagctcaga?aggtgcagca?gatgaaacag????120
cgtctgaatg?agaatgaacg?taaaagaaaa?cgtccacgcc?tgactgacac?c?????????????171
<210>4
<211>170
<212>DNA
＜213〉people
<400>4
acaggatttc?ctctaaaatg?caagatacaa?tggaggagaa?cagtgagagt?gctctacgga?????60
aacgtattcg?agaggacaga?aaggccacca?cagctcagaa?ggtgcagcag?atgaaacaga????120
ggctaaatga?gaatgaacga?aaaagaaaaa?ggccacgcct?gactgacacc???????????????170
<210>5
<211>174
<212>DNA
＜213〉artificial sequence
<400>5
gaattcgttt?ctgtggaaaa?gcgccaggct?cctggcaaac?gctctgagtc?tggctcccct?????60
tctgctggtg?gccacagcaa?acctcctcac?agcccactgg?tcctcaagcg?ctgccacgtc????120
tccacacatc?agcacaacta?cgcagcgcct?ccgtccactc?gcaaggacca?tatg??????????174
<210>6
<211>161
<212>DNA
＜213〉people
<400>6
gtttctgtgg?aaaagaggca?ggctcctggc?aaaaggtcag?agtctggatc?accttctgct?????60
ggaggccaca?gcaaacctcc?tcacagccca?ctggtcctca?agaggtgcca?cgtctccaca????120
catcagcaca?actacgcagc?gcctcctcca?ctggcaagga?c????????????????????????161
<210>7
<211>148
<212>PRT
＜213〉artificial sequence
<400>7
Arg?Ser?Thr?Gly?Leu?Ser?Ser?Lys?Met?Gln?Asp?Thr?Met?Glu?Glu?Asn
1???????????????5???????????????????10??????????????????15
Ser?Glu?Ser?Ala?Leu?Arg?Lys?Arg?Ile?Arg?Glu?Asp?Arg?Lys?Ala?Thr
20??????????????????25??????????????????30
Thr?Ala?Gln?Lys?Val?Gln?Gln?Met?Lys?Gln?Arg?Leu?Asn?Glu?Asn?Glu
35??????????????????40??????????????????45
Arg?Lys?Arg?Lys?Arg?Pro?Arg?Leu?Thr?Asp?Thr?Phe?Lys?Thr?Gly?Leu
50??????????????????55??????????????????60
Ser?Ser?Lys?Met?Gln?Asp?Thr?Met?Glu?Glu?Asn?Ser?Glu?Ser?Ala?Leu
65??????????????????70??????????????????75??????????????????80
Arg?Lys?Arg?Ile?Arg?Glu?Asp?Arg?Lys?Ala?Thr?Thr?Ala?Gln?Lys?Val
85??????????????????90??????????????????95
Gln?Gln?Met?Lys?Gln?Arg?Leu?Asn?Glu?Asn?Glu?Arg?Lys?Arg?Lys?Arg
100?????????????????105?????????????????110
Pro?Arg?Leu?Thr?Asp?Thr?Glu?Phe?Met?Val?Pro?Leu?Pro?Ala?Gly?Gly
115?????????????????120?????????????????125
Gly?Thr?Val?Leu?Thr?Lys?Met?Tyr?Pro?Arg?Gly?Asn?His?Trp?Ala?Val
130?????????????????135?????????????????140
Gly?His?Leu?Met
145
<210>8
<211>456
<212>DNA
＜213〉artificial sequence
<400>8
ggatccacag?gtctttcctc?taaaatgcaa?gatacaatgg?aggagaacag?tgagagtgct?????60
ctgcgcaaac?gtattcgaga?ggacagaaag?gccaccacag?ctcagaaggt?gcagcagatg????120
aaacagcgtc?tgaatgagaa?tgaacgtaaa?agaaaacgtc?cacgcctgac?tgacaccttt????180
aaaacaggtc?tttcctctaa?aatgcaagat?acaatggagg?agaacagtga?gagtgctctg????240
cgcaaacgta?ttcgagagga?cagaaaggcc?accacagctc?agaaggtgca?gcagatgaaa????300
cagcgtctga?atgagaatga?acgtaaaaga?aaacgtccac?gcctgactga?caccgaattt????360
atggtcccgc?tgcctgcggg?cggtggtacc?gtgctgacca?agatgtaccc?gcgcggcaac????420
