CN1443850A - Method for preparing recombinant human CTLA4 extracellular region protein, and obtained product and application - Google Patents
Method for preparing recombinant human CTLA4 extracellular region protein, and obtained product and application Download PDFInfo
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Abstract
The present invention relates to a production method for producing CTLA4 extracellular region protein by using high-expression yeast engineering bacterium strain, in which said engineering bactrium strain contains the gene segment as shown in sequence 1, and the CTLA4 extracellular region protein produced by utilizing above-mentioned gene engineering method and the application in preparation of medicine for ucring some diseases related to the over activation of T cell.
Description
Technical field
The present invention relates to the genetically engineered field.More specifically, the present invention relates to use the high expression level Yeast engineering bacterium strain to produce the production method of CTLA4 extracellular region protein, wherein this project bacterial strain contains just like gene fragment shown in the sequence 1; And the CTLA4 extracellular region protein produced of said gene engineering method and be used for the treatment of purposes in the medicine of some and T cell transition activation diseases associated in preparation.
Background technology
Cytotoxic T lymphocyte antigen 4 (Cytotoxic T Lymphocyte Antigen 4, hereinafter to be referred as " CTLA4 ") be the 4th the specific antigens molecule of in T cell otherness clone, finding, its by signal peptide, extracellular region, stride the film district and intracellular region is formed, the major function of extracellular region is to combine with the corresponding part of CTLA4 such as B7 molecule, CTLA4 antibody etc.Because of B7-CD28 is a most important subsidiary signal approach in the T cell activation, and CTLA4 is bigger 100 times than CD28 molecule with the bonding force of B7 molecule, so when CTLA4 with after the B7 molecule combines, the B7 molecule can not combine with CD28, thereby blocked this approach, and then blocking t cell activation, thereby to significant treatment and prophylactic effect (Lenschow DJ being arranged with T cell transition activation diseases associated such as various autoimmune disease and transplanting back immunological rejection etc., Walunas TL, the common CD28/B7 system (CD28/B7 system of T cell costimulation) that stimulates of Bluestone JA.T cell. immunology yearbook (Annu Rev Immunol.) 1996; 14:233-58; Thompson CB, AllisonJP.CTLA4 is as the negative effect of adjusting (The emerging role of CTLA4 as animmune attenuator) of immunity. immunity (Immunity) 1997; 7 (4): 445-450 and WaterhouseP etc., CTLA-4, a kind of negative conditioning agent of T-lymphocyte activation (CTLA-4, a negativeregulator of T-lymphocyte activation), immunology summary (Immunol.Rew.) 1996; 153; 183-207).
But, so far, do not find this fragment is developed as the genetically engineered medicine.In fact, abroad some company has for other reasons with this fragment and part IgG fusion carrying out drug development program.And to understand as those of ordinary skills, the superiority that gene engineering method production medicine compares to traditional method is conspicuous.
Therefore, press for a kind of can production steadily in the long term in the prior art and have the method for the recombinant human CTLA 4 extracellular region protein of identical biologic activity, to solve the technical problem of the required a large amount of CTLA4 extracellular region proteins of various diseases that clinical treatment caused because of the activation of T cell transition with natural CTLA4 extracellular region.
Summary of the invention
At above-mentioned needs of the prior art, the inventor is through long-term exploration and a large amount of research, finally successfully invent the method that a kind of preparation can be applicable to clinical recombinant human CTLA 4 extracellular region protein, thereby solved long-term puzzlement those skilled in the art's technical barrier.
Therefore, first aspect of the present invention, a kind of a kind of method for preparing recombinant human CTLA 4 extracellular region protein of method for preparing recombinant human CTLA 4 extracellular region protein is provided, has it is characterized in that using the high expression level Yeast engineering bacterium strain of the nucleic acid that contains SEQ ID No.1 that genetically engineered makes up to carry out.
