CN1513878A - Antitumour medicine containing recombination bacterial toxin protein complex substance - Google Patents
Antitumour medicine containing recombination bacterial toxin protein complex substance Download PDFInfo
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Abstract
An antineoplastic medicine containing the recombinant bacteriotoxin protein composition TPCB whose key components are recombinant proteins IP33 and LP14 is disclosed. Under the action of the particular enzyme generated by tumor cells, the non-toxic protein can be changed into toxic protein to directly kill tumor cells specifically.
Description
Technical field
The present invention relates to a kind of medicine for the treatment of tumour, particularly relate to a kind of bio-pharmaceutical for the treatment of tumour.
Background of invention
Scientific research in recent years finds, nearly all human tumor cell all overexpression uPA (uPA) and uPA acceptor (uPAR) [Int.J.Cancer 1997 for Andreasen, P.A. etc.; 72:1-22]; And in healthy tissues, the expression of uPA and uPAR is in low-down level.The initial secreted form of uPA is active very low strand enzyme.UPA strand enzyme is converted into highly active uPA streptokinase-streptodornase under the effect of plasmin, and highly active uPA streptokinase-streptodornase plasminogen activation generates plasmin, impels more highly active uPA streptokinase-streptodornase to produce then.This has constituted the circulation path of a positive regeeration, and this Profibrinolysin is the positive feedback conditioning agent of circulation path, and it and uPA precursor after the receptors bind of cell surface, are regulated the entire reaction path by different assembling modes.Therefore, the overexpression of uPA and uPAR is used as sign [Dano, K. etc., the Act Pathol.Microbiol.Immunol.Scand.1999 that tumour worsens usually; 107:120-127].Research at present confirms that the overexpression of uPA and uPAR can be used as the deterioration sign of following tumour: lung cancer, colorectal carcinoma, mammary cancer, cancer of the stomach, carcinoma of the pancreas, tumor of head and neck, skin carcinoma, uterus carcinoma, ovarian cancer, brain tumor, melanoma, soft tissue sarcoma, monocytic leukemia etc.
The anthrax bacillus toxin protein is that anthrax bacillus makes the lethal main virulence factor of people, forms [Dixon T.C. etc., N.Engl.J.Med1999 by protective antigen (PA), lethal gene (LF) and edema factor albumen such as (EF); 341:815-826].But three kinds of albumen of this that separate itself are nontoxic.[Nature 2001 for Bradley, K.A. etc. for tumor endothelial cell marker protein 8 (TEM8); 414:225-229], be a kind of acceptor that extensively has cell surface, can combine with PA, TEM8 is especially remarkable in the expression of tumor cell surface.PA is with after acceptor TEM8 combines, under the proteolytic enzyme effect of Furin or Furin sample, PA is cut into two polypeptide fragments [Klimpel, K.R. etc., Proc.Natl.Acad.Sci.USA1992 of 20KD and 63KD size at " arginine Methionin Methionin arginine (RKKR) " sequence place; 89:10277-10281], shearing site has the specificity of height.The polypeptide fragment (PA63) of the carboxyl terminal 63KD size of the PA formation polymer that can mutually combine wherein, and can be in conjunction with termolecular LF and the common eight aggressiveness mixtures that form of EF, LF and EF can enter cell cytosol via this eight aggressiveness mixture transhipment, thus the performance cytotoxic effect.And do not have this function after complete PA albumen and the receptors bind, so the formation that is sheared contratoxin mixture of PA in the RKKR site is vital.In nearest research, scientists just once attempted to replace RKKR with some specific sequence, reached specific purpose [Liu, S. etc., Cancer Res.2000 in the hope of activating the toxin protein mixture in certain location; 60:6061-6067], as utilize specific MMPs to shear sequence and replace RKKR, in the hope of reaching the purpose of treatment tumour.
Goal of the invention
The object of the invention is to provide a kind of recombinant bacteria toxin protein; The object of the invention also is to provide a kind of medicine for the treatment of tumour and preparation method thereof.
Summary of the invention
The present invention utilizes specific uPA to shear sequence and replaces RKKR, produces the PA albumen of sudden change.The PA albumen of sudden change is assembled into eight aggressiveness mixtures at tumor cell surface under the effect of uPA, carry edema factor EF and the bacterial exotoxin fusion rotein enters in the cell.[Nature 001 for Bradley, K.A. etc. under the effect of endoplasmic reticulum; 414:225-229; Cunningham, Proc.Natl.Acad.Sci.USA such as K. 2002; 99:7049-7053; Mogridge, Proc.Natl.Acad.Sci.USA such as J. 2002; 99:7045-7048], after EF and bacterial exotoxin fusion rotein enter endochylema, performance cytotoxic effect, thereby kill tumor cell.
The present invention is merged protective antigen PA binding fragment and the middle adenosine diphosphate ribose base fragment of Pseudomonas aeruginosa exotoxin A (ETA) among the edema factor EF by the method for gene clone, obtains recombinant protein LP14; Utilize the RKKR aminoacid sequence in specific uPA (uPA) the shearing sequence application splicing overlap extension replacement protective antigen (PA), obtain the PA albumen of sudden change, i.e. recombinant protein IP33.Bacteriotoxin albumen composition (TPCB), i.e. 1: 3 mole ratio mixture of recombinant protein LP14 and IP33.After the TEM8 receptors bind that recombinant protein IP33 and tumor cell surface are rich in, under the uPA effect that tumor cell surface efficiently expresses, be cut into two polypeptide fragments of 20KD and 63KD, the polypeptide fragment of 63KD size can combine the back with LP14 and be advanced tumour cell by transhipment, LP14 brings into play cytotoxic effect in endochylema, thus the kill tumor cell.
The present invention is achieved through the following technical solutions:
One, the clone of anthrax bacillus protective antigen PA encoding gene
The present invention is being protected property of a clone antigen encoding gene from the anthrax bacillus genome.
The present invention uses SDS (sodium lauryl sulphate)/CTAB (cetyltriethylammonium bromide) method to extract genome deoxyribonucleotide (DNA) from the anthrax bacillus of a large amount of cultivations, and genomic dna is standby as template behind the purifying repeatedly through phenol/chloroform/primary isoamyl alcohol.According to known anthrax bacillus protective antigen gene sequence (Genebank numbering: NC_001496) design a pair of primer; upstream primer is formed (SEQ IDNO:1) by 30 DNA that comprise initiator codon; downstream primer is formed (SEQ ID NO:2) by 30 DNA that comprise terminator codon; clone through PCR; 80 ℃ of complete sex change 2 minutes; 95 ℃ 30 seconds; 55 ℃ 30 seconds; 68 ℃ 2 minutes; circulate 35 times; last 68 ℃ were extended 5 minutes, being protected property antigen (PA) encoding gene, and with the corresponding KpnI of PA encoding gene fragment warp; the NotI restriction endonuclease sites inserts in the pcDNA3 carrier (purchasing the company in Stratagen).
Two, the clone of anthrax bacillus edema factor EF encoding gene
The present invention is that the clone obtains the edema factor encoding gene from the anthrax bacillus genome.
The present invention uses in the technique scheme resulting anthrax bacillus genomic dna as template, according to known anthrax bacillus edema factor EF gene order (Genebank numbering: NC001496) design a pair of primer, upstream primer is formed (SEQ ID NO:3) by 30 DNA that comprise initiator codon, downstream primer is formed (SEQ ID NO:4) by 30 DNA that comprise terminator codon, obtain the EF encoding gene through the PCR clone, and EF encoding gene fragment is inserted in the pCDNA3 carrier through corresponding KpnI, NotI restriction endonuclease sites.
