CN101597616A - A kind of method that improves expression quantity of gene recombinant human coagulation factor 8 - Google Patents
A kind of method that improves expression quantity of gene recombinant human coagulation factor 8 Download PDFInfo
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- CN101597616A CN101597616A CNA2008100385047A CN200810038504A CN101597616A CN 101597616 A CN101597616 A CN 101597616A CN A2008100385047 A CNA2008100385047 A CN A2008100385047A CN 200810038504 A CN200810038504 A CN 200810038504A CN 101597616 A CN101597616 A CN 101597616A
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Abstract
The present invention passes through Protocols in Molecular Biology, structure contains internal ribosome entry site (the internal ribosome entry site of reduction, abbreviate " IRES " as) new expression vector of sequence and dual selection markers, this carrier can be used to recombinant expressed human blood coagulation factor VII I, by comprehensive screening of stably express strain, can improve the expression amount of blood coagulation factor VIII in mammalian cell effectively.
Description
Technical field
The present invention relates to a kind of method that improves gene recombinant human blood coagulation factor VIII expression amount, relate in particular to a kind of method that in mammalian cell, improves gene recombinant human blood coagulation factor VIII expression amount.
Background technology
Blood coagulation factor VIII (Factor VIII-related Antigen, abbreviate " FVIII " as) be a blood plasma polymer glycoprotein, form by 2332 amino-acid residues, blood coagulation factor VIII has played crucial effect in the intrinsic coagulation system, it is the cofactor that activates plasma thromboplastin component.The FVIII assignment of genes gene mapping is in Xq28, and total length 186kb is made up of 26 exons and 25 introns.The quantity not sufficient or the abnormal quality of body intravascular coagulation Factor IX will cause haemophilia A.Haemophilia A is a kind of common X sex chromosome linkage inheritance hemorrhagic diseases, and the sickness rate in the male sex is 1: 10,000.Point mutation, little genetically deficient cause that slight FVIII lacks; Terminator codon, big genetically deficient, insertion, displacement, nonsense mutation often cause that serious FVIII lacks and clinical phenotypes.Confirmed that FVIII 22 intron inversions sudden changes causes that the FVIII famine is the molecule pathogenesis of 45% heavy hemophilia A, also finds the sudden change of FVIII introne 1 inversion recently in 1% heavy hemophilia A.
Main methods of treatment to this disease is the concentrated FVIII preparation that extracts from human plasma at present.But often import blood products, the patient is communicable disease such as infective virus hepatitis and AIDS very easily, and medical expense is also very expensive, and in developed country, the medical expense in patient every year is usually at U.S. dollar more than 10,000 yuan.Simultaneously, because FVIII is extremely unstable, and the content in blood plasma is very little, 1 liter of blood plasma only contains 150 microgram FVIII, and the efficient of extracting FVIII from refrigerated plasma is not high, and therefore the dense preparation of FVIII that provides at present only can be used for treatment, and seldom as conventional prophylactic.In addition, some patient produces acute allergic reaction to the dense preparation of FVIII easily, and has 15% patient can produce FVIII antibody, has increased the treatment difficulty more.
Since FVIII cDNA in 1984 cloned, respectively at CHO, successfully obtaining in the systems such as 3T3 had bioactive rFVIII, FDA to ratify to be applied to clinical.Preliminary clinical trial shows: reorganization FVIII has identical biochemistry, immunity and the pharmacological characteristics of FVIII with the blood source, the bleeding tendency that can effectively correct the haemophiliac, the clinical result who has obtained satisfaction.
FVIII is starkly lower than gene kin with it at external expression amount, has only 1% of FIX as the expression level of FVIII, has the negative regulation mechanism that FVIII is expressed in this explanation cell, makes FVIII remain on a lower state in intracellular level.This may be the reflection of body to the FVIII demand, but to being a major obstacle by in-vitro recombination expression FVIII.In addition, at present in the world to finding also in the trial of haemophiliachemophiliac gene therapy that the expression amount of keeping FVIII has bigger difficulty at treatment level, its reason also be in the artificial constructed FVIII expression system FVIII expression amount can not to satisfy treatment required. this has caused very big difficulty to of fine quality, the inexpensive FVIII of large-scale commercial production.