Second aspect of the present invention, a kind of Yeast engineering bacterium strain that provides has been integrated multiple copied recombinant human CTLA 4 extracellular region cDNA ceneme in this strain gene group.This yeast strain can express recombinant human CTLA 4 extracellular region protein, and the sequence of this recombinant protein is as described in the SEQ ID No.2.
The 3rd aspect of the present invention, the recombinant human CTLA 4 extracellular region protein of being produced by aforesaid method is provided, its sequence has identical biological function shown in SEQ ID No.2 and with the CTLA4 extracellular region, promptly with B7 molecule bonded function, thereby treatment and prophylactic effect are arranged because of T cell transition reactivity disease to various.
The 4th aspect of the present invention provides the purposes of Yeast engineering bacterium strain in the suitability for industrialized production recombinant human CTLA 4 extracellular region protein of above-mentioned " second aspect ".
The 5th aspect of the present invention, the recombinant human CTLA 4 extracellular region protein that above-mentioned " the 3rd aspect " be provided is used for the treatment of and prevents purposes in the medicine of those and T cell transition activation diseases associated in preparation.
The 6th aspect of the present invention, the recombinant human CTLA 4 extracellular region protein that above-mentioned " the 3rd aspect " be provided is in treatment and prevent those and T cell transition to activate purposes in the diseases associated.
But in following " detailed Description Of The Invention ", especially on the basis of the disclosure of " embodiment " part, other aspects and advantages of the present invention are conspicuous to those skilled in the art.
Description of drawings
Fig. 1. the structure of pIC9-400 expression vector of the present invention.
Fig. 2. the structure of pPIC9K-400 expression vector of the present invention.
Fig. 3. identify the photo of Western Blotting of the expression of target protein of the present invention.
Fig. 4. CTLA4 extracellular region protein of the present invention is to the restraining effect of the CTLL-2 cell proliferation of IL-2 stimulation.
Fig. 5. CTLA4 extracellular region of the present invention is to the restraining effect of cell proliferation in the mixed lymphocyte reacion.
Fig. 6. CTLA4 extracellular region of the present invention is to producing the influence of IL-2 in the mixed lymphocyte reacion.
Fig. 7. CTLA4 extracellular region of the present invention and commercialization CTLA4Ig suppress the comparison of mixed lymphocytes proliferative response.
Fig. 8. CTLA4 extracellular region of the present invention to allotransplantation heart, skin, kidney dis current influence.
Detailed Description Of The Invention
In the context of the present specification, unless specialize, otherwise used any of this specification Technical term has the implication that those of ordinary skills understand in the art usually, and does not annotate The experimental technique of bright actual conditions is according to the normal experiment method, translates molecular cloning such as Jin Dongyan etc. The experiment guide second edition, Science Press, Beijing, 1992; Wu Naihu etc., Principles of Gene Engineering, Science Press, Beijing, 2000; The thick plinth of Zhu etc. is translated, protein purification and identification experiment guide, Science Press, Beijing, 1999; With the Li Yuyang chief editor, gene expression technique, Science Press, Beijing, 2000 described carry out; Or carry out according to the operational manual that supplier advises.
Disclosed in this invention is how to prepare a kind of can be applicable to outside the clinical recombinant human CTLA 4 born of the same parents The method of district's albumen. The human CTLA 4 total length has 237 amino acid, by 41 amino acid of signal peptide, 125 amino acid of extracellular region, stride 34 amino acid of film district 37 amino acid and cytoplasmic domain and form, this Invent related CTLA4 extracellular region and be actually the part of CTLA4 extracellular region, i.e. extracellular region Totally 124 amino acid whose preparations of the 1st to the 124th.
The invention provides a kind of a kind of preparation heavily of method for preparing recombinant human CTLA 4 extracellular region protein Organize the method for human CTLA 4 extracellular region protein, it is characterized in that using the SEQ that contains of genetic engineering structure The high expressed Yeast engineering bacterium strain of the cDNA of ID No.1 carries out.