cactgggcgg?tgggtcacct?gatgtgataa?ggatcc??????????????????????????????456
<210>9
<211>438
<212>DNA
＜213〉artificial sequence
<400>9
acaggtcttt?cctctaaaat?gcaagataca?atggaggaga?acagtgagag?tgctctgcgc?????60
aaacgtattc?gagaggacag?aaaggccacc?acagctcaga?aggtgcagca?gatgaaacag????120
cgtctgaatg?agaatgaacg?taaaagaaaa?cgtccacgcc?tgactgacac?ctttaaaaca????180
ggtctttcct?ctaaaatgca?agatacaatg?gaggagaaca?gtgagagtgc?tctgcgcaaa????240
cgtattcgag?aggacagaaa?ggccaccaca?gctcagaagg?tgcagcagat?gaaacagcgt????300
ctgaatgaga?atgaacgtaa?aagaaaacgt?ccacgcctga?ctgacaccga?atttatggtc????360
ccgctgcctg?cgggcggagg?gaccgtgctg?accaagatgt?acccgcgcgg?caaccactgg????420
gcggtggggc?acttaatg??????????????????????????????????????????????????438
<210>10
<211>203
<212>PRT
＜213〉artificial sequence
<400>10
Arg?Ser?Thr?Gly?Leu?Ser?Ser?Lys?Met?Gln?Asp?Thr?Met?Glu?Glu?Asn
1???????????????5???????????????????10??????????????????15
Ser?Glu?Ser?Ala?Leu?Arg?Lys?Arg?Ile?Arg?Glu?Asp?Arg?Lys?Ala?Thr
20??????????????????25??????????????????30
Thr?Ala?Gln?Lys?Val?Gln?Gln?Met?Lys?Gln?Arg?Leu?Asn?Glu?Asn?Glu
35??????????????????40??????????????????45
Arg?Lys?Arg?Lys?Arg?Pro?Arg?Leu?Thr?Asp?Thr?Phe?Lys?Thr?Gly?Leu
50??????????????????55??????????????????60
Ser?Ser?Lys?Met?Gln?Asp?Thr?Met?Glu?Glu?Asn?Ser?Glu?Ser?Ala?Leu
65??????????????????70??????????????????75??????????????????80
Arg?Lys?Arg?Ile?Arg?Glu?Asp?Arg?Lys?Ala?Thr?Thr?Ala?Gln?Lys?Val
85??????????????????90??????????????????95
Gln?Gln?Met?Lys?Gln?Arg?Leu?Asn?Glu?Asn?Glu?Arg?Lys?Arg?Lys?Arg
100?????????????????105?????????????????110
Pro?Arg?Leu?Thr?Asp?Thr?Glu?Phe?Val?Ser?Val?Glu?Lys?Arg?Gln?Ala
115?????????????????120?????????????????125
Pro?Gly?Lys?Arg?Ser?Glu?Ser?Gly?Ser?Pro?Ser?Ala?Gly?Gly?His?Ser
130?????????????????135?????????????????140
Lys?Pro?Pro?His?Ser?Pro?Leu?Val?Leu?Lys?Arg?Cys?His?Val?Ser?Thr
145?????????????????150?????????????????155?????????????????160
His?Gln?His?Asn?Tyr?Ala?Ala?Pro?Pro?Ser?Thr?Arg?Lys?Asp?His?Met
165?????????????????170?????????????????175
Val?Pro?Leu?Pro?Ala?Gly?Gly?Gly?Thr?Val?Leu?Thr?Lys?Met?Tyr?Pro
180?????????????????185?????????????????190
Arg?Gly?Asn?His?Trp?Ala?Val?Gly?His?Leu?Met
195?????????????????200
<210>11
<211>621
<212>DNA
＜213〉artificial sequence
<400>11
ggatccacag?gtctttcctc?taaaatgcaa?gatacaatgg?aggagaacag?tgagagtgct?????60
ctgcgcaaac?gtattcgaga?ggacagaaag?gccaccacag?ctcagaaggt?gcagcagatg????120
aaacagcgtc?tgaatgagaa?tgaacgtaaa?agaaaacgtc?cacgcctgac?tgacaccttt????180
aaaacaggtc?tttcctctaa?aatgcaagat?acaatggagg?agaacagtga?gagtgctctg????240
cgcaaacgta?ttcgagagga?cagaaaggcc?accacagctc?agaaggtgca?gcagatgaaa????300