Three, the clone of Pseudomonas aeruginosa exotoxin A (ETA) encoding gene
The present invention is that the clone obtains the exotoxin A encoding gene from the Pseudomonas aeruginosa genome.
The present invention uses SDS (sodium lauryl sulphate)/CTAB (cetyltriethylammonium bromide) method to extract (detailed process is seen embodiment 3) genome deoxyribonucleotide (DNA) from the Pseudomonas aeruginosa of a large amount of cultivations, genomic dna is through the extracting repeatedly of phenol/chloroform/primary isoamyl alcohol, at last with upper phase with 70% isopropanol precipitating after and be dissolved among the TE standby as template.According to known Pseudomonas aeruginosa exotoxin A gene sequence (Genebank numbering: K01397) design a pair of primer, upstream primer is by 30 DNA based compositions that comprise initiator codon (SEQ ID NO:5), downstream primer is by 30 DNA based compositions that comprise terminator codon (SEQ ID NO:6), obtain the ETA encoding gene through the PCR clone, and ETA encoding gene fragment is inserted in the pcDNA3 carrier through corresponding HindIII, NotI restriction endonuclease sites.
Four, the structure of recombination fusion protein LP14 encoding gene
The present invention is that the method for using gene engineering is fused to recombinant protein LP14 encoding gene with adenosine diphosphate ribose base functional domain encoding gene in protective antigen combined function territory encoding gene and the Pseudomonas aeruginosa exotoxin A among the edema factor EF.
The present invention uses PCR method, 80 ℃ of complete sex change 2 minutes, 95 ℃ 30 seconds, 55 ℃ 30 seconds, 68 ℃ 1 minute, circulate 35 times, last 68 ℃ of extensions obtained edema factor encoding gene 5 ' end 1～735 gene fragment (L1) in 5 minutes respectively, with Pseudomonas aeruginosa exotoxin A encoding gene 3 ' end 1092～1842 gene fragments (L2), at last (L1, L2) two gene fragments are connected to the reorganization encoding gene through the HindIII restriction endonuclease sites, i.e. LP14 encoding gene (SEQ ID NO:14).L1 gene amplification is template with CDNA3/ef, and upstream primer is by 30 DNA based compositions that comprise initiator codon (SEQ ID NO:3), and downstream primer is by 30 DNA based compositions that comprise HindIII restriction enzyme base (SEQ ID NO:7); L2 gene amplification is template with pCDNA3/eta, and upstream primer is by 30 DNA based compositions that comprise HindIII restriction enzyme base (SEQ ID NO:8), and downstream primer is by 30 DNA based compositions that comprise terminator codon (SEQ ID NO:6).
Five, the structure of recombination mutation protein I P33 encoding gene
The present invention is that the method for using gene engineering produces IP33 recombinant protein encoding gene with natural protective antigen PA encoding gene sudden change.
The present invention uses the Overlap PCR method with in the natural protective antigen PA encoding gene " AGAAAAAAGC GA " ' the deoxyribonucleotide base mutation is " CCAGGAAGTGGAAAATCAGCA " deoxyribonucleotide base, thus clone's (detailed process is seen embodiment 5) obtains IP33 encoding gene (SEQ ID NO:12).In the process of Overlap PCR, with pCDNA3/pa is template, at first amplify two gene fragments that overlap with aminoterminal and two pairs of primers of carboxyl terminal (SEQ ID NO:1,9 and SEQ ID NO:10,2) respectively, and then be template with the mixture of these two gene fragments, with the primer that comprises initiator codon is upstream primer (SEQ ID NO:1), the primer that comprises terminator codon is downstream primer (SEQ ID NO:2), obtains the IP33 encoding gene through PCR.
Six, the expression and purification of recombinant protein LP14 and IP33
The present invention has bioactive recombinant protein LP14 and IP33 in external a large amount of preparations.
The LP14 gene fragment is downcut from the pCNDA3 recombinant plasmid with KpnI, NotI restriction enzyme, and subclone obtains the Yeast expression carrier of LP14 to the corresponding multiple clone site of (detailed process is seen embodiment 6) Yeast expression carrier pPICZ α A (available from Invitrogen company).The IP33 gene fragment is downcut from the pCNDA3 recombinant plasmid with KpnI, NotI restriction enzyme, and subclone (detailed process is seen embodiment 6) obtains the Yeast expression carrier of IP33 to the corresponding multiple clone site of Yeast expression carrier pPICZ α A (available from Invitrogen company).The secreting signal peptide of pPICZ α A can efficiently be transported to recombinant protein in the yeast culture liquid with soluble form, therefore has the great expression recombinant protein, and is easy to the advantage of purifying.Recombinant expression plasmid is converted among the yeast GS115 (available from Invitrogen company) after the linearizing of SacI enzyme, and screening growth on the flat board that contains on the Zeocin (100ug/ml).Getting one is cloned in the 10ml conditioned medium [1% yeast extract (purchasing the company in Gibco)), 2% peptone, 2% glucose, 100ug/ml Zeocin] 28 ℃ to grow to the OD595 absorbance is 12, centrifugal then collection yeast, and be resuspended in the 10ml methyl alcohol substratum [1% yeast extract, 2% peptone, 100mM PH6.0 potassium phosphate buffer, 0.5% methyl alcohol, 0.00001% vitamin H (purchasing the company in Gibco), the basic nitrogen of 1.3% yeast are former], and in 28 ℃ of growths 2 days.Yeast in 1600g centrifugal 20 minutes is at last got supernatant and is stored in-80 ℃ rapidly, and yeast sedimentation is resuspended in the fresh culture standby.Get 20ul culture supernatant row 15%SDS-PAGE electrophoresis, obtain LP14, IP33 respectively, see Figure 10 and Figure 11.
Seven, the preparation of LP14 and IP33 mixture (TPCB)
According to the binding characteristic of IP33 and LP14 in theory, with the above-mentioned reorganization LP14 for preparing and IP33 albumen freeze-drying powder according to a certain percentage, the best is 1: 3 mol ratio (being equivalent to mass ratio is 1: 3.8) uniform mixing, every of 2mg be stored in-80 ℃ standby; Face with before being dissolved among the PBS, mother liquid concentration is 2mg/ml, and is diluted to different final concentrations according to different test objectives.
Experimental result shows that bacteriotoxin albumen composition of the present invention (TPCB) is the kill tumor cell directly, and reaches the purpose of eradicating tumour in a short time.TPCB has the specificity of height to the effect of tumour cell, and other normal cell is not had any cytotoxicity, and normal experiment mice is not had obvious detrimentally affect yet, is a kind of effective medicine for treating tumor thing.
Recombinant bacteria toxin protein mixture TCPB is to the effect of oncotherapy
This research is by the inhibition experiment of recombinant bacteria toxin protein mixture TCPB to the fibrosarcoma growth, inhibition experiment to melanotic tumor B6F10 growth, inhibition experiment to the hepatoma growth, directly kill tumor cell of recombinant bacteria toxin protein mixture TCPB is described, has the effect that suppresses tumor growth significantly.And recombinant bacteria toxin protein mixture is to mouse normal tissue cell and external Normocellular growth and have no effect, illustrating that the cytotoxic effect of recombinant bacteria toxin protein mixture has the tumor tissues specificity of height, is safe to normal tissue cell.
