CN1854295A - Production of specific micro-antibody for oarium cancer - Google Patents
Production of specific micro-antibody for oarium cancer Download PDFInfo
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- CN1854295A CN1854295A CN 200510025361 CN200510025361A CN1854295A CN 1854295 A CN1854295 A CN 1854295A CN 200510025361 CN200510025361 CN 200510025361 CN 200510025361 A CN200510025361 A CN 200510025361A CN 1854295 A CN1854295 A CN 1854295A
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- idiotype
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Abstract
The invention discloses a ribotide sequence to code specific typed micro-antibody of ovary cancer and manufacturing method and constructing, expressing and purifying technology of relative engineering cell, which is fit for expressing in the escherichia coli and optimizes combined expressing plasmid and host bacteria and fermenting technology to improve expressing quantity.
Description
Technical field
The present invention relates to the genetically engineered field.More specifically, the invention provides a kind of method of High-efficient Production ovary cancer anti-idiotype micro-antibody, and the structure of relevant engineering cell, ovary cancer anti-idiotype micro-antibody expression and purifying process.
Background technology
Jerne in 1974 has proposed famous immune network theory according to the understanding of modern immunology antagonist molecule idiotype on the basis of Burnet " clonal selection theory ".This theory is thought, all exists idiotype on any antibody molecule or the lymphocytic antigen receptor, and they can be stimulated by other lymphocyte identifications in the body brings out the generation antiidiotype.With idiotype with antiidiotype be identified as the basis mutually, constitute " network " contact in the immunity system, in immunomodulatory, play an important role.According to immune network theory, antiidiotypic antibody (anti-Id or Ab
2β) (produce antibody behind the antigenic stimulation body, this antibody A b
1Stimulate body to produce anti-antibody Ab once more
2, anti-antibody is divided into three kinds, and wherein a kind of is antiidiotypic antibody, the Ab in the antiidiotypic antibody
2β has the effect of analogue antigen).Have the simulation original antigen and induce body to produce, thereby can suppress Ab at antigenic specific antibody of initiating or cellullar immunologic response
1Association reaction with corresponding antigens.Ab
2β has sequence of amino acid similar to exotic antigen or sterie configuration, and it can simulate the antigenic effect of initiating in vivo, uses Ab
2This characteristic of β can on purpose prepare Ab
2β is used for basis and clinic study with it as antigenic surrogate.Simultaneously, Ab
2β not only can excite at exotic antigen or autoantigen Ab1 and produce precursor cell, makes body to intruding into intravital antigen immunne response take place rapidly, and relevant with the existence of memory cell.After the immunne response that antigen is brought out by it is removed, because memory cell identification antigen receptor has higher avidity, thereby Ab
2β substitutes propagation and the survival that original antigen may enough stimulate memory cell.To Ab
2The antigenic Mechanism Study of β self simulation shows Ab
2β not only has the effect of analogue antigen, is again important immune-regulating factor simultaneously, participates in immunoreactive adjusting:
(1) Ab
2β can activate special cell clone by the sequence or the structure of analogue antigen, thereby enlarges the humoral immune reaction to specific antigen.
(2) body also can be with Ab
2β offers the cell to T with the form of antiidiotype peptide, makes CD
4+The T cell is bred in a large number, participates in cellular immunization, has experiment to show that this kind reaction might not limited by MHC.
Owing to bringing out immune response in human body, mouse source antibody protein limited it in the intravital application of people.Therefore, just begin one's study from the mid-80 people and the mouse monoclonal antibody is transformed or humanization with engineered method.Wherein small molecular antibody is one of important method.1993, Italian scholar Pessi at first used miniantibody (minibody) speech in its article, and that names a kind of synthetic only has 61 residues, but has the active antibody of bond.There is the investigator to carry out complete IgG at tumor-bearing mice, F (ab ') 2, the immune imaging results of scFv and miniantibody and the comparative studies of result of treatment found that the imaging results of miniantibody is best, and are good with complete IgG and miniantibody in result of treatment.