Summary of the invention
The objective of the invention is the reconstruction by gene, improve the expression amount of the FVIII in mammalian cell effectively, thereby make up novel reorganization FVIII expression plasmid, set up expression system, the line stabilization of going forward side by side is expressed comprehensive screening of strain.
In order to achieve the above object, the present invention adopts following technical scheme:
A kind of method that improves recombinant human blood coagulation factor VII I expression amount comprises the steps:
With the internal ribosome entry site sequence IRES processing that weakens;
Described sequence one end after the reduction connects goal gene, and the other end connects the selection markers gene, makes up the expression vector that contains dual selection markers, wherein, and the shared promotor of described goal gene and selection markers gene.
Preferably, described selection markers gene is dihydrofolate reductase gene or glutamine synthetase gene, and described expression vector is pIRES-DHFR or pIRES-GS.
Further, also comprise gene by methotrexate or MSX pressurization amplification selection markers gene both wings.
Further, described reduction processing is meant by the bicistronic mRNA carrier selection markers gene dihydrofolate reductase gene or glutamine synthetase gene is connected in 3 of internal ribosome entry site sequence IRES ' end that selection markers expression of gene level is reduced.
Further, also comprise and make up different blood coagulation factor VIII expression systems, comprise B domain disappearance but comprise the VIII factor [blood coagulation factor VIII (Δ B)] of novel Linker sequence, with the VIII factor [blood coagulation factor VIII (HL)] of blood coagulation factor VIII weight, light chain coexpression, and therefrom screen suitable engineering cell strain.
Internal ribosome entry site (the internal ribosome entry site of the present invention by containing reduction, abbreviating " IRES " as) expression vector that contains dual selection markers of sequence comes express recombinant FVIII, greatly simplify screening process, improve screening efficiency, improve the acquisition high expression level and stablize the probability of strain, promptly through a mono-clonal screening, can obtain a large amount of stable expression cell strains, the positive rate of monoclonal cell reaches 95%.
Description of drawings
Fig. 1 is the expression vector pIRES-DHFR/GS synoptic diagram that has dual selection markers among the present invention.
Fig. 2 is the structure synoptic diagram of different FVIII expression plasmid.
Fig. 3 is that different FVIII expression plasmids is cloned the synoptic diagram that enters pIRES-DHFR/GS respectively.
Fig. 4 is a FVIII-HC/FVIII-LC double expression plasmid synoptic diagram.
Embodiment
Around the low problem of FVIII expression level, people have carried out a large amount of experimental studies, in the hope of improving its expression amount.FVIII and derivative thereof show in the eukaryotic expression result of study: the expression amount of FVIII (Δ B) in the Cos-7 cell is higher than the FVIII of full molecule, and is higher than the foreign literature newspaper; The expression of FVIII has certain tissue specificity, and the expression amount in hepatoma cell strain is the highest, and COS-7 and Chinese hamster ovary celI take second place, and the expression amount in pneumonocyte is minimum; FVIII is heavy, the coexpression of light chain molecule in eukaryotic cell CHO, and the FVIII expression amount is significantly increased, and is 3 times of FVIII (Δ B); With FVIII (Δ B) and vWF co expression, the expression amount of FVIII also is significantly increased.
The reduction technology, promptly by there being the purpose transformation to make the translation initiation efficient of selectable marker gene in external source purpose expressing gene 3 ' end downstream reduce greatly, genetic expression is weakened.Therefore, improve the expression of exogenous gene level and can express, improve the foreign gene level of amplification and reach by the reduction selectable marker gene.
Therefore, improve the expression of exogenous gene level selectable marker gene that can weaken and express, improve the foreign gene level of amplification and reach.The expression of reduction selectable marker gene can make that selective marker dhfr gene copy number is higher when using the selective pressure identical with conventional expression vector, and the expression of exogenous gene level is also corresponding to be improved.As a kind of bicistronic mRNA carrier the dhfr gene is connected in 3 of foreign gene ' end, thereby the translation initiation efficient that is positioned at the dhfr gene in foreign gene 3 ' end downstream reduces greatly, dhfr genetic expression is weakened.