Following experimental example is used to further specify the present invention.
Experimental example 1: recombinant bacteria toxin protein mixture TPCB is to the restraining effect of fibrosarcoma growth
The purpose of this experiment is the restraining effect of research TPCB to the fibrosarcoma growth.
With fibrosarcoma cell strain-MM45T.Li cell at 37 ℃, 5%CO
2, cultivate in the 60% humidity incubator, when cell quantity is enough, become individual cells with trysinization, and counting, (PBS) is diluted to cell suspending liquid (1 * 10 with phosphate buffered saline buffer
6Individual/milliliter) standby.Choose 25 of male nude mouses (60 grams ± 5 grams), every in the cell suspension 1ml of back center line subcutaneous injection preparation.After two weeks, the tumer positive mouse is divided into two groups at random, every group each 10, one group at abdominal injection 1ml PBS, (LP14 and IP33 ratio are 1: 3 to another group in the mixture at the PBS solution of same position injection 1ml recombinant bacteria toxin protein mixture TPCB, total concn is 20ug/ml), injection is once weekly.Same time every day is with the major diameter of vernier caliper measurement lump and minor axis, and according to formula:
Gross tumor volume=4/3 Л (major diameter/2) (minor axis/2) (major diameter+minor axis)/4
Calculating gross tumor volume size stops experiment when death appears in injection PBS group mouse.Found that, recombinant bacteria toxin protein TPCB can strongly inhibited the growth of fibrosarcoma in nude mice (see Table 1 and Fig. 1).
Experimental example 2: recombinant bacteria toxin protein mixture TPCB is to the restraining effect of hepatoma growth
The purpose of this experiment is the restraining effect of research TPCB to the hepatoma growth.
With hepatoma cells strain-Hepa 1-6 cell at 37 ℃, 5%CO
2, cultivate in the 60% humidity incubator, when cell quantity is enough, become individual cells with trysinization, and counting, (PBS) is diluted to cell suspending liquid (1 * 10 with phosphate buffered saline buffer
6Individual/milliliter) standby.Choose 25 of male nude mouses (60 grams ± 5 grams), every in the cell suspension 1ml of back center line subcutaneous injection preparation.After two weeks, the tumer positive mouse is divided into two groups at random, every group each 10, one group at abdominal injection 1ml PBS, (LP14 and IP33 ratio are 1: 3 to another group in the mixture at the PBS solution of same position injection 1ml recombinant bacteria toxin protein mixture TPCB, total concn is 20ug/ml), injection is once weekly.Same time every day is with the major diameter of vernier caliper measurement lump and minor axis, and according to formula:
Gross tumor volume=4/3 Л (major diameter/2) (minor axis/2) (major diameter+minor axis)/4
Calculating gross tumor volume size stops experiment behind the fortnight, observe tumour profile size, the results are shown in Figure 12; And get tumor tissues and do the section analysis, through the dyeing of TUNEL method, the results are shown in Figure 13.Found that, recombinant bacteria toxin protein TPCB can strongly inhibited the growth of hepatoma in nude mice (see Table 1 and Fig. 2).
Embodiment 3: recombinant bacteria toxin protein mixture TPCB is to the restraining effect of melanoma growth
The purpose of this experiment is the restraining effect of research TPCB to the melanotic tumor growth.
With melanoma cell strain-B6F12 cell at 37 ℃, 5%CO
2, cultivate in the 60% humidity incubator, when cell quantity is enough, become individual cells with trysinization, and counting, (PBS) is diluted to cell suspending liquid (1 * 10 with phosphate buffered saline buffer
6Individual/milliliter) standby.Choose 25 of male nude mouses (60 grams ± 5 grams), every in the cell suspension 1ml of back center line subcutaneous injection preparation.After two weeks, the tumer positive mouse is divided into two groups at random, every group each 10, one group at abdominal injection 1ml PBS, (LP14 and IP33 ratio are 1: 3 to another group in the mixture at the PBS solution of same position injection 1ml recombinant bacteria toxin protein mixture TPCB, total concn is 20ug/ml), injection is once weekly.Same time every day is with the major diameter of vernier caliper measurement lump and minor axis, and according to formula:
Gross tumor volume=4/3 Л (major diameter/2) (minor axis/2) (major diameter+minor axis)/4
Calculating gross tumor volume size stops experiment when death appears in injection PBS group mouse.Found that, recombinant bacteria toxin protein TPCB can strongly inhibited the growth of melanoma in nude mice (see Table 1 and Fig. 3).
Table 1 is respectively organized the mean tumour volume (mean ± standard deviation) of mouse
| Gross tumor volume (CM 3) | Control group | Melanoma treatment group | Fibrosarcoma treatment group | Hepatoma treatment group |
| The 1st day | ??0.0097±0.0015 | ??0.0097±0.0012 | ??0.0097±0.0018 | ??0.0097±0.0008 |
| The 2nd day | ??0.3542±0.0073 | ??0.0223±0.0054 | ??0.0233±0.0145 | ??0.0189±0.0024 |
| The 3rd day | ??0.7832±0.0241 | ??0.1434±0.0134 | ??0.1434±0.0242 | ??0.0934±0.0142 |
| The 4th day | ??1.5638±0.1326 | ??0.3915±0.0752 | ??0.3915±0.0894 | ??0.2453±0.0764 |
| The 5th day | ??3.0758±0.5634 | ??0.3936±0.0932 | ??0.3936±0.0984 | ??0.3936±0.1623 |
| The 6th day | ??5.4322±0.8542 | ??0.2879±0.3127 | ??0.2879±0.1442 | ??0.3542±0.2831 |
| The 7th day | ??7.1324±1.0463 | ??0.2432±0.8432 | ??0.2432±0.1842 | ??0.3123±0.1943 |
| The 8th day | ??6.9954±1.1243 | ??0.3674±0.6643 | ??0.1768±0.3422 | ??0.3674±0.2413 |
| The 9th day | ??8.7843±1.1142 | ??0.2435±0.5422 | ??0.1656±0.2842 | ??0.4253±0.2314 |
| The 10th day | ??10.2344±1.2064 | ??0.3974±0.6032 | ??0.1433±0.3462 | ??0.5024±0.3412 |
| The 11st day | ??12.1453±1.0246 | ??0.4312±0.5832 | ??0.1324±0.4248 | ??0.4983±0.3132 |
| The 12nd day | ??13.9873±1.365 | ??0.2543±0.4824 | ??0.1232±0.4835 | ??0.4853±0.4313 |
| The 13rd day | ??15.3342±1.4634 | ??0.2845±0.4264 | ??0.1311±0.3346 | ??0.4821±0.1342 |
| The 14th day | ??17.0123±1.3246 | ??0.3124±0.4842 | ??0.1289±0.4732 | ??0.4623±0.2431 |
Experimental example 4: the acute toxicity test of recombinant bacteria toxin protein TPCB
The purpose of this experiment is the acute toxicity of research recombinant bacteria toxin protein TPCB.
Previously prepared recombinant bacteria toxin protein TPCB is dissolved in the PBS solution, and final concentration is 200ug/ml.Healthy Balb/C mouse, random packet, every group each 10, the heavy dose of TPCB of TPCB group abdominal injection (effective dose 10 times, be 2mg/Kg), control group abdominal injection PBS solution observed for 1 week, and symptom mouse number appears in each group of statistics.Found that symptom (the results are shown in Table 2) does not appear in TPCB group mouse in a large number, illustrates that TPCB is a safe enough in effective dosage ranges.