Ovarian tumor is gynaecology's kinds of tumor, accounts for 32% of female sex organ tumour.It can betide any age, can see every year until old from the infant, but the sickness rate of child-bearing age is the highest.Because ovary is positioned at pelvic cavity, their early stage seldom has symptom, and its malignant tumour very easily spreads and ascites occurs.Still lack practical method of early diagnosis at present both at home and abroad, and in a single day find, often belonged to late period.Although the treatment for ovarian cancer has had considerable progress in recent years, 5 years survival rates of ovarian cancer are still paced up and down about 30%, and case fatality rate surpasses cervical cancer and carcinoma of corpus uteri sum, the numerous women's of serious harm health.American National Instrument CancerInstitute statistic data in 1999 shows that the sickness rate of ovarian cancer is the 5th of women's cancer morbidity.Wherein the highest age bracket of morbidity is 30-54 year in white race women, Japan, Spain and the Philippines.
Because the tumour antigen of having found at present is few, and is difficult to a large amount of preparations.Thereby utilize Ab
2The characteristic of β prepares tumor vaccine extremely to be paid attention to, and is called " antiidiotype vaccine ".Wagner etc. with antiidiotypic antibody ACA125 as vaccine therapy the epithelial ovarian cancer of 16 example late periods or recurrence.These patients have accepted 3 to 9 times ACA125 immunotherapy, and wherein 9 routine patients induce and produced Ab
3(Ab
2β injects the animal of lotus tumour or patient and can induce and be referred to as to resist-resist-Ab of idiotype antibody
3) and specific cell immune response, 3 routine patient IFN-y raise.Be 11 months these patients' the lifetime that on average gets nowhere, and the negative group of Ab3 is 8 months.Remartz etc. carry out active immunity treatment with ACA125 to the ovarian cancer patients of 45 example late periods or recurrence, IFN-r in the T Cells in Peripheral Blood from Patients,, IL-2, IL-4 increases.
The COC166-9 mouse monoclonal antibody has specificity preferably to epithelial ovarian cancer, immunohistochemical staining confirms 80% epithelial ovarian cancer stained positive, and less with other malignant tissue's cross reactions, in the healthy tissues that had detected, do not find cross reaction as yet.With COC166-9 immunity homology mouse, the antiidiotypic antibody 6B11 for preparing has the function of simulation ovarian cancer antigen OC166-9, inducing producing specificity ovarian cancer resistance humoral immunization and cell immune response in vivo and in vitro.We construct the small molecular antibody gene with 6B11scFv (strand variable region) with the heavy chain CH3 fragment that has human IgG1's hinge area, i.e. anti-idiotype micro-antibody (anti-Idminibody) gene, and express.Because this antibody by merging with CH3, has increased molecular weight and stability, has realized the part humanization.When using in vivo, have scFv fragment and the incomparable superiority of other fusion roteins.
But because 6B11V
LV
HThe hC gene is an eukaryotic gene, present disclosed 6B11V
LV
HThe G and the C content of hC gene 5 ' end homing sequence are higher, contain more colibacillary rare codon, and the expression amount in intestinal bacteria is very low, is not suitable for scale operation.
System of the present invention provides a kind of method of High-efficient Production ovary cancer anti-idiotype micro-antibody.By optimizing the nucleotide sequence of coding ovary cancer anti-idiotype micro-antibody, optimization expression carrier/host bacterium combination improves zymotechnique simultaneously, improves the expression level of ovary cancer anti-idiotype micro-antibody.Easy purifying process simultaneously obtains high expression level, high yield, has a kind of ovary cancer anti-idiotype micro-antibody production method of industrialization value.
Summary of the invention
Purpose of the present invention just provides a kind of method of efficient and/or easy production ovary cancer anti-idiotype micro-antibody.