Fig. 1 is the expression vector pIRES-DHFR/GS synoptic diagram that has dual selection markers among the present invention, as shown in Figure 1, the present invention adopts and contains internal ribosome entry site (internal ribosome entrysite, IRES) expression vector that dual selection markers the is arranged---pIRES-DHFR/GS of sequence, the IRES of this carrier utilization reduction connects goal gene and selection markers gene-dihydrofolate reductase gene (DHFR) or glutamine synthetase gene (gs), two shared promotors of gene, two fragment genes obtain translation simultaneously, rrna is to first gene termination codon period of the day from 11 p.m. to 1 a.m in translation process, disintegrate down from mRNA, but owing to there is the IRES sequence, rrna again can be automatically in conjunction with mRNA, directly translates the gene behind the IRES.The IRES sequence of using owing to this carrier is handled through reduction simultaneously, second expression of gene level (DHFR or gs) is that previous gene is 1/10 of a goal gene, if clone will be survived in the substratum that contains MTX or MSX, must high level expression dihydrofolate reductase gene (DHFR) or glutamine synthetase gene (gs), the corresponding high-caliber expression of goal gene before Ruo Hua the IRES like this.In addition, the gene of DHFR or gs both wings because MTX or MSX pressurization can be increased through too much taking turns pressurization and improving pressurization screening concentration, can further improve the expression amount of goal gene.
Fig. 2 is the structure synoptic diagram of different FVIII expression plasmid, Fig. 3 is that different FVIII expression plasmids is cloned the synoptic diagram that enters pIRES-DHFR/GS respectively, Fig. 4 is a FVIII-HC/FVIII-LC double expression plasmid synoptic diagram, as Fig. 2, Fig. 3, shown in Figure 4, make up different FVIII expression plasmids, comprise that Bdomain disappearance and FVIII are heavy, the light chain co-expression plasmid, clone's FVIII full-length cDNA (FVIII-W), on this basis, structure is finished multiple molecule reconstruction body, the FVIII (FVIII-Δ B) that comprises B structural domain disappearance, the heavy chain part (FVIII-HC) of FVIII and the light chain part (FVIII-LC) of FVIII, and it is cloned the A multiple clone site that enters pIRES-DHFR/GS respectively, the clone is entered FVIII-HC and the FVIII-LC of pIRES-DHFR/GS, enzyme cutting clone makes up double expression plasmid in the pBudCE4.1 plasmid respectively.
Expression in mammalian cell has tissue specificity in view of the FVIII gene in the expression of FVIII different reconstruction body in the histological types cell, expression amount in liver, nephrocyte is higher, thereby selects for use more BHK expression system of present use and widely used CHO expression system to carry out expression study respectively.The expression vector difference transfection that the clone is good is in BHK or Chinese hamster ovary celI.Went down to posterity according to 1: 15 behind the transfection 48h, add corresponding screening of medicaments MTX or MSX screening stable expression cell strain behind the 24h, and measure FVIII antigenic content and the biological activity of expressing in the supernatant.
Claims (5)
1. method that improves gene recombinant human blood coagulation factor VIII expression amount is characterized in that: comprise the steps: the processing that weakens with internal ribosome entry site sequence IRES; Described sequence one end after the reduction connects goal gene, and the other end connects the selection markers gene, makes up the expression vector that contains dual selection markers, wherein, and the shared promotor of described goal gene and selection markers gene.
2. a kind of method that improves gene recombination blood coagulation factor VIII expression amount as claimed in claim 1, it is characterized in that: described selection markers gene is dihydrofolate reductase gene or glutamine synthetase gene, and described expression vector is pIRES-DHFR or pIRES-GS.
3. a kind of method that improves gene recombination blood coagulation factor VIII expression amount as claimed in claim 1 is characterized in that: also comprise the gene by methotrexate or MSX pressurization amplification selection markers gene both wings.