The heavy dose of TPCB of table 2 is to the influence of mouse growth
| Group | Number of animals | Asymptomatic number | The symptom rate appears |
| The TPCB | 12 | 9 | ????25％ |
| The PBS | 10 | ????0 | ????0％ |
Experimental example 5: recombinant bacteria toxin protein TPCB is to the influence of normal endothelial cell growth
This experiment purpose is the influence of research recombinant bacteria toxin protein TPCB to normal cell growth.
Previously prepared recombinant bacteria toxin protein TPCB is dissolved in the PBS solution, and final concentration is 200ug/ml.With the ox huve cell at 37 ℃, 5%CO
2, cultivate in the incubator of 60% humidity.When treating that cell has sufficient amount, by 1 * 10
6Evenly be inoculated in overnight incubation in two six orifice plates.(concentration is respectively 0,25,50,100,200,400ug/ml) or PBS solution to add the recombinant bacteria toxin T PCB solution of different concns in second day respectively in two six different orifice plates.Cell continues to cultivate 3 days in incubator, measures cell survival rate through violet staining on the 4th day.Found that drug treating group and control group, the cells survival rate is the same, and promptly TPCB does not have obvious cytotoxic effect to normal endothelial cell.Experimental result is seen Fig. 4.
Description of drawings:
Fig. 1 TPCB is to the influence of fibrosarcoma growth
Fig. 2 TPCB is to the influence of hepatoma growth
Fig. 3 TPCB is to the influence of melanoma growth
Fig. 4 TPCB is to the influence of the normal tissue cell growth of vitro culture
Fig. 5 pcDNA
3/ pa is at the postdigestive agarose gel electrophoresis picture of restriction enzyme KpnI, NotI
Fig. 6 pcDNA
3/ ef is at the postdigestive agarose gel electrophoresis picture of restriction enzyme KpnI, NotI
Fig. 7 pcDNA
3/ eta is at the postdigestive agarose gel electrophoresis picture of restriction enzyme HindIII, NotI
Fig. 8 pBluescript/lp14 is at the postdigestive agarose gel electrophoresis picture of restriction enzyme KpnI, NotI
Fig. 9 pcDNA
3/ ip33 is at the postdigestive agarose gel electrophoresis picture of restriction enzyme KpnI, NotI
The proteic SDS-PAGE photo of Figure 10 IP33
The proteic SDS-PAGE photo of Figure 11 LP14
Figure 12 TPCB is to the influence of tumor-bearing mice tumor growth size
Figure 13 tumor tissues frozen section (dyeing of TUNEL method)
The amino acid code table is as follows:
A Ala L-Ala; M Met methionine(Met); C Cys halfcystine; N Asn l-asparagine; D Asp aspartic acid
P Pro proline(Pro); E Glu L-glutamic acid; Q Gln glutamine; F Phe phenylalanine; R Arg arginine
G Gly glycine; S Ser Serine; H His Histidine; T Thr Threonine; I Ile Isoleucine; V Val Xie Ansuan
K Lys Methionin; W Trp tryptophane; L Leu leucine; Y Tyr tyrosine
Embodiment:
Embodiment 1The clone of anthrax bacillus protective antigen PA encoding gene
Be being protected property of clone antigen encoding gene from the anthrax bacillus genome, anthrax bacillus is cultured to state of saturation in 100ml SOC nutrient solution (0.5% yeast extract/2% tryptone/10mM sodium-chlor/2.5mM Repone K/10mM magnesium chloride/20mM sal epsom/20mM glucose), after centrifugal bacterial precipitation is resuspended among the 9.5ml TE, and add the sodium lauryl sulphate (SDS) of 0.5ml 10% and the Proteinase K (purchasing Man) of 50ul 20mg/ml in Bao Ling, mix afterwards in 37 ℃ of incubations 1 hour; Add 1.8ml 5M sodium-chlor and mix, 65 ℃ of incubations 1 hour; Add equal-volume phenol/chloroform/primary isoamyl alcohol (25: 24: 1 mixed) extracting 2 times then, and with 0.6 times of volume isopropanol precipitating, at last precipitation is dissolved in the TE solution, it is standby to obtain the anthrax bacillus genomic dna.According to known anthrax bacillus protective antigen gene sequence (Genebank numbering: NC001496) design a pair of primer; upstream primer is formed (SEQ ID NO:1) by 30 DNA that comprise initiator codon; downstream primer is formed (SEQ ID NO:2) by 30 DNA that comprise terminator codon, through PCR clone, and 80 ℃ of complete sex change 2 minutes; 95 ℃ 30 seconds; 55 ℃ 30 seconds, 68 ℃ 2 minutes, circulate 35 times; last 68 ℃ were extended being protected property antigen encoding gene cDNA 5 minutes.PA gene PCR product agarose gel electrophoresis is seen Fig. 5.5 ' end and 3 ' end at the PA encoding gene of cloning are introduced KpnI, NotI restriction enzyme site respectively, behind electrophoresis, reclaim respective segments, reclaim respective segments after KpnI, NotI enzyme are cut, at T
4Following 20 ℃ of 8 hours subclones of dna ligase effect are gone up on the corresponding multiple clone site to pCDNA3 carrier (Stratagen company), choose recombinant plasmid and carry out sequencing analysis, and inferring the amino acid of coded by said gene by resulting dna sequence dna, the result is consistent with the sequence that Genebank has announced.
Embodiment 2The clone of anthrax bacillus edema factor EF encoding gene
For clone from the anthrax bacillus genome obtains the edema factor encoding gene, anthrax bacillus is cultured to state of saturation in 100ml SOC nutrient solution (0.5% yeast extract/2% tryptone/10mM sodium-chlor/2.5mM Repone K/10mM magnesium chloride/20mM sal epsom/20mM glucose), after centrifugal bacterial precipitation is resuspended among the 9.5ml TE, and add the SDS of 0.5ml 10% and the Proteinase K of 50ul 20mg/ml, mix the back in 37 ℃ of incubations 1 hour; Add 1.8ml5M sodium-chlor and mix, 65 ℃ of incubations 1 hour; Add equal-volume phenol/chloroform/primary isoamyl alcohol extracting then 2 times, and with 0.6 times of volume isopropanol precipitating, at last precipitation is dissolved in the TE solution, it is standby to obtain the anthrax bacillus genomic dna.According to known anthrax bacillus edema factor gene order (Genebank numbering: NC001496) design a pair of primer; upstream primer is formed (SEQ ID NO:3) by 30 DNA that comprise initiator codon; downstream primer is formed (SEQ ID NO:4) by 30 DNA that comprise terminator codon, through PCR clone, and 80 ℃ of complete sex change 2 minutes; 95 ℃ 30 seconds; 55 ℃ 30 seconds, 68 ℃ 2 minutes, circulate 35 times; last 68 ℃ were extended being protected property antigen encoding gene cDNA 5 minutes.EF gene PCR product agarose gel electrophoresis is seen Fig. 6.5 ' end and 3 ' end at the EF encoding gene of cloning are introduced KpnI, NotI restriction enzyme site respectively, behind electrophoresis, reclaim respective segments, reclaim respective segments after KpnI, NotI enzyme are cut, at T
4Following 20 ℃ of 8 hours subclones of dna ligase effect are gone up on the corresponding multiple clone site to pCDNA3 carrier (Stratagen company), choose recombinant plasmid and carry out sequencing analysis, and inferring the amino acid of coded by said gene by resulting dna sequence dna, the result is consistent with the sequence that Genebank has announced.