Another object of the present invention just provides the encoding sequence of ovary cancer anti-idiotype micro-antibody and is used for the expression vector and the engineering cell of this method.
In a first aspect of the present invention, just provided a kind of nucleotide sequence of the ovary cancer anti-idiotype micro-antibody of encoding, it is characterized in that, aminoacid sequence shown in the described nucleotide sequence coded SEQ ID NO:2, and nucleotides sequence is shown 95% above homogeny shown in described nucleotide sequence coded district and the SEQ ID NO:1.
In another preference, described nucleotide sequence contains the nucleotide sequence shown in the SEQ ID NO:1.
In a second aspect of the present invention, just provided the optimum combination of a kind of expression vector and host bacterium, it is characterized in that described expression vector contains the described nucleotide sequence of claim 1.
In another preference, described expression vector is pTrcHisA/6B11V
LV
HHC.
In a third aspect of the present invention, a kind of engineering cell is provided, it is characterized in that it is integrated with the described expression vector of claim 3.
In a fourth aspect of the present invention, a kind of method of producing ovary cancer anti-idiotype micro-antibody is provided, the method comprising the steps of:
A) under the expression condition that is fit to, cultivate host cell as claimed in claim 4, thereby give expression to ovary cancer anti-idiotype micro-antibody;
B) separation and purification goes out the ovary cancer anti-idiotype micro-antibody of expression.
In another preference of the present invention, the culture condition of described step (a) comprising:
(i) nitrogenous source contains organic nitrogenous source and inorganic nitrogen-sourced, wherein organic nitrogen source such as peptone, yeast powder, or the mixture of the two, total concn 0.1-3%; Inorganic nitrogen-sourced as ammoniacal liquor or NH
4Cl, (NH
4)
2SO
4Deng ammonium salt, concentration 0-1.5%.
(ii) inorganic salt comprise phosphoric acid salt, Chinese holly hydrochloride, Mg
2+Salt etc.Preferably, phosphate buffer concentration 20-200mM, pH5-8; Mg
2+Salt 0-5mM.
(iii) in substratum, add VITMAIN B1 VITAMIN as a supplement, concentration 1～1000PPM.
(iv) according to M
9Trace element formula in the substratum, trace element can add 0.1-2ml/L training liquid.
(v) carbon source comprises glycerine, glucose, lactose etc.Can be single carbon source, also can be mixed carbon source.Preferably, carbon source is selected from down group: glycerol concentration is 0.1-3%; Lactose concn is 0.1-3%; Glucose concn is 0.1-1.5%.
In another preference of the present invention, the separation condition of described step (b) comprising:
(vi) fermented sample is taken out fermented supernatant fluid by centrifugal and/or filter type, obtain thalline;
(vii) smudge cells obtains broken bacterium liquid;
(viii) broken bacterium liquid is carried out centrifugal to remove brokenly the bacterium supernatant liquor, the acquisition precipitation;
(ix) precipitation is become renaturation;
(x) by saltout and/after preliminary stratification is carried out in ultrafiltration, or, reach 95% pure product thereby obtain purity directly by the chromatography purification method.
Description of drawings
Fig. 1 has shown the building process of expression plasmid pTrcHisA/6B11VLVHhC.
Accompanying drawing 2 is that pcr amplification and the electrophoresis of human ovary cancer anti-idiotype micro-antibody gene 6B11VLVHhC identifies that wherein swimming lane 1 is 1kb DNA Ladder, and swimming lane 2 is the PCR product of 6B11VLVHhC gene.
Embodiment
Extensive studies by the optimization design of gene coded sequence, changes the ovary cancer anti-idiotype micro-antibody encoding sequence over to intestinal bacteria to the inventor by going deep into, thereby has realized high level expression, has finished the present invention on this basis.
In another preference,, further improved the expression level of ovary cancer anti-idiotype micro-antibody by the optimum combination of expression plasmid and host bacterium.