4. a kind of method that improves gene recombination blood coagulation factor VIII expression amount as claimed in claim 1, it is characterized in that: described reduction processing is meant by the bicistronic mRNA carrier selection markers gene dihydrofolate reductase gene or glutamine synthetase gene is connected in 3 of internal ribosome entry site sequence IRES ' end that selection markers expression of gene level is reduced.
5. a kind of method that improves gene recombinant human blood coagulation factor VIII expression amount as claimed in claim 1, it is characterized in that: also comprise making up the blood coagulation factor VIII expression system that contains described expression vector, comprise that the VIII factor that contains the Linker sequence [blood coagulation factor VIII (Δ B)] of Bdomain disappearance, blood coagulation factor VIII weigh, the VIII factor [blood coagulation factor VIII (HL)] of light chain coexpression.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321668A (en) * | 2011-07-06 | 2012-01-18 | 中国人民解放军军事医学科学院野战输血研究所 | A kind of method of express recombinant human blood coagulation factors VII and dedicated carrier thereof |
| CN102719472A (en) * | 2011-06-24 | 2012-10-10 | 四川大学 | Ternary expression element, eukaryotic expression vector containing same and use thereof |
| CN103201382A (en) * | 2010-11-08 | 2013-07-10 | 日本化学研究株式会社 | Novel expression vector |
| CN104593406A (en) * | 2015-01-08 | 2015-05-06 | 山西医科大学 | PIRES/TgDHFR-TS eukaryotic expression recombinant plasmid as well as construction and application thereof |
| CN104805107A (en) * | 2014-01-26 | 2015-07-29 | 中国人民解放军军事医学科学院生物工程研究所 | GS screening system-based animal cell high efficiency expression vector and application thereof |
| CN107427557A (en) * | 2014-08-13 | 2017-12-01 | 费城儿童医院 | For packing and expressing variant Factor VIII component is expressed to treat haemophiliachemophiliac improvement |
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2008
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103201382A (en) * | 2010-11-08 | 2013-07-10 | 日本化学研究株式会社 | Novel expression vector |
| EP2639303A1 (en) * | 2010-11-08 | 2013-09-18 | JCR Pharmaceuticals CO., LTD. | Novel expression vector |
| EP2639303A4 (en) * | 2010-11-08 | 2014-05-28 | Japan Chem Res | Novel expression vector |
| JP5913122B2 (en) * | 2010-11-08 | 2016-04-27 | Ｊｃｒファーマ株式会社 | New expression vector |
| KR101890446B1 (en) * | 2010-11-08 | 2018-08-21 | 제이씨알 파마 가부시키가이샤 | Novel expression vector |
| CN102719472A (en) * | 2011-06-24 | 2012-10-10 | 四川大学 | Ternary expression element, eukaryotic expression vector containing same and use thereof |
| CN102719472B (en) * | 2011-06-24 | 2015-07-15 | 四川大学 | Ternary expression element, eukaryotic expression vector containing same and use thereof |
| CN102321668A (en) * | 2011-07-06 | 2012-01-18 | 中国人民解放军军事医学科学院野战输血研究所 | A kind of method of express recombinant human blood coagulation factors VII and dedicated carrier thereof |
| CN104805107A (en) * | 2014-01-26 | 2015-07-29 | 中国人民解放军军事医学科学院生物工程研究所 | GS screening system-based animal cell high efficiency expression vector and application thereof |
| CN104805107B (en) * | 2014-01-26 | 2018-03-23 | 中国人民解放军军事医学科学院生物工程研究所 | It is a kind of based on the zooblast efficient expression vector of GS screening systems and application |
| CN107427557A (en) * | 2014-08-13 | 2017-12-01 | 费城儿童医院 | For packing and expressing variant Factor VIII component is expressed to treat haemophiliachemophiliac improvement |
| CN104593406A (en) * | 2015-01-08 | 2015-05-06 | 山西医科大学 | PIRES/TgDHFR-TS eukaryotic expression recombinant plasmid as well as construction and application thereof |
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Open date: 20091209 |