Embodiment 3The clone of Pseudomonas aeruginosa exotoxin A (ETA) encoding gene
For clone from the Pseudomonas aeruginosa genome obtains the exotoxin A encoding gene, Pseudomonas aeruginosa is cultured to state of saturation in 100ml SOC nutrient solution (0.5% yeast extract/2% tryptone/10mM sodium-chlor/2.5mM Repone K/10mM magnesium chloride/20mM sal epsom/20mM glucose), after centrifugal bacterial precipitation is resuspended among the 9.5ml TE, and add the SDS of 0.5ml 10% and the Proteinase K of 50ul 20mg/ml, mix the back in 37 ℃ of incubations 1 hour; Add 1.8ml5M sodium-chlor and mix, 65 ℃ of incubations 1 hour; Add equal-volume phenol/chloroform/primary isoamyl alcohol extracting then 2 times, and with 0.6 times of volume isopropanol precipitating, at last precipitation is dissolved in the TE solution, it is standby to obtain the Pseudomonas aeruginosa genomic dna.According to known Pseudomonas aeruginosa exotoxin A gene sequence (Genebank numbering: K01397) design a pair of primer, upstream primer is formed (SEQ ID NO:5) by 30 DNA that comprise initiator codon, downstream primer is formed (SEQ ID NO:6) by 30 DNA that comprise terminator codon, through PCR clone, and 80 ℃ of complete sex change 2 minutes, 95 ℃ 30 seconds, 55 ℃ 30 seconds, 68 ℃ 2 minutes, circulate 35 times, last 68 ℃ were extended 5 minutes, and obtained exotoxin A encoding gene cDNA.ETA gene PCR product agarose gel electrophoresis is seen Fig. 7.5 ' end and 3 ' end at the ETA encoding gene of cloning are introduced HindIII, NotI restriction enzyme site respectively, behind electrophoresis, reclaim respective segments, reclaim respective segments after HindIII, NotI enzyme are cut, at T
4Following 20 ℃ of 8 hours subclones of dna ligase effect are gone up on the corresponding multiple clone site to pcDNA3 carrier (Stratagen company), choose recombinant plasmid and carry out sequencing analysis, and inferring the amino acid of coded by said gene by resulting dna sequence dna, the result is consistent with the sequence that Genebank has announced.
Embodiment 4The structure of recombination fusion protein LP14 encoding gene
For making up recombinant protein LP14 encoding gene plasmid, the method for using gene engineering is formed by connecting adenosine diphosphate ribose base functional domain encoding gene in protective antigen combined function territory encoding gene and the Pseudomonas aeruginosa exotoxin A among the edema factor EF.At first the PCR clone obtains edema factor encoding gene 5 ' end 1～735 gene fragment L1 and Pseudomonas aeruginosa exotoxin A encoding gene 3 ' end 1092～1842 gene fragment L2.L1 gene amplification is template with pCDNA3/ef, and introduces the HindIII restriction endonuclease sites at 3 ' end, 80 ℃ of complete sex change 2 minutes, 95 ℃ 30 seconds, 55 ℃ 30 seconds, 68 ℃ 1 minute, circulate last 68 ℃ of extensions 5 minutes 35 times.Upstream primer is by 30 DNA based compositions that comprise initiator codon (SEQ ID NO:3), and downstream primer is by 30 DNA based compositions that comprise HindIII restriction enzyme base (SEQ ID NO:7).L2 gene amplification is template with pCDNA3/eta, and introduces the HindIII restriction endonuclease sites at 5 ' end, 80 ℃ of complete sex change 1 minute, 95 ℃ 30 seconds, 55 ℃ 30 seconds, 68 ℃ 2 minutes, circulate last 68 ℃ of extensions 5 minutes 35 times.Upstream primer is by 30 DNA based compositions that comprise HindIII restriction enzyme base (SEQ ID NO:8), and downstream primer is by 30 DNA based compositions that comprise terminator codon (SEQ ID NO:6).At last with two gene fragments of L1, L2 through the HindIII restriction endonuclease sites at T
4The effect of DNA enzyme obtains the encoding gene of recombinating, i.e. LP14 encoding gene (SEQ ID NO:13), and with LP14 encoding gene fragment again through T
4The DNA enzyme connects 8 hours subclones to the pBluescript carrier for 20 ℃.See Fig. 8.
Embodiment 5The structure of recombination mutation protein I P33 encoding gene
For making up IP33 recombinant protein encoding gene plasmid; use the Overlap PCR method that " AGAAAAAAGCGA " deoxyribonucleotide base mutation in the natural protective antigen PA encoding gene is obtained for " CCAGGAAGTGGAA AATCAGCA ", Dui Ying amino acid sports " PGSGKSA " by " RKKR " with it.In Overlap PCR, with pCDNA3/pa is template, at first amplify the gene fragment M1 and the M2 of two sequences that overlap respectively with aminoterminal and two pairs of primers of carboxyl terminal (SEQ ID NO:1,9 and SEQ ID NO:10,2), the PCR reaction conditions is, 80 ℃ of complete sex change 2 minutes, 95 ℃ 30 seconds, 55 ℃ 30 seconds, 68 ℃ 2.5 minutes, circulate 35 times, last 68 ℃ were extended 5 minutes.The PCR product is diluted in (test kit is purchased the company in Qiagen) behind the 150ul PB solution, is transferred in the microcentrifugation post, after 70% ethanol cleans, is dissolved among the TE.These two gene fragments of M1, M2 are mixed the back as template, 80 ℃ of complete sex change 2 minutes, 95 ℃ 30 seconds, 55 ℃ 30 seconds, 68 ℃ 3 minutes, circulate 35 times, last 68 ℃ were extended 5 minutes, with the primer that comprises initiator codon is upstream primer (SEQ ID NO:1), and the primer that comprises terminator codon is downstream primer (SEQ ID NO:2), obtains IP33 encoding gene (SEQ ID NO:11) through PCR.The PCR product downcuts the purpose fragment and is dissolved in (purified reagent is purchased the company in Qiagen) in the QG damping fluid, and be transferred in the microcentrifugation post behind agarose gel electrophoresis, after 70% ethanol cleans, is dissolved in TE and obtains target gene fragment; Target gene fragment is again through 37 ℃ of digestion of KpnI and NotI restriction enzyme after 2 hours, through T
4The DNA enzyme connects 8 hours for 20 ℃, and subclone is to the pcDNA3 carrier.See Fig. 9.