In another preference, also, not only improved the ovary cancer anti-idiotype micro-antibody expression amount, and simplified separation purifying technique by the optimization of zymotechnique.
The high level expression of foreign gene, at least relate to the mutual relationship between host, carrier and the clone gene three, also be subjected to the influence of its residing envrionment conditions, this research is by the multiple expression vector of optimized choice, as pBADHisA, pBAD/gIIIA, pTrcHisA etc., obtain the optimum combination of expression vector/host bacterium.With the 6B11V after modifying
LV
HHC inserts different expression vectors, transformed into escherichia coli, and by expressing experimental result, preferred vector pTrcHisA-host bacterium JF1125 combination has realized efficiently expressing in intestinal bacteria, expression amount accounts for about 50% of total protein of cell.Plasmid pTrcHisA copy number height has trc promotor, rrnBanti-termination region and rrnB transcription terminator, can make foreign gene obtain high efficiency stable expression; Simultaneously, host bacterium JF1125 is not vulnerable to infecting of microorganism during the fermentation.
For the extensive ovary cancer anti-idiotype micro-antibody that obtains, need in fermentor tank, express cultivation.The present invention has studied pilot scale fermentation technology, and the expression level after the optimization reaches 1500mg/L.
Through experiment repeatedly of the present invention, the expression condition after the optimization is as follows:
(1) substratum: high-density improvement M9 substratum.
(2) fermentation condition: 28～40 ℃ of culture temperature are more preferred from 30～37 ℃; PH6.0～8.0 are more preferred from 6.5～7.5; Inductor IPTG concentration is 0.1～2mM, is more preferred from 0.2～1mM.
Behind fermentation expression, the ovary cancer anti-idiotype micro-antibody inclusion body protein of expressing is separated.Usually, fermented sample is earlier removed fermented liquid supernatant in modes such as centrifugal, filtrations, obtains thalline.Behind the buffered soln suspension thalline, carry out broken fully bacterium, obtain broken bacterium liquid in modes such as ultrasonic disruption, high-pressure machinery fragmentations.Then broken bacterium liquid is carried out centrifugal to remove brokenly the bacterium supernatant liquor, the acquisition precipitation.Precipitation contains target protein matter ovary cancer anti-idiotype micro-antibody.Broken bacterium precipitation is become renaturation, and the activated ovary cancer anti-idiotype micro-antibody of acquisition descends the step purifying again.Purifying generally comprises two steps, and the first step employing is saltoutd and/or preliminary purification is carried out in ultrafiltration, and second step was adopted the chromatography purification method.Be applicable to that chromatographic technique of the present invention comprises cation-exchange chromatography, anion-exchange chromatography, gel permeation chromatography, affinity chromatography etc.
Compare through difference change renaturation and chromatography process, the purification process of optimization comprises:
1. adopt the mode of the broken thalline of high-pressure compressing, obtain thick inclusion body.
2. adopt the washings purifying inclusion body contain under certain denaturing agent condition, obtain purity greater than 85% inclusion body.
3. adopt the denaturing agent dissolving inclusion body that contains high density.
4. adopt the mode of dilution refolding, renaturation miniantibody albumen obtains activated miniantibody target protein.
5. adopt the mode of ultrafiltration to replace buffer system, make sample be fit to carry out chromatography purification.
6. adopt the mode of a step anion-exchange chromatography, and select the mode of certain gradient elution, obtain purity, removed other impurity such as nucleic acid simultaneously greater than 95% pure product of miniantibody.
Through purifying of last step, can obtain the pure product of ovary cancer anti-idiotype micro-antibody, the purifying yield is more than 40%, and purity is more than 95%, the about 500mg/L fermented liquid of pure product yield.
Ovary cancer anti-idiotype micro-antibody can be made various formulations with routine techniques behind the purifying.