Preferably, method of the present invention is characterised in that following steps:
1) in human peripheral, clones human CTLA 4 extracellular region cDNA, its sequence such as SEQ ID No.1;
2) this cDNA is inserted in the suitable carrier, make up single copy expression vector;
3) 2) basis structure multicopy expression vector;
4) constructed expression vector electricity is transformed in the saccharomycete, filters out and contain the genes of interest fragment The high expressed bacterial strain;
5) positive colony is cultivated, expressed target protein; With
6) isolate recombinant human CTLA 4 extracellular region protein, its sequence is shown in SEQ ID No.2.
More preferably, this method comprises: 1) clone human CTLA 4 extracellular region cDNA in human peripheral, its sequence such as SEQ ID No.1; 2) this cDNA is inserted in the pIC9 plasmid, make up single copy expression vector; 3) 2) make up pIC9K multiple copied expression vector on the basis; 4) constructed expression vector electricity is transformed in the GS115 yeast, filters out the high expression level bacterial strain that contains target gene fragment; 5) positive colony is cultivated, expressed target protein; With 6) isolate recombinant human CTLA 4 extracellular region protein, its sequence such as SEQ ID No.2.
Wherein, after having obtained human CTLA 4 extracellular region cDNA, can be inserted in the suitable carriers, so that transform or identify.Be applicable to that carrier of the present invention comprises various carrier known in the art.For example select commercially available various carriers for use, the human CTLA 4 extracellular region cDNA that the present invention cloned can be inserted the multiple clone site of carrier, the integrated expression vector of reorganization can take place with the yeast genes group in formation." integrated expression vector " be meant and can the DNA reorganization take place with the host yeast genome, thereby the exogenous gene expression unit integration is gone into carrier in the host bacterium genome.Suitable integrating vector includes, but is not limited to: pIC9, pIC9k, p α ADH2 etc.
Host cell of the present invention is preferably pichia pastoris phaff (pichia pastoris) GS115, but can also be intestinal bacteria, insect cell or mammalian cell.
The various methods that foreign gene is imported host cell all are suitable for the present invention, and these methods include, but is not limited to: electroporation, particle bombardment, protoplasm body, lyticase method and chemical process etc.
Behind constructed expression vector importing host cell, can screen according to corresponding resistant maker gene, for example in substratum, add penbritin, G418 etc., thereby filter out the positive colony of high expression level.
The inventor filters out a large amount of Yeast engineering bacterium strains that reaches purpose of the present invention by aforesaid method.Its expression level is at least the 50mg/L culture, is preferably the 100mg/L culture, is most preferably the 200mg/L culture.
One of Yeast engineering bacterium strain that the present invention sieves is pichia pastoris phaff (pichiapastoris), it is preserved in BeiJing ZhongGuanCun China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on February 8th, 2002, and preserving number is CGMCC NO.0714.And this yeast expression level is the 100mg/L culture.
Integrated the yeast clone of multiple copied recombinant human CTLA 4 extracellular region ceneme in the genome that the expression level of engineering strain filters out, may under the condition that is fit to this recombinant human CTLA 4 extracellular region protein of expression, cultivate this yeast strain, to express target protein.Because expression vector of the present invention is an excretion vector, so the recombinant human CTLA 4 extracellular region protein of expressing is secreted in culture supernatant.
After having obtained to contain recombinant human CTLA 4 extracellular region protein yeast supernatant, can use ordinary method known in the art and carry out separation and purification, thereby obtain highly purified recombinant human CTLA 4 extracellular region protein.Suitable separation method comprises (but being not limited only to): albumen precipitation, ion exchange chromatography, molecule chromatography, affinity chromatography, HPLC etc.