Embodiment 6The expression and purification of recombinant protein LP14 and IP33
PcDNA3/lp14 under the effect of KpnI, NotI restriction enzyme 37 ℃ digestion 2 hours, after the gelose gel electrophoresis separates, downcut the purpose fragment and be dissolved in (purified reagent is purchased the company in Qiagen) in the QG damping fluid, and be transferred in the microcentrifugation post, after 70% ethanol cleans, be dissolved in TE and obtain the lp14 target gene fragment; Target gene fragment is again through T
48 hours subclones of 20 ℃ of effects of dna ligase obtain the Yeast expression carrier of LP14 to the corresponding KpnI of Yeast expression carrier pPICZ α A (available from Invitrogen company), NotI restriction endonuclease sites.Identical method is downcut with KpnI, NotI restriction enzyme the IP33 gene fragment from the pcDNA3 recombinant plasmid, and subclone obtains the Yeast expression carrier of IP33 to the corresponding KpnI of Yeast expression carrier pPICZ α A (available from Invitrogen company), NotI restriction endonuclease sites.Recombinant expression plasmid is respectively through 37 ℃ of SacI restriction enzymes digestion 2 hours, make its linearizing after, be converted among the yeast GS115 (available from Invitrogen company), and screening growth on the flat board that contains on the Zeocin (100ug/ml).Getting one respectively is cloned in the 10ml conditioned medium (1% yeast extract, 2% peptone, 2% glucose, 100ug/ml Zeocin) 28 ℃ to grow to OD595 is 12, centrifugal then collection yeast, and be resuspended in the 10ml methyl alcohol substratum (1% yeast extract, 2% peptone, 100mM PH6.0 potassium phosphate buffer, 0.5% methyl alcohol, 0.00001% vitamin H, the basic nitrogen of 1.3% yeast are former), and in 28 ℃ of growths 2 days.Yeast in 1600g centrifugal 20 minutes is at last got supernatant and is stored in-80 ℃ rapidly, and yeast sedimentation is resuspended in the fresh culture standby.Get 20ul culture supernatant row 15%SDS-PAGE electrophoresis, see Figure 10 and Figure 11.
Embodiment 7The preparation of LP14 and IP33 mixture (TPCB)
According to the binding characteristic of IP33 and LP14 in theory, reorganization LP14 that the foregoing description 6 is prepared and IP33 albumen freeze-drying powder be according to 1: 3 mol ratio (being equivalent to mass ratio is 1: 3.8) uniform mixing, every of 2mg be stored in-80 ℃ standby; Face with before being dissolved among the PBS, mother liquid concentration is 2mg/ml, and is diluted to different final concentrations according to different test objectives.
Sequence table
SEQ?ID?NO：1
Deoxyribonucleotide several 30
Molecule type DNA
The source reorganization
The sequence description of SEQ ID NO:1
ttt?ggt?acc?atg?aaa?aaa?cga?aaa?gtg?tta
SEQ?ID?NO：2
Deoxyribonucleotide several 30
Molecule type DNA
The source reorganization
The sequence description of SEQ ID NO:2
ttt?gcg?gcc?gct?tat?cct?atc?tca?tag?cct
SEQ?ID?NO：3
Deoxyribonucleotide several 30
Molecule type DNA
The source reorganization
The sequence description of SEQ ID NO:3
ttt?ggt?acc?atg?aat?gaa?cat?tac?act?gag
SEQ?ID?NO：4
Deoxyribonucleotide several 30
Molecule type DNA
The source reorganization
The sequence description of SEQ ID NO:4
ttt?gcg?gcc?gct?tat?ttt?tca?tca?ata?att
SEQ?ID?NO：5
Deoxyribonucleotide several 30
Molecule type DNA
The source reorganization
The sequence description of SEQ ID NO:5
ttt?aag?ctt?atg?cac?ctg?ata?ccc?cat?tgg
SEQ?ID?NO：6
Deoxyribonucleotide several 30
Molecule type DNA
The source reorganization
The sequence description of SEQ ID NO:6
ttt?gcg?gcc?gct?tac?ttc?agg?tcc?tcg?cgc
SEQ?ID?NO：7
Deoxyribonucleotide several 30
Molecule type DNA
The source reorganization
The sequence description of SEQ ID NO:7
ttt?aag?ctt?ttt?ctc?aaa?tcc?ccc?ttt?ttc
SEQ?ID?NO：8
Deoxyribonucleotide several 30
Molecule type DNA
The source reorganization
The sequence description of SEQ ID NO:8
ttt?aag?ctt?gcg?gcc?aac?gcc?gac?gtg?gtg
SEQ?ID?NO：9
Deoxyribonucleotide several 43
Molecule type DNA
The source reorganization
The sequence description of SEQ ID NO:9
tgc?tga?tct?tcc?act?tcc?tgg?tga?gtt?cga?aga?ttt?ttg?ttt?t
SEQ?ID?NO：10
Deoxyribonucleotide several 45
Molecule type DNA
The source reorganization
The sequence description of SEQ ID NO:10
cca?gga?agt?gga?aga?tca?gca?agt?aca?agt?gct?gga?cct?acg?gtt
SEQ?ID?NO：11
Amino acid no 739
Molecule type amino acid
The source reorganization
The sequence description of SEQ ID NO:11
MEVKQENRLLNESESSSQGLLGYYFSDLNFQAPMVVTSSTTGDLSIPSSELENIPSENQYFQSAIWSGFIKVKKSD
EYTFATSADNHVTMWVDDQEVINKASNSNKIRLEKGRLYQIKIQYQRENPTEKGLDFKLYWTDSQNKKEVISSDNL
QLPELKQKSSNSPGSGRSASTSAGPTVPDRDNDGIPDSLEVEGYTVDVKNKRTFLSPWISNIHEKKGLTKYKSSPE
KWSTASDPYSDFEKVTGRIDKNVSPEARHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTISKNTSTSRTHT
SEVHGNAEVHASFFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTGTAPIYN
VLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQLRLDT
DQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTARIIFNGKDLNLVERRIAAVNPSDPLETTKPDMTLKEALK
IAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIA
VGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKK
YNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG
SEQ?ID?NO：12
Few nucleotide 2220
Molecule type DNA
The source reorganization
The sequence description of SEQ ID NO:12
1?atg?gaa?gtt?aaa?cag?gag?aac?cgg?tta?tta?aat?gaa?tca?gaa?tca?agt?tcccag?ggg?tta
M???E??V???K??Q???E??N???R??L???L??N???E??S???E??S???S??S???Q??G???L
61?cta?gga?tac?tat?ttt?agt?gat?ttg?aat?ttt?caa?gca?ccc?atg?gtg?gtt?acctct?tct?act
L???G??Y???Y??F???S??D???L??N???F??Q???A??P???M??V???V??T???S??S???T