cagcgtctga?atgagaatga?acgtaaaaga?aaacgtccac?gcctgactga?caccgaattc????360
gtttctgtgg?aaaagcgcca?ggctcctggc?aaacgctctg?agtctggctc?cccttctgct????420
ggtggccaca?gcaaacctcc?tcacagccca?ctggtcctca?agcgctgcca?cgtctccaca????480
catcagcaca?actacgcagc?gcctccgtcc?actcgcaagg?accatatggt?cccgctgcct????540
gcgggcggtg?gtaccgtgct?gaccaagatg?tacccgcgcg?gcaaccactg?ggcggtcggt????600
cacctgatgt?gataactcga?g??????????????????????????????????????????????621
<210>12
<211>602
<212>DNA
＜213〉artificial sequence
<400>12
acaggtcttt?cctctaaaat?gcaagataca?atggaggaga?acagtgagag?tgctctgcgc?????60
aaacgtattc?gagaggacag?aaaggccacc?acagctcaga?aggtgcagca?gatgaaacag????120
cgtctgaatg?agaatgaacg?taaaagaaaa?cgtccacgcc?tgactgacac?ctttaaaaca????180
ggtctttcct?ctaaaatgca?agatacaatg?gaggagaaca?gtgagagtgc?tctgcgcaaa????240
cgtattcgag?aggacagaaa?ggccaccaca?gctcagaagg?tgcagcagat?gaaacagcgt????300
ctgaatgaga?atgaacgtaa?aagaaaacgt?ccacgcctga?ctgacaccga?attcgtttct????360
gtggaaaaga?ggcaggctcc?tggcaaaagg?tcagagtctg?gatcaccttc?tgctggaggc????420
cacagcaaac?ctcctcacag?cccactggtc?ctcaagaggt?gccacgtctc?cacacatcag????480
cacaactacg?cagcgcctcc?tccactggca?aggaccatat?ggtcccgctg?cctgcgggcg????540
gagggaccgt?gctgaccaag?atgtacccgc?gcggcaacca?ctgggcggtg?gggcacttaa????600
tg???????????????????????????????????????????????????????????????????602
<210>13
<211>213
<212>PRT
＜213〉artificial sequence
<400>13
Met?Leu?Gly?Asp?Gly?Gly?Asp?Val?Ser?Phe?Ser?Thr?Arg?Gly?Thr?Gln
1???????????????5???????????????????10??????????????????15
Asn?Trp?Thr?Val?Glu?Arg?Leu?Leu?Gln?Ala?His?Arg?Gln?Leu?Glu?Glu
20??????????????????25??????????????????30
Arg?Gly?Tyr?Val?Phe?Val?Gly?Tyr?His?Gly?Thr?Phe?Leu?Glu?Ala?Ala
35??????????????????40??????????????????45
Gln?Ser?Ile?Val?Phe?Gly?Gly?Val?Arg?Ala?Arg?Ser?Gln?Asp?Leu?Asp
50??????????????????55??????????????????60
Ala?Ile?Trp?Arg?Gly?Phe?Tyr?Ile?Ala?Gly?Asp?Pro?Ala?Leu?Ala?Tyr
65??????????????????70??????????????????75??????????????????80
Gly?Tyr?Ala?Gln?Asp?Gln?Glu?Pro?Asp?Ala?Arg?Gly?Arg?Ile?Arg?Asn
85??????????????????90??????????????????95
Gly?Ala?Leu?Leu?Arg?Val?Tyr?Val?Pro?Arg?Ser?Ser?Leu?Pro?Gly?Phe
100?????????????????105?????????????????110
Tyr?Arg?Thr?Ser?Leu?Thr?Leu?Ala?Ala?Pro?Glu?Ala?Ala?Gly?Glu?Val
115?????????????????120?????????????????125
Glu?Arg?Leu?Ile?Gly?His?Pro?Leu?Pro?Leu?Arg?Leu?Asp?Ala?Ile?Thr
130?????????????????135?????????????????140
Gly?Pro?Glu?Glu?Glu?Gly?Gly?Arg?Leu?Glu?Thr?Ile?Leu?Gly?Trp?pro
145?????????????????150?????????????????155?????????????????160
Leu?Ala?Glu?Arg?Thr?Val?Val?Ile?Pro?Ser?Ala?Ile?Pro?Thr?Asp?Pro