Recombinant human CTLA 4 extracellular region protein that the present invention is prepared and engineering strain have following advantage: the expression level height, normal conditions are assigned the 50-100mg/L culture, and can reach the expression level of 200mg/L culture, and the expressing protein biologic activity of purifying obviously is better than commercial similar reagent recombinant C TLA4Ig (for more than the latter's the twice) at aspects such as suppressing mixed lymphocyte reacion.In addition, it also has the following advantages:
1) the recombinant human CTLA 4 extracellular region ceneme is integrated in the yeast cell genome, thereby can express the target protein recombinant human CTLA 4 extracellular region protein steadily in the long term.
2) recombinant human CTLA 4 extracellular region protein is an abduction delivering in the engineering yeast strain, is that application of pure methyl alcohol carries out abduction delivering in one embodiment of the present of invention.
3) the prepared recombinant human CTLA 4 extracellular region protein of the present invention has identical biologic activity with natural CTLA4 extracellular region, can be used for the various diseases that clinical treatment is caused because of the activation of T cell transition.
In brief, the recombinant human CTLA 4 extracellular region protein engineering strain expression level that the present invention obtains is up to the 200mg/L culture, and expression is very stable, and adjustable, and product has the biological function identical with natural fragment.
Another aspect of the present invention, the recombinant human CTLA 4 extracellular region protein of being produced by aforesaid method is provided, its sequence has identical biological function shown in SEQ ID No.2 and with the CTLA4 extracellular region, promptly with B7 molecule bonded function, thereby treatment and prophylactic effect are arranged because of T cell transition reactivity disease to various.
In addition, the invention provides the pharmaceutical composition that can be applicable to clinical recombinant human CTLA 4 extracellular region protein, it contains recombinant human CTLA 4 extracellular region protein and the pharmaceutically acceptable carrier or the vehicle for the treatment of effective dose, and suitable carriers includes, but is not limited to: salt solution, damping fluid, glucose, water, glycerine, N.F,USP MANNITOL, ethanol and combination thereof.
Simultaneously, the invention provides above-mentioned recombinant human CTLA 4 extracellular region protein is used for the treatment of and prevents purposes in the medicine of those and T cell transition activation diseases associated in preparation.Based on being complementary with administering mode, pharmaceutical composition of the present invention can be made into various pharmaceutical preparations.For example, recombinant human CTLA 4 extracellular region protein of the present invention can be made into dry powder injection form, and carries out the ordinary method preparation by the injection liquid etc. of using physiological saline, water for injection or containing glucose; Also can be made into oral preparations such as tablet, capsule; Also can be made into application such as sprays, liniment.The dosage of active ingredient is a dose therapeutically effective, for example the 10ug/kg body weight.
In addition, the present invention also provides the purposes of the Yeast engineering bacterium strain that the present invention screened in producing recombinant human CTLA 4 extracellular region protein.
At last, the present invention's recombinant human CTLA 4 extracellular region protein that gained of the present invention also is provided is in treatment with prevent those and T cell transition to activate purposes in the diseases associated.
Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.In the following example, the experimental technique of unreceipted actual conditions carries out according to the normal experiment method usually, or carries out according to the operation instructions that manufacturer advises.
Embodiment 1
With RT-PCR method clone human CTLA 4 extracellular region cDNA
Get normal people's peripheral blood 5ml, conventional gradient centrifugation separation of human mononuclearcell, the mRNA that provides with Qigan company extracts test kit extraction mRNA wherein, as template, with the capable RT-PCR of random primer, be template with this product again, there is justice 5 ' GGCTCGAGAAAAGAATGCATGTGGCACAGCCTGCT (SEQ No.3) antisense 5 '-GCGAATTCTTAGTCAGAATCTGGGCACGGTTCTGG (SEQ No.4) to carry out PCR with following implementation sequence primer, amplifies the purpose fragment of 400bp.
To amplify product among the embodiment 1, results are purified into the purpose fragment of 400bp size after agarose electrophoresis.With this fragment EcoR I, Xho I enzyme is cut in the directed pIC9 of the insertion expression vector of back, screens in importing the host bacterium, extracts the correct pIC9-400 expression plasmid (Fig. 1) that contains human CTLA 4 extracellular region cDNA that inserted.