121?aca?ggg?gat?tta?tct?att?cct?agt?tct?gag?tta?gaa?aat?att?cca?tcg?gaaaac?caa?tat
T???G??D???L??S???I??P???S??S???E??L???E??N???I??P???S??E???N??Q???Y
181?ttt?caa?tct?gct?att?tgg?tca?gga?ttt?atc?aaa?gtt?aag?aag?agt?gat?gaatat?aca?ttt
F???Q??S???A??I???W??S???G??F???I??K???V??K???K??S???D??E???Y??T???F
241?gct?act?tcc?gct?gat?aat?cat?gta?aca?atg?tgg?gta?gat?gac?caa?gaa?gtgatt?aat?aaa
A???T??S???A??D???N??H???V??T???M??W???V??D???D??Q???E??V???I??N???K
301?gct?tct?aat?tct?aac?aaa?atc?aga?tta?gaa?aaa?gga?aga?tta?tat?caa?ataaaa?att?caa
A???S??N???S??N???K??I???R??L???E??K???G??R???L??Y???Q??I???K??I???Q
361?tat?caa?cga?gaa?aat?cct?act?gaa?aaa?gga?ttg?gat?ttc?aag?ttg?tac?tggacc?gat?tct
Y???Q??R???E??N???P??T???E??K???G??L???D??F???K??L???Y??W???T??D???S
421?caa?aat?aaa?aaa?gaa?gtg?att?tct?agt?gat?aac?tta?caa?ttg?cca?gaa?ttaaaa?caa?aaa
Q???N??K???K??E???V??I???S??S???D??N???L??Q???L??P???E??L???K??Q???K
481?tct?tcg?aac?tca?cca?gga?agt?gga?aga?tca?gca?agt?aca?agt?gct?gga?cct
acg?gtt?cca
S???S??N???S??P???G??S???G??R???S??A???S??T???S??A???G??P???T??V???P
541?gac?cgt?gac?aat?gat?gga?atc?cct?gat?tca?tta?gag?gta?gaa?gga?tat?acggtt?gat?gtc
D???R??D???N??D???G??I???P??D???S??L???E??V???E??G???Y??T???V??D???V
601?aaa?aat?aaa?aga?act?ttt?ctt?tca?cca?tgg?att?tct?aat?att?cat?gaa?aagaaa?gga?tta
K???N??K???R??T???F??L???S??P???W??I???S??N???I??H???E??K???K??G???L
661?acc?aaa?tat?aaa?tca?tct?cct?gaa?aaa?tgg?agc?acg?gct?tct?gat?ccg?tacagt?gat?ttc
T???K??Y???K??S???S??P???E??K???W??S???T??A???S??D???P??Y???S??D???F
721?gaa?aag?gtt?aca?gga?cgg?att?gat?aag?aat?gta?tca?cca?gag?gca?aga?cacccc?ctt?gtg
E???K??V???T??G???R??I???D??K???N??V???S??P???E??A???R??H???P??L???V
781?gca?gct?tat?ccg?att?gta?cat?gta?gat?atg?gag?aat?att?att?ctc?tca?aaaaat?gag?gat
A???A??Y???P??I???V??H???V??D???M??E???N??I???I??L???S??K???N??E???D
841?caa?tcc?aca?cag?aat?act?gat?agt?caa?acg?aga?aca?ata?agt?aaa?aat?acttct?aca?agt
Q???S??T???Q??N???T??D???S??Q???T??R???T??I???S??K???N??T???S??T???S
901?agg?aca?cat?act?agt?gaa?gta?cat?gga?aat?gca?gaa?gtg?cat?gcg?tcg?ttcttt?gat?att
R???T??H???T??S???E??V???H??G???N??A???E??V???H??A???S??F???F??D???I
961?ggt?ggg?agt?gta?tct?gca?gga?ttt?agt?aat?tcg?aat?tca?agt?acg?gtc?gcaatt?gat?cat
G???G??S???V??S???A??G???F??S???N??S???N??S???S??T???V??A???I??D???H
1021?tca?cta?tct?cta?gca?ggg?gaa?aga?act?tgg?gct?gaa?aca?atg?ggt?tta?aatacc?gct?gat
S???L??S???L??A???G??E???R??T???W??A???E??T???M??G???L??N???T??A???D
1081?aca?gca?aga?tta?aat?gcc?aat?att?aga?tat?gta?aat?act?ggg?acg?gct?ccaatc?tac?aac
T???A??R???L??N???A??N???I??R???Y??V???N??T???G??T???A??P???I??Y???N
1141?gtg?tta?cca?acg?act?tcg?tta?gtg?tta?gga?aaa?aat?caa?aca?ctc?gcg?acaatt?aaa?gct
V???L??P???T??T???S??L???V??L???G??K???N??Q???T??L???A??T???I??K???A
1201?aag?gaa?aac?caa?tta?agt?caa?ata?ctt?gca?cct?aat?aat?tat?tat?cct?tctaaa?aac?ttg
K???E??N???Q??L???S??Q???I??L???A??P???N??N???Y??Y???P??S???K??N???L
1261?gcg?cca?atc?gca?tta?aat?gca?caa?gac?gat?ttc?agt?tct?act?cca?att?acaatgaat?tac
A???P??I???A??L???N??A???Q??D???D??F???S??S???T??P???I??T???M??N???Y
1321?aat?caa?ttt?ctt?gag?tta?gaa?aaa?acg?aaa?caa?tta?aga?tta?gat?acg?gatcaa?gta?tat
N???Q??F???L??E???L??E???K??T???K??Q???L??R???L??D???T??D???Q??V???Y
1381?ggg?aat?ata?gca?aca?tac?aat?ttt?gaa?aat?gga?aga?gtg?agg?gtg?gat?acaggc?tcg?aac
G???N??I???A??T???Y??N???F??E???N??G???R??V???R??V???D??T???G??S???N
1441?tgg?agt?gaa?gtg?tta?ccg?caa?att?caa?gaa?aca?act?gca?cgt?atc?att?tttaat?gga?aaa
W???S??E???V??L???P??Q???I??Q???E??T???T??A???R??I???I??F???N??G???K
1501?gat?tta?aat?ctg?gta?gaa?agg?cgg?ata?gcg?gcg?gtt?aat?cct?agt?gat?ccatta?gaa?acg
D???L??N???L??V???E??R???R??I???A??A???V??N???P??S???D??P???L??E???T
1561?act?aaa?ccg?gat?atg?aca?tta?aaa?gaa?gcc?ctt?aaa?ata?gca?ttt?gga?tttaac?gaa?ccg
T???K??P???D??M???T??L???K??E???A??L???K??I???A??F???G??F???N??E???P
1621?aat?gga?aac?tta?caa?tat?caa?ggg?aaa?gac?ata?acc?gaa?ttt?gat?ttt?aatttc?gat?caa
N???G??N???L??Q???Y??Q???G??K???D??I???T??E???F??D???F??N???F??D???Q
1681?caa?aca?tct?caa?aat?atc?aag?aat?cag?tta?gcg?gaa?tta?aac?gca?act?aacata?tat?act
Q???T??S???Q??N???I??K???N??Q???L??A???E??L???N??A???T??N???I??Y???T
1741?gta?tta?gat?aaa?atc?aaa?tta?aat?gca?aaa?atg?aat?att?tta?ata?aga?gataaa?cgt?ttt
V???L??D???K??I???K??L???N??A???K??M???N??I???L??I???R??D???K??R???F
1801?cat?tat?gat?aga?aat?aac?ata?gca?gtt?ggg?gcg?gat?gag?tca?gta?gtt?aaggag?gct?cat
H???Y??D???R??N???N??I???A??V???G??A???D??E???S??V???V??K???E??A???H
1861?aga?gaa?gta?att?aat?tcg?tca?aca?gag?gga?tta?ttg?tta?aat?att?gat?aaggat?ata?aga
R???E??V???I??N???S??S???T??E???G??L???L??L???N??I???D??K???D??I???R
1921?aaa?ata?tta?tca?ggt?tat?att?gta?gaa?att?gaa?gat?act?gaa?ggg?ctt?aaagaa?gtt?ata
K???I??L???S??G???Y??I???V??E???I??E???D??T???E??G???L??K???E??V???I
1981?aat?gac?aga?tat?gat?atg?ttg?aat?att?tct?agt?tta?cgg?caa?gat?gga?aaaaca?ttt?ata
N???D??R???Y??D???M??L???N??I???S??S???L??R???Q??D???G??K???T??F???I
2041?gat?ttt?aaa?aaa?tat?aat?gat?aaa?tta?ccg?tta?tat?ata?agt?aat?ccc?aattat?aag?gta
D???F??K???K??Y???N??D???K??L???P??L???Y??I???S??N???P??N???Y??K???V
2101?aat?gta?tat?gct?gtt?act?aaa?gaa?aac?act?att?att?aat?cct?agt?gag?aatggg?gat?act
N???V??Y???A??V???T??K???E??N???T??I???I??N???P??S???E??N???G??D???T
2161?agt?acc?aac?ggg?atc?aag?aaa?att?tta?atc?ttt?tct?aaa?aaa?ggc?tat?gagata?gga?taa
S???T??N???G??I???K??K???I??L???I??F???S??K???K??G???Y??E???I??G
SEQ?ID?NO：13
Amino acid acid number 499
Molecule type amino acid
The source reorganization
The sequence description of SEQ ID NO:13
MNEHYTESDIKRNHKTEKNKTEKEKFKDSINNLVKTEFTNETLDKIQQTQDLLKKIPKDVLEIYSELGGEIYFTDI
DLVEHKELQDLSEEEKNSMNSRGEKVPFASRFVFEKKRETPKLIINIKDYAINSEQSKEVYYEIGKGISLDIISKD