165?????????????????170?????????????????175
Arg?Asn?Val?Gly?Gly?Asp?Leu?Asp?Pro?Ser?Ser?Ile?Pro?Asp?Lys?Glu
180?????????????????185?????????????????190
Gln?Ala?Ile?Ser?Ala?Leu?Pro?Asp?Tyr?Ala?Ser?Gln?Pro?Gly?Asp?Pro
195?????????????????200?????????????????205
Pro?Lys?Asp?Glu?Leu
210
<210>14
<211>216
<212>DNA
＜213〉artificial sequence
<400>14
ctcgagtgga?gagcatgggg?agacccggga?ccctgctggg?tttctctgtc?acaaaggaaa?????60
ataatccccc?tggtgtgaca?gacccaagga?cagaacacag?cagaggtcag?cactggggaa????120
gacaggttgt?cctcccaggg?gatgggggtc?catccacctt?gccgaaaaga?tttgtctgag????180
gaactgaaaa?tagaagggaa?aaaagaggag?aagctt??????????????????????????????216
<210>15
<211>351
<212>DNA
＜213〉artificial sequence
<400>15
catatgagcg?ataaaattat?tcacctgact?gacgacagtt?ttgacacgga?tgtactcaaa?????60
gcggacgggg?cgatcctcgt?cgatttctgg?gcagagtggt?gcggtccgtg?caaaatgatc????120
gccccgattc?tggatgaaat?cgctgacgaa?tatcagggca?aactgaccgt?tgcaaaactg????180
aacatcgatc?aaaaccctgg?cactgcgccg?aaatatggca?tccgtggtat?cccgactctg????240
ctgctgttca?aaaacggtga?agtggcggca?accaaagtgg?gtgcactgtc?taaaggtcag????300
ttgaaagagt?tcctcgacgc?taacctggcc?ggttctggtt?ctggcggatc?c?????????????351
<210>16
<211>477
<212>DNA
＜213〉artificial sequence
<400>16
ggatccggcg?ttcgtggtcc?gcgttctaca?ggtctttcct?ctaaaatgca?agatacaatg?????60
gaggagaaca?gtgagagtgc?tctgcgcaaa?cgtattcgag?aggacagaaa?ggccaccaca????120
gctcagaagg?tgcagcagat?gaaacagcgt?ctgaatgaga?atgaacgtaa?aagaaaacgt????180
ccacgcctga?ctgacacctt?taaaacaggt?ctttcctcta?aaatgcaaga?tacaatggag????240
gagaacagtg?agagtgctct?gcgcaaacgt?attcgagagg?acagaaaggc?caccacagct????300
cagaaggtgc?agcagatgaa?acagcgtctg?aatgagaatg?aacgtaaaag?aaaacgtcca????360
cgcctgactg?acaccgaatt?tatggtcccg?ctgcctgcgg?gcggtggtac?cgtgctgacc????420
aagatgtacc?cgcgcggcaa?ccactgggcg?gtgggtcacc?tgatgtgata?actcgag???????477
Claims (23)
1, a kind of non-virus carrier, it comprises a kind of target fusion rotein that is rich in positive amino acid whose polypeptide and a kind of receptors ligand polypeptide successively for holding from N to C end, wherein
It is described that to be rich in positive amino acid whose polypeptide be the fragment of the DNA of Protein-tyrosine-phosphatase PTP 332-388 amino acids in conjunction with section,
Described receptors ligand polypeptide be can with gastrin releasing peptide receptor specificity bonded gastrin release peptide GRP or its associated clip, analogue or mutant,
2, non-virus carrier as claimed in claim 1, it is P2G, it holds the DNA of PTP 332-388 amino acids representative that is followed successively by two polyphones to C end in conjunction with section and the gastrin release peptide GRP that is made up of 27 amino acid from N, and its aminoacid sequence is shown in SEQ ID NO:7.