Embodiment 3
Contain the preparation of the multiple copied expression vector pIC9K-400 of human CTLA 4 extracellular region cDNA
PIC9-400 is the directed pIC9K that inserts after Bam HI, Sal I enzyme are cut, and obtains pIC9K-400.Sac I enzyme is cut the back electricity changes GS115, and the G418 gradient filters out positive transformed bacteria (Fig. 2).
In the YPD nutrient solution, 30 ℃ of oscillating growths spend the night with pichia pastoris phaff (pichia pastoria) GS115 bacterial classification inoculation, to bacterium liquid OD
600Reach between the 1.1-1.3, will in the bacterium liquid after the pIC9K-400 plasmid after the Sac I enzymic digestion adds washing, get in an amount of adding conductivity cell, carry out electricity with 1500V/25 μ F/400 Ω and transform.The yeast-inoculated that electricity is transformed is in the MD culture medium flat plate, place 30 ℃ of incubators to cultivate 3-5 days, again positive colony is connected in the G418 culture plate that contains the different concns gradient, cultivate after 3-5 days, that selects in the highest G418 concentration flat board goes out positive colony, goes into 30 ℃ of shaking culture of MD substratum 36 hours.Extract DNA and identify, contain human CTLA 4 extracellular region cDNA in the conclusive evidence institute selected clone.Height copy is cloned in the culturing bottle cultivates, behind methanol induction, with the capable immunoblotting of putting of anti-human CTLA 4 antibody, with the expression of further conclusive evidence goal gene.One of engineering strain of being screened is preserved in BeiJing ZhongGuanCun China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on February 8th, 2002, and preserving number is CGMCC NO.0714.
Embodiment 5
The expression of recombinant human CTLA 4 extracellular region protein
Institute's selected clone is inoculated in the 25ml BMGY nutrient solution, spends the night in 30 ℃ of shaking culture, change over to again in the 1000ml YPD nutrient solution and cultivated about 20 hours, to OD with 250-300rpm speed
600Be 4.0-6.0.Be inoculated in the 30L fermentor tank with 1: 10, it is 30 ℃ that leavening temperature is set, and regulates rotating speed and oxygen-supply quantity, and cell is well grown, and works as OD
600(need 20-30 hour approximately) when increasing to 80-100, slowly continue to add an amount of methyl alcohol, induce the expression of target protein, continue to cultivate after about 72 hours following jar.And after cultivation, put mutually 0,24,48,72,96 etc. the time sample thief a little, with 10000rpm centrifugal after, supernatant is frozen in-80 ℃, in order to analyzing and purifying is used.
Embodiment 6
The evaluation of the target protein of expressing SDS-PAGE
The poly-propionic acid amide gel of preparation 12.5%SDS-is whenever gone up sample 30ul on the sample hole.Behind the electrophoresis at sized molecules amount place, the 28000 dalton left and right sides visible target protein band (contrast blank bacterium liquid and not inducing in the bacterium liquid, do not have this band) clearly, size conforms to design.This protein band prolonged and strengthens to some extent with expression time.
Immunoblotting (western blot) detects
The same SDS-PAGE that carries out takes off gel behind the electrophoresis, electricity forwards to pvdf membrane, uses the mouse-anti human CTLA 4 monoclonal antibody that has Biotin and hybridizes, and develops the color in conjunction with back DAB with Avidin-HRP again.As seen visible stronger positive band (contrast blank bacterium liquid and do not induce in the bacterium liquid, do not have this band) is (Fig. 3) at sized molecules amount place, the 28000 dalton left and right sides.