KSLDPEFLNLIKSLSDDSDSSDLLFSQKFKEKLELNNKSIDINFIKENLTEFQHAFSLAFSYYFAPDHRTVLELYA
PDMFEYMNKLEKGGFEKKLAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWT
VERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIR
NGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSA
IPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK
SEQ?ID?NO：14
Deoxyribonucleotide several 1500
Molecule type DNA
The source reorganization
The sequence description of SEQ ID NO:14
1???atgaatgaac?attacactga?gagtgatatt?aaaagaaacc?ataaaactga?aaaaaataaa
61??actgaaaaag?aaaaatttaa?agacagtatt?aataacttag?ttaaaacaga?atttaccaat
121?gaaactttag?ataaaataca?gcagacacaa?gacttattaa?aaaagatacc?taaggatgta
181?cttgaaattt?atagtgaatt?aggaggagaa?atctatttta?cagatataga?tttagtagaa
241?cataaggagt?tacaagattt?aagtgaagaa?gagaaaaata?gtatgaatag?tagaggtgaa
301?aaagttccgt?ttgcatcccg?ttttgtattt?gaaaagaaaa?gggaaacacc?taaattaatt
361?ataaatatca?aagattatgc?aattaatagt?gaacaaagta?aagaagtata?ttatgaaatt
421?ggaaagggga?tttctcttga?tattataagt?aaggataaat?ctctagatcc?agagttttta
481?aatttaatta?agagtttaag?cgatgatagt?gatagtagcg?accttttatt?tagtcaaaaa
541?tttaaagaga?agctagaatt?gaataataaa?agtatagata?taaattttat?aaaagaaaat
601?ttaactgaat?ttcagcatgc?gttttcttta?gcgttttctt?attattttgc?acctgaccat
661?agaacggtat?tagagttata?tgcccccgac?atgtttgagt?atatgaataa?gttagaaaaa
721?gggggatttg?agaaaaagct?tgcggccaac?gccgacgtgg?tgagcctgac?ctgcccggtc
781?gccgccggtg?aatgcgcggg?cccggcggac?agcggcgacg?ccctgctgga?gcgcaactat
841?cccactggcg?cggagttcct?cggcgacggc?ggcgacgtca?gcttcagcac?ccgcggcacg
901?cagaactgga?cggtggagcg?gctgctccag?gcgcaccgcc?aactggagga?gcgcggctat
961?gtgttcgtcg?gctaccacgg?caccttcctc?gaagcggcgc?aaagcatcgt?cttcggcggg
1021?gtgcgcgcgc?gcagccagga?cctcgacgcg?atctggcgcg?gtttctatat?cgccggcgat
1081?ccggcgctgg?cctacggcta?cgcccaggac?caggaacccg?acgcacgcgg?ccggatccgc
1141?aacggtgccc?tgctgcgggt?ctatgtgccg?cgctcgagcc?tgccgggctt?ctaccgcacc
1201?agcctgaccc?tggccgcgcc?ggaggcggcg?ggcgaggtcg?aacggctgat?cggccatccg
1261?ctgccgctgc?gcctggacgc?catcaccggc?cccgaggagg?aaggcgggcg?cctggagacc
1321?attctcggct?ggccgctggc?cgagcgcacc?gtggtgattc?cctcggcgat?ccccaccgac
1381?ccgcgcaacg?tcggcggcga?cctcgacccg?tccagcatcc?ccgacaagga?acaggcgatc
1441?agcgccctgc?cggactacgc?cagccagccc?ggcaaaccgc?cgcgcgagga?cctgaagtaa
Claims (10)
1, a kind of reorganization biological gene albumen comprises the activeconstituents with aminoacid sequence shown in the SEQ ID NO:11.
2, biological gene albumen as claimed in claim 1, aminoacid sequence SEQ ID NO:11 is wherein inferred out by the cDNA sequence of SEQ ID NO:12 representative.
3, a kind of reorganization biological gene albumen comprises the activeconstituents with aminoacid sequence shown in the SEQ ID NO:13.
4, biological gene albumen as claimed in claim 3, aminoacid sequence SEQ ID NO:13 is wherein inferred out by the cDNA sequence of SEQ ID NO:14 representative.
5, biological gene albumen as claimed in claim 3 is formed by protective antigen combined function territory in the anthrax bacillus edema factor and the fusion of bacterial exotoxin functional domain; Wherein, edema factor protective antigen combined function territory is by at least 245 amino acid of edema factor aminoterminal, and the proteic aminoacid sequence of no more than at the most complete edema factor constitutes.
6, a kind of recombinant bacteria toxin protein mixture, the nucleus of this mixture comprise the reorganization biological gene albumen of the activeconstituents with aminoacid sequence shown in the SEQID NO:11 and have the reorganization biological gene albumen of the activeconstituents of aminoacid sequence shown in the SEQ IDNO:13.
7, recombinant bacteria toxin protein mixture as claimed in claim 6 is characterized in that having the reorganization biological gene albumen of activeconstituents of aminoacid sequence shown in the SEQID NO:11 and the reorganization biological gene albumen freeze-drying powder of activeconstituents with aminoacid sequence shown in the SEQ IDNO:13 according to 1: 3 mol ratio uniform mixing.
8, a kind of pharmaceutical composition, the albumen composition that it is characterized in that containing the albumen of claim 1 or 3 or claim 6 is as activeconstituents.
9, the application in the preparation antitumor drug as claim 6 or 7 described recombinant bacteria toxin protein mixtures.
10, the application in the preparation antitumor drug as claim 1 or 3 described albumen.
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| CN 03143142 CN1249088C (en) | 2003-06-13 | 2003-06-13 | Antitumour medicine containing recombination bacterial toxin protein complex substance |
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| CN 03143142 CN1249088C (en) | 2003-06-13 | 2003-06-13 | Antitumour medicine containing recombination bacterial toxin protein complex substance |
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| CN1513878A true CN1513878A (en) | 2004-07-21 |
| CN1249088C CN1249088C (en) | 2006-04-05 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112500469A (en) * | 2020-12-16 | 2021-03-16 | 熊猫乳品集团股份有限公司 | Bioactive polypeptide AAPAAPAAAPPAE, and preparation method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112500469A (en) * | 2020-12-16 | 2021-03-16 | 熊猫乳品集团股份有限公司 | Bioactive polypeptide AAPAAPAAAPPAE, and preparation method and application thereof |
| CN112500469B (en) * | 2020-12-16 | 2022-03-29 | 熊猫乳品集团股份有限公司 | Bioactive polypeptide AAPAAPAAAPPAE, and preparation method and application thereof |
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| Publication number | Publication date |
|---|---|
| CN1249088C (en) | 2006-04-05 |
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