3, a kind of non-virus carrier, its for hold from N to C end comprise successively a kind of polypeptide that is rich in positive amino acid whose polypeptide, a kind of concentration of DNA and a kind of receptors ligand polypeptide and the target fusion rotein, wherein
Described receptors ligand polypeptide be can with gastrin releasing peptide receptor specificity bonded gastrin release peptide GRP or its associated clip, analogue or mutant,
It is described that to be rich in positive amino acid whose polypeptide be the fragment of the DNA of Protein-tyrosine-phosphatase PTP 332-388 amino acids in conjunction with section.
The proteic non-specific DNA of the polypeptide c-myc of described concentration of DNA is in conjunction with the fragment of the 265-318 amino acids of section.
4, non-virus carrier as claimed in claim 3, it is P2CG, its from N hold be followed successively by two polyphones to C end the DNA of PTP 332-388 amino acids representative in conjunction with the non-specific DNA of section, the proteic 265-318 amino acids of c-myc representative in conjunction with section and the gastrin release peptide GRP that forms by 27 amino acid, its aminoacid sequence is shown in SEQ ID NO:10.
5, a kind of fusion gene, each non-virus carrier of its coding claim 1-4.
6, fusion gene as claimed in claim 5, the non-virus carrier P2G of its coding claim 2 has the nucleotide sequence shown in the SEQ ID NO:8.
7, fusion gene as claimed in claim 5, the non-virus carrier P2G of its coding claim 2 has the nucleotide sequence shown in the SEQ ID NO:9.
8, fusion gene as claimed in claim 5, the non-virus carrier P2CG of its coding claim 4 has the nucleotide sequence shown in the SEQ ID NO:11.
9, fusion gene as claimed in claim 5, the non-virus carrier P2CG of its coding claim 4 has the nucleotide sequence shown in the SEQ ID NO:12.
10, the carrier that comprises each described fusion gene of claim 5-9.
11, carrier as claimed in claim 10, it is as shown in Figure 3 pET28a-P2G or pET28a-P2CG.
12, carrier as claimed in claim 10, it is pET30a-TrxA-Th-P2G or pET30a-TrxA-Th-P2CG.
13, by the host cell of carrier conversion, transfection or the transduction of claim 10.
14, each the method for non-virus carrier of a kind of production claim 1-4 is included in the host cell of cultivating claim 13 under the condition of suitable expression, and separation and purifying are by the described non-virus carrier of described host cell expression.