Embodiment 7
The purifying of target protein recombinant human CTLA 4 extracellular region protein
10000rpm is centrifugal with fermented liquid, gets supernatant, concentrates with the 5k hollow fiber column ultrafilter, last sample is crossed the QFF ion exchange column, collects elution peak, after the molecular sieve gel post, collecting molecular weight is 28000 daltonian components, concentrates the highly purified recombinant human CTLA 4 extracellular region protein of preparation again through dialysing.
Embodiment 8
The mensuration of target protein expression amount and yield
Target protein through above-mentioned process expression and purifying obtain shows that through the Xylene Brilliant Cyanine G concentration determination target protein yield that this technology is expressed and purifying obtains is respectively 100mg/L culture and 40mg/L culture.
Embodiment 9
External and the interior effect detection of body of recombinant human CTLA 4 extracellular region protein Interaction in vitro detects
1) can express the CTLL-2 cell inoculation of B7 and CD28 molecule simultaneously in 96 well culture plates, add the different concns recombinant human CTLA 4 extracellular region protein of being produced, detect the restraining effect of recombinant human CTLA 4 extracellular region protein with mtt assay after 3 days the growth of CTLL-2 cell.Find that recombinant human CTLA 4 extracellular region protein can obviously suppress the propagation (Fig. 4) of CTLL-2 cell.
2) in mixed lymphocytes is cultivated, add prepared different concns recombinant human CTLA 4 extracellular region protein, find that it can obviously suppress cell proliferation and IL-2 generation (Fig. 5 and 6) in the mixed lymphocytes.
3) similar reagent C TLA4Ig with commercialization (Chimerigen company, Britain) relatively, find the resulting CTLA4 extracellular region protein of this technology to mixed lymphocytes activatory inhibiting rate than the former high about 2 times (Fig. 7).
Effect detects in the body
In transplantation models such as rat allosome/xenogenesis skin, kidney, heart, tracheae, add with prepared various dose recombinant human CTLA 4 extracellular region protein, find that it can obviously prolong the survival of transplanting, and be certain dose-dependence (Fig. 8).
All documents that occur in this specification sheets all are incorporated by reference in this text in this application and examine, and just quote as a reference separately as each piece document.Should be understood that in addition those of ordinary skills can be used for various modifications and revise the present invention after reading the above-mentioned instruction content of this specification sheets, these equivalent form of values fall within this specification sheets appended claims institute restricted portion equally.
＜110〉＜120〉CTLA4＜130〉I020058＜160〉4＜170〉PatentIn version 3.1＜210〉1＜211〉372＜212〉DNA＜213〉Homo sapiens＜400〉1atgcatgtgg cacagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360ccagattctg at 372＜210〉2＜211〉124＜212〉PRT＜213〉Homo sapiens＜400〉2Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile1 5 10 15Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30Arg?Val?Thr?Val?Leu?Arg?Gln?Ala?Asp?Ser?Gln?Val?Thr?Glu?Val?Cys
35 40 45Ala?Ala?Thr?Tyr?Met?Met?Gly?Asn?Glu?Leu?Thr?Phe?Leu?Asp?Asp?Ser
50 55 60Ile?Cys?Thr?Gly?Thr?Ser?Ser?Gly?Asn?Gln?Val?Asn?Leu?Thr?Ile?Gln65 70 75 80Gly?Leu?Arg?Ala?Met?Asp?Thr?Gly?Leu?Tyr?Ile?Cys?Lys?Val?Glu?Leu
85 90 95Met?Tyr?Pro?Pro?Pro?Tyr?Tyr?Leu?Gly?Ile?Gly?Asn?Gly?Thr?Gln?Ile
100 l05 110Tyr?Val?Ile?Asp?Pro?Glu?Pro?Cys?Pro?Asp?Ser?Asp
115 120<210>3<211>35<212>DNA<213>Artificial<400>3ggctcgagaa?aagaatgcat?gtggcacagc?ctgct 35<210>4<211>35<212>DNA<213>Artificial<400>4gcgaattctt?agtcagaatc?tgggcacggt?tctgg 35
Claims (17)
1. a method for preparing recombinant human CTLA 4 extracellular region protein is characterized in that the high expression level Yeast engineering bacterium strain that contains the Nucleotide shown in the SEQ ID No.1 that uses genetically engineered to make up carries out.