In an example of the present invention, the acquisition of ovary cancer anti-idiotype micro-antibody gene and the structure of expression plasmid are provided, the gene after the optimization makes the expression amount of expressing ovary cancer anti-idiotype micro-antibody improve.
In another example of the present invention,, further improved the expression amount of ovary cancer anti-idiotype micro-antibody through the Combinatorial Optimization of expression vector and host bacterium.
In another example of the present invention, the optimization of technology has by fermentation further improved the expression amount of ovary cancer anti-idiotype micro-antibody.
In another example of the present invention,, can obtain pure product 500mg/L through purifying.
Stoste adds suitable auxiliary material behind the purifying, makes the powder ampoule agent for injection of ovary cancer anti-idiotype micro-antibody.The preliminarily stabilised test shows that this powder injection is stable.
Pilot scale research shows that product manufacture is stable, and is simple to operate, and the cycle is short, and cost is low, and every liter of fermented liquid can obtain the pure product 500mg of ovary cancer anti-idiotype micro-antibody.Be fit to industrialization production.
The invention has the advantages that:
(1) the ovary cancer anti-idiotype micro-antibody gene after the optimization is highly suitable for expression in escherichia coli, has the characteristics of high expression level, high stable.
(2) combination of optimized expression vectors and host bacterium has further improved expression amount, by controlling crucial technological condition for fermentation, makes expression level reach 1500mg/L.
(3) change renaturation technology is simple, protein renaturation rate height.
(4) purifying process is easy, and rate of recovery height makes the scale operation ovary cancer anti-idiotype micro-antibody become possibility.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The acquisition of goal gene and the structure of expression plasmid
The acquisition of goal gene
The human ovary cancer anti-idiotype micro-antibody gene source is in Beijing people hospital gynecological tumor research centre, and we have designed primer it is optimized, and the gene order after the optimization is shown in SEQ ID NO:1.Design a pair of primer, gene 5 ' terminal sequence of not optimizing is carried out pointed decoration, introduce NcoI, EcoRI restriction enzyme site simultaneously with the method for PCR.
The structure of expression plasmid
Select intestinal bacteria JF1125 as the host bacterium, and this bacterial strain is transformed, with the expression plasmid pTrcHisA/6B11V that builds
LV
HHC transformed into escherichia coli JF1125.From containing the LB flat board difference picking mono-clonal (JF1125/pTrcHisA/6B11V of 100 μ g/ml penbritins and 34 μ g/ml paraxin
LV
HHC), be inoculated in the 3ml LB liquid nutrient medium, 300rpm shaken overnight on 37 ℃ of constant temperature shaking tables is inoculated in 20mL LB (penbritin concentration is 100 μ g/ml and 34 μ g/ml paraxin) nutrient solution, at 37 ℃, is cultured to OD under the 250rpm condition
600=0.5-1.0, this moment, cell was in logarithmic phase; It is 1mM to final concentration that aseptic condition adds IPTG down, and continuation is at 37 ℃, and abduction delivering under the 250rpm condition was taken a sample after 3 hours, and SDS-PAGE detects expression.The result shows a band of expression near 41KD, the theoretical molecular basically identical of molecular weight and miniantibody.
Embodiment 2
The optimum combination of expression vector and host bacterium
Select intestinal bacteria JF1125 as the host bacterium, and this bacterial strain is transformed, with the expression plasmid pTrcHisA/6B11V that builds
LV
HHC, pBADHisA/6B11V
LV
HHC, pBAD/gIIIA/6B11V
LV
HHC is transformed into escherichia coli JF1125 respectively.From containing the LB flat board difference picking mono-clonal of 100 μ g/ml penbritins and 34 μ g/ml paraxin, be inoculated in the 3ml LB liquid nutrient medium, 300rpm shaken overnight on 37 ℃ of constant temperature shaking tables, be inoculated in 20mL LB (penbritin concentration is 100 μ g/ml and 34 μ g/ml paraxin) nutrient solution, at 37 ℃, be cultured to OD under the 250rpm condition
600=0.5-1.0, this moment, cell was in logarithmic phase; Then under aseptic condition to bacterial strain JF1125/pTrcHisA/6B11V
LV
HThe nutrient solution of hC add IPTG to final concentration be 1mM, bacterial strain JF1125/JF1125/pTrcHisA/6B11V
LV
HHC and JF1125/JF1125/pBAD/gIIIA/6B11V
LV
HAdd pectinose in the hC nutrient solution to final concentration 0.002%, continue at 37 ℃, abduction delivering under the 250rpm condition, sampling after 3 hours, SDS-PAGE detects expression.