15, a kind of pharmaceutical composition that is used for the target therapy of tumor, described pharmaceutical composition comprise claim 1-4 each non-virus carrier and a kind of mixture of expression type recombinant chou, described expression type recombinant chou comprises a kind of proteinic gene that pair cell has kill capability of expressing.
16, pharmaceutical composition as claimed in claim 15, the protein that wherein said pair cell has kill capability is pseudomonal toxin, diphtheria toxin, Phytolacca acinosa, Ricin, or other all pair cells have the albumen from human body, animals and plants or microorganism of kill capability, or their active function zone, mutant or analogue; And the protein or the polypeptide that have kill capability according to the pair cell of synthetic.
17, the protein that pharmaceutical composition as claimed in claim 16, wherein said pair cell have kill capability is pseudomonal toxin, or its second and the 3rd functional domain.
18, the protein that pharmaceutical composition as claimed in claim 16, wherein said pair cell have kill capability is the 3rd functional domain or its mutant of pseudomonal toxin, and the aminoacid sequence of described mutant is shown in SEQ ID NO.:13.
19, pharmaceutical composition as claimed in claim 15, wherein said described expression type recombinant chou are the recombinant expressed type recombinant chou of the eucaryon pGL3-CEA-PEIIImut that is subjected to the promotor control of CEA, and wherein the DNA sequences encoding of CEA is shown in SEQ ID NO.:14.
20, pharmaceutical composition as claimed in claim 15, wherein said expression type recombinant chou are to be subjected to the promotor of CEA and the recombinant expressed type recombinant chou of the eucaryon DYPII of second intron regulation and control of rabbit globin.
21, pharmaceutical composition as claimed in claim 15, it is selected from P2CG/pGL3-CEA-PEIIImut, P2CG/DYPII, P2G/pGL3-CEA-PEIIImut, and P2G/DYPII.
22, each pharmaceutical composition of claim 15-21 is as the application to the accurate targeted drug of malignant tumour.
23, the application of claim 22, wherein said malignant tumour are GRP-R
+CEA
+Cancer of the stomach, lung cancer, mammary cancer, colorectal carcinoma and carcinoma of the pancreas.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031239412A CN100553682C (en) | 2003-06-11 | 2003-06-11 | G type non-virus carrier and the pharmaceutical composition that comprises it |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031239412A CN100553682C (en) | 2003-06-11 | 2003-06-11 | G type non-virus carrier and the pharmaceutical composition that comprises it |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1566348A true CN1566348A (en) | 2005-01-19 |
| CN100553682C CN100553682C (en) | 2009-10-28 |
Family
ID=34468968
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB031239412A Expired - Fee Related CN100553682C (en) | 2003-06-11 | 2003-06-11 | G type non-virus carrier and the pharmaceutical composition that comprises it |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100553682C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429523B (en) * | 2007-11-07 | 2011-04-06 | 中国医学科学院基础医学研究所 | Type T non--viral vector and composite medicament containing the same |
| CN101134110B (en) * | 2006-09-01 | 2012-11-07 | 中国医学科学院基础医学研究所 | Q type non-viral vector and pharmaceutical composition containing the same |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1181422A (en) * | 1996-10-31 | 1998-05-13 | 上海市肿瘤研究所 | Gene transfer carrier structured by polypeptide in conjunction with growth factor receptor |
-
2003
- 2003-06-11 CN CNB031239412A patent/CN100553682C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101134110B (en) * | 2006-09-01 | 2012-11-07 | 中国医学科学院基础医学研究所 | Q type non-viral vector and pharmaceutical composition containing the same |
| CN101429523B (en) * | 2007-11-07 | 2011-04-06 | 中国医学科学院基础医学研究所 | Type T non--viral vector and composite medicament containing the same |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100553682C (en) | 2009-10-28 |
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