2. the method for claim 1 is characterized in that following steps:
1) in human peripheral, clones human CTLA 4 extracellular region cDNA, its sequence such as SEQ ID No.1;
2) this cDNA is inserted in the suitable carrier, make up single copy expression vector;
3) 2) make up the multiple copied expression vector on the basis;
4) constructed expression vector electricity is transformed in the yeast, filters out the high expression level bacterial strain that contains target gene fragment;
5) positive colony is cultivated, expressed target protein; With
6) isolate recombinant human CTLA 4 extracellular region protein, its sequence is shown in SEQ ID No.2.
3. claim 1 or 2 method is characterized in that said cDNA is inserted in the pIC9 plasmid, make up single copy expression vector.
4. claim 1 or 2 method is characterized in that wherein constructed multiple copied expression vector is a pIC9K multiple copied expression vector.
5. claim 1 or 2 method is characterized in that wherein the yeast as host cell is the GS115 yeast.
6. claim 1 or 2 method, it is characterized in that high expression level bacterial strain wherein is the pichia pastoris phaff engineering strain, it is preserved in BeiJing ZhongGuanCun China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on February 8th, 2002, has preserving number CGMCC NO.0714.
7. claim 1 or 2 method, the expression that it is characterized in that Yeast engineering bacterium strain wherein is an abduction delivering.
8. the method for claim 7 is characterized in that wherein abduction delivering carries out with methyl alcohol.
9. expression vector, it contains the nucleotide sequence shown in the SEQ ID No.1.
10. gene engineering microzyme strain, it contains the expression vector of claim 9.
11. the engineering yeast strain of claim 10, it is a pichia pastoris phaff, and it is preserved in BeiJing ZhongGuanCun China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on February 8th, 2002, has preserving number CGMCC NO.0714.
12. a recombinant human CTLA 4 extracellular region protein, its be produce by each method among the claim 1-8 and its sequence shown in SEQ ID No.2.
13. a pharmaceutical composition contains recombinant human CTLA 4 extracellular region protein and the pharmaceutical carrier or the vehicle of claim 12.
14. the pharmaceutical composition of claim 13, it is used for the treatment of and prevents those and T cell transition to activate diseases associated.
15. the purposes of engineering yeast strain in the suitability for industrialized production recombinant human CTLA 4 extracellular region protein of claim 10 or 11.
16. the recombinant human CTLA 4 extracellular region protein of claim 12 is used for the treatment of and prevents purposes in the medicine of those and T cell transition activation diseases associated in preparation.
17. the recombinant human CTLA 4 extracellular region protein of claim 12 is in treatment and prevent those and T cell transition to activate purposes in the diseases associated.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1322122C (en) * | 2003-11-03 | 2007-06-20 | 中国科学院微生物研究所 | Xylanase, production and special producing strain thereof |
| CN104672333A (en) * | 2013-11-27 | 2015-06-03 | 深圳先进技术研究院 | Human-derived CTLA4 fusion protein and its preparation method and use |
| CN113372451A (en) * | 2010-02-19 | 2021-09-10 | Xencor公司 | Novel CTLA4-IG immunoadhesins |
-
2002
- 2002-03-08 CN CN 02107034 patent/CN1443850A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1322122C (en) * | 2003-11-03 | 2007-06-20 | 中国科学院微生物研究所 | Xylanase, production and special producing strain thereof |
| CN113372451A (en) * | 2010-02-19 | 2021-09-10 | Xencor公司 | Novel CTLA4-IG immunoadhesins |
| CN104672333A (en) * | 2013-11-27 | 2015-06-03 | 深圳先进技术研究院 | Human-derived CTLA4 fusion protein and its preparation method and use |
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