As follows according to SDS-PAGE collection of illustrative plates scanning result:
| Bacterial strain | JF1125/pTrcHisA/6B11V L V HhC | JF1125/pBAD/gIIIA/6B11 V LV HhC | JF1125/pBAD HisA/6B11V LV HhC |
| Expression level (expression product accounts for the percentage ratio of bacterial protein) | 51％ | 18％ | 12％ |
Compare by experiment, adopt bacterial strain JF1125/pTrcHisA/6B11V
LV
HThe human ovary cancer anti-idiotype micro-antibody expression amount that hC obtains is the highest, and effect is obvious.
Embodiment 3
The ovary cancer anti-idiotype micro-antibody fermentation
Preparation LB fermentation seed liquid 300ml and M9 fermention medium 2.7L prepare 50% glucose and 5%CA feed supplement 100ml.Be placed in the 250ml Erlenmeyer flask.Sterilization.Seed liquor cooling back inoculation.After reaching OD 0.6～1, stop to cultivate jar in the inoculation.37 ℃ of controlled temperature, pH=7.0 is cultured to OD to 3.0, adds 0.6g IPTG and induces, receive after 3 hours jar, during flow feeding keep bacteria growing.Centrifugal collection thalline, SDS-PAGE electrophoresis result show that the target protein expression level reaches 50%.
Embodiment 4
The ovary cancer anti-idiotype micro-antibody purifying
| Program | Buffer system | Purpose |
| The thalline squeezing | 40mM TBS(pH9.0)+1mM EDTA | Obtain inclusion body |
| ↓ | ||
| The inclusion body washing | 40mM TBS (pH9.0)+1mM EDTA+2M urea+0.1 %Tween80 | Remove some lipids and nucleic acid class impurity |
| ↓ | ||
| Solubilization of inclusion bodies | 40mM TBS (pH9.0)+1mM EDTA+6M Guanidinium hydrochloride+0.5% mercaptoethanol | Obtain the inclusion body of solubility |
| ↓ |
| Snapback | 40mM TBS (pH9.0)+0.1% mercaptoethanol+2% sucrose+0.1%Tween80+0.4M Guanidinium hydrochloride | Obtain stable renaturation intermediate |
| ↓ | ||
| Dilution ultrafiltration renaturation | 40mM TBS (pH9.0)+0.1% mercaptoethanol | Obtain renaturation sample completely |
| ↓ | ||
| The purifying of Q post | Buffer A: 40mM TBS (pH9.0)+0.1% mercaptoethanol buffer B: 40mM TBS (pH9.0)+0.1% mercaptoethanol+1M NaCl | Albumen after the consummate renaturation |
Through above purifying process, can get the pure product 500mg/L of albumen at last.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Xinshengyuan Medicine Research Co., Ltd.
＜120〉production method of ovary cancer anti-idiotype micro-antibody
<160>2
<210>1
<211>1143
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)…(1143)
＜223〉the human ovary cancer anti-idiotype micro-antibody gene of You Huaing
<400>1
ATGGGAGATA TCGAACTTAC TCAATCACCG CTGTCCCTGC CTGTCAGTCT 50
TGGAGATCAA GCCTCCATCT CTTGCAGATC TAGTCAGAAC CTTGTACACA 100
GTAATGGAAA CACCTATTTA CATTGGTACC TGCAGAAGCC AGGCCAGTCT 150
CCAAAGCTCC TGATCTACATA GTTTCCAACC GATTTTCTGG GGTCCCAGAC 200
AGGTTCAGTG GCAGTGGATC AGGGACAGAT TTCACACTCA AGATCAGCAG 250
AGTGGAGGCT GATGATCTGG GAGTTTATTT CTGCTCTCAG AGTACACATT 300
TTCCGTACAC GTTCGGAGGG GGGACAAAGT TGGAAATAAA ACGGGGTGGA 350
GGCGGTTCAG GCGGAGGTGG CTCTGGCGGT GGCGGATCGC AGGTCAAACT 400
GCAGGAGTCT GGCCCTGGGA TATTGCAGCC CTCCCAGACC CTCAGTCTGA 450
CTTGTTCTTT CTCTGGGCTT TCACTGCACA CTTATGGTAT AGGAGTAGGC 500
TGGATTCGTC AGCCTTCAGG GAAGGGTCTG GAGTGGCTGG CACACATTTG 550
GTGGAATAAT AATAAGTACT ATAACACAGC CCTGAAGAGC CGGCTCACTG 600
TCTCCAAGGA CGCCTCCAAC AACCAGGTAT TCCTCAACAT CGCCAGTGTG 650
GACACTGCAG ATACTGCCAC ATACTACTGT GCTCGAATAG CCCTTATTAC 700
TACGAAAATA GCGTGGTACT TCGATGTCTG GGGCCAAGGC ACCACGGTCA 750
CCGTCTCCTC AGGATCCGAG TCCAAATCTT GTGACAAAAC TCACACATGC 800
CCACCGTGCC CACTCGAGGG GCAGCCCCGA GAACCACAGG TGTACACCCT 850
GCCCCCATCC CGGGATGAGC TGACCAAGAA CCAGGTCAGC CTGACCTGCC 900
TGGTCAAAGG CTTCTATCCC AGCGACATCG CCGTGGAGTG GGAGAGCAAT 950
GGGCAGCCGG AGAACAACTA CAAGACCACG CCTCCCGTGC TGGACTCCGA 1000
CGGCTCCTTC TTCCTCTACA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC 1050
AGCAGGGGAA CGTCTTCTCA TGCTCCGTGA TGCATGAGGC TCTGCACAAC 1100
CACTACACGC AGAAGAGCCT CTCCCTGTCT CCGGGTAAAT GA 1142
<210>2
<211>380
<212>PRT
＜213〉homo sapiens
<400>2
MET Gly Asp Ile Glu Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Ser
1 5 10 15
Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Leu Val
20 25 30
His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly
35 40 45
Gln Ser Pro Lys Leu Leu Ile Tyr Ile Val Ser Asn Arg Phe Ser Gly
50 55 60
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
65 70 75 80
Lys Ile Ser Arg Val Glu Ala Asp Asp Leu Gly Val Tyr Phe Cys Ser
85 90 95
Gln Ser Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu
100 105 110
Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gln Val Lys Leu Gln Glu Ser Gly Pro Gly Ile Leu Gln Pro
130 135 140
Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Leu Ser Leu His
145 150 155 160
Thr Tyr Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly
165 170 175
Leu Glu Trp Leu Ala His Ile Trp Trp Asn Asn Asn Lys Tyr Tyr Asn
180 185 190
Thr Ala Leu Lys Ser Arg Leu Thr Val Ser Lys Asp Ala Ser Asn Asn
195 200 205
Gln Val Phe Leu Asn Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr
210 215 220
Tyr Tyr Cys Ala Arg Ile Ala Leu Ile Thr Thr Lys Ile Ala Trp Tyr
225 230 235 240
Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser
245 250 255
Glu Ser Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Leu
260 265 270
Glu Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
275 280 285
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
290 295 300
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
305 310 315 320
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
325 330 335
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
340 345 350
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
355 360 365
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
370 375 380
Claims (8)
1. the nucleotide sequence of the ovary cancer anti-idiotype micro-antibody of encoding, it is characterized in that, aminoacid sequence shown in the described nucleotide sequence coded SEQ ID NO:2, and nucleotides sequence is shown 95% above homogeny shown in described nucleotide sequence coded district and the SEQ ID NO:1.
2. nucleotide sequence as claimed in claim 1 is characterized in that, contains the aminoacid sequence shown in the SEQ ID NO:1.
3. the optimum combination of expression vector and host bacterium is characterized in that described expression vector contains the described nucleotide sequence of claim 1.
4. an engineering cell is characterized in that, it is integrated with the described expression vector of claim 3.
5. engineering cell as claimed in claim 4 is characterized in that it is intestinal bacteria.
6. method of producing ovary cancer anti-idiotype micro-antibody is characterized in that the method comprising the steps of:
(a) under the expression condition that is fit to, cultivate engineering cell as claimed in claim 4, thereby give expression to ovary cancer anti-idiotype micro-antibody;
(b) separation and purification goes out the ovary cancer anti-idiotype micro-antibody of expression.
7. method as claimed in claim 6 is characterized in that, the culture condition of described step (a) comprising: 28～40 ℃ of culture temperature are more preferred from 30～37 ℃; PH6.0～8.0 are more preferred from 6.5～7.5; Inductor IPTG concentration is 0.1～2mM, is more preferred from 0.2～1mM.
8. method as claimed in claim 6 is characterized in that, the separation condition of described step (b) comprising:
(i) fermented sample is taken out fermented supernatant fluid by centrifugal and/or filter type, obtain thalline;
(ii) smudge cells obtains broken bacterium liquid;
(iii) broken bacterium liquid is carried out centrifugal to remove brokenly the bacterium supernatant liquor, the acquisition precipitation;
(iv) precipitation is become renaturation;
(, or, reach 95% pure product thereby obtain purity directly by the chromatography purification method v) by saltouing and/or preliminary purification is carried out in ultrafiltration.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510025361 CN1854295A (en) | 2005-04-25 | 2005-04-25 | Production of specific micro-antibody for oarium cancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510025361 CN1854295A (en) | 2005-04-25 | 2005-04-25 | Production of specific micro-antibody for oarium cancer |
Publications (1)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102753194A (en) * | 2009-12-02 | 2012-10-24 | 伊麦吉纳博公司 | J591 minibodies and cys-diabodies for targeting human prostate specific membrane antigen (psma) and methods for their use |
| US11254744B2 (en) | 2015-08-07 | 2022-02-22 | Imaginab, Inc. | Antigen binding constructs to target molecules |
| US11266745B2 (en) | 2017-02-08 | 2022-03-08 | Imaginab, Inc. | Extension sequences for diabodies |
-
2005
- 2005-04-25 CN CN 200510025361 patent/CN1854295A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102753194A (en) * | 2009-12-02 | 2012-10-24 | 伊麦吉纳博公司 | J591 minibodies and cys-diabodies for targeting human prostate specific membrane antigen (psma) and methods for their use |
| US8772459B2 (en) | 2009-12-02 | 2014-07-08 | Imaginab, Inc. | J591 minibodies and Cys-diabodies for targeting human prostate specific membrane antigen (PSMA) and methods for their use |
| US11180570B2 (en) | 2009-12-02 | 2021-11-23 | Imaginab, Inc. | J591 minibodies and cys-diabodies for targeting human prostate specific membrane antigen (PSMA) and methods for their use |
| US11254744B2 (en) | 2015-08-07 | 2022-02-22 | Imaginab, Inc. | Antigen binding constructs to target molecules |
| US11266745B2 (en) | 2017-02-08 | 2022-03-08 | Imaginab, Inc. | Extension sequences for diabodies |
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