CN105779490A - Construction method of Pichia pastoris expressed by OCH1 defect anti-CD20 tetravalent antibody - Google Patents
Construction method of Pichia pastoris expressed by OCH1 defect anti-CD20 tetravalent antibody Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/84—Pichia
Abstract
The invention discloses a construction method of Pichia pastoris expressed by an OCH1 defect anti-CD20 tetravalent antibody. The method comprises the following steps: knocking out the alpha-1,6-mannosyl transferase OCH1 of a strain JC308, connecting reconstructed och1 having no expression ability and a screening label to pPICZalphaA, introducing the obtained pPICZalphaA to wild Pichia pastoris JC308, carrying out homologous recombination, screening to obtain an OCH1 defect strain denoted as detaoch1, connecting the sequence of a synthesized anti-CD20 tetravalent antibody with a Pichia pastoris expression plasmid pPIC9, and introducing the obtained product to the OCH1 defect strain detaoch1 to obtain the Pichia pastoris. The Pichia pastoris alpha-1,6-mannosyl transferase (OCH1) gene is knocked out to prevent formation of high-mannose carbohydrate chains, so glycosylation difference between yeast expression proteins and human natural proteins is shortened, and the safety of medicinal proteins is guaranteed.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of OCH1 gene defection type anti-CD 20 tetravalent antibody and express the construction method of Pichi strain.
Background technology
Pichia yeast expression system, it is presently the most one of successful exogenous protein expression system, compared with other expression system existing, pichia pastoris phaff the processing of expression product, outer point of secret, post translational modification and glycosylation modified etc. in have obvious advantage, be now widely used in the expression of foreign protein.But, Pichia sp. is unsuitable for the expression of glycoprotein, and its expression product is high mannose type, it is easy to produces immunoreation in human body, limits the Pichia sp. use as Host Strains.α-1 of OCH1 gene code, the existence of 6-mannose transferase, is the major reason causing high mannose to be formed.To knocking out of OCH1 gene, can effectively blocking yeast and high mannoseization after the translation of glycoprotein is modified, the glycoprotein obtained is low mannose structures, can evade Pichia sp. high mannose and modify drawback, is the follow-up glycosylation engineered work basis of Pichia sp. simultaneously.
CD20 antigen is a kind of B cell differentiation antigen.Owing to CD20 only expresses in pre-B lymphocyte, immature B lymphocyte, ripe bone-marrow-derived lymphocyte, activation bone-marrow-derived lymphocyte, and at plasma cell, lymph pluripotent stem cell and other tissue all without the expression of CD20, also without the existence of Free form CD2 0 in human serum.Therefore CD20 can as a fine therapy target of bone-marrow-derived lymphocyte tumor treatment.The preparation method disclosing a kind of anti-CD 20 tetravalent antibody in patent CN101544694A, but owing to the expression vector of its use is escherichia coli, it is possible to its application as medicine albumen is affected containing endotoxin.
Pichia sp. has the N-glycosylation metabolic pathway similar to human body cell as eukaryotic cell, but the N-glycosylation process of Pichia sp. and people there is also different.The N-of Pichia sp. is glycosylation modified for high mannose type N-glycosylation (Heavymannosetype), and N-in human body cell is glycosylation modified is mainly complexity N-glycosylation (Complextype) and heterozygous N-glycosylation (Hybridtype).The N-of the high mannose type produced in Pichia sp. is glycosylation modified, can change activity and the reaction kinetic characteristics of therapeutic glycoprotein on the one hand, may result in anaphylaxis after entering human body on the other hand.Therefore, not engineered Pichia sp., it is not suitable for the production of most of pharmaceutical glycoprotein.
Majority pharmaceutical albumen all utilizes mammalian cell to produce absolutely at present.But, the problems such as production cost is too high, yield is on the low side because mammalian expression systems itself there is also, be easily contaminated in incubation, limit its large-scale application in medicine albumen field.In order to solve this problem, research worker begins to seek to express these albumen in the prokaryotic micro-organisms such as escherichia coli and bacillus cereus earlier.But the modification system of processing of eukaryotic protein, the incorrect foreign protein of endogenous protein enzymatic degradation space conformation, periplasmic are contained within the production that the features such as miscellaneous endotoxin are medicine albumen and bring inconvenience owing to lacking the renaturation function to eukaryotic protein, lacking by prokaryote.Adopt and belong to eukaryotic cell together and have the Pichia sp. of stronger secretory protein ability as medicine protein expression system, be solve current albumen to be difficult to the optimum selection of low cost, mass production.
Summary of the invention
The technical problem to be solved in the present invention is to overcome existing defect, it is provided that a kind of OCH1 gene defection type anti-CD 20 tetravalent antibody expresses the construction method of Pichi strain: comprise the following steps:
One, the structure of recombinant bacterium Δ och1OCH1 bacterial strain
1), structure is fused into gene och1
With pichia pastoris X-33 genome for template, two sections of homologous sequences of upstream and downstream of clone's OCH1 gene, two sections of homologous sequences of upstream and downstream of OCH1 gene are fused into gene och1, described fusion gene lacks the open reading frame of OCH1 gene；
2), clone Orotidine-5 '-phosphate decarboxylase gene URA3；
3), by 1) in gained fusion gene och1 and 2) in gained URA3 import pichia vector pPICZ α A, build and obtain pPICZ α A-URA3-och1 plasmid；
4), by pPICZ α A-URA3-och1 Plastid transformation Pichi strain JC308, through genome screening and phenotypic screen, positive colony Δ och1-bacterial strain is obtained；
Two, the structure of anti-CD 20 tetravalent antibody expression strain
1), C is built2B8Single-chain antibody ScFv gene and people's IgG antibody1The fusion gene C of Fc2B8(ScFvHL)2Fc
Clone CD20 monoclonal antibody C2B8Single-chain antibody ScFv gene and people's IgG antibody1Fc gene；By two C2B8After single-chain antibody ScFv gene tandem and with people's IgG antibody1Fc gene connects, and obtains fusion gene C2B8(ScFvHL)2Fc；
2), by step 1) in the fusion gene cloning that obtains to yeast expression vector pPIC9, constitute recombinant expression carrier pPIC9 (C2B8(ScFvHL)2Fc)；
3), by step 2) in the recombinant expression carrier that obtains convert Pichia yeast engineering Δ och1, screening positive clone.
Domestic at present realize the expression at eukaryotic expression system of the CD20 monoclonal antibody.Zhong Xin state build the eukaryotic expression utilizing Chinese hamster ovary celI to achieve CD20 monoclonal antibody and anti-CD 20 tetravalent antibody, experiment in vitro (CDC and ADCC) and the experiment of internal mouse-borne tumor in and all achieves good effect.But owing to eukaryotic cell toxigenic capacity is high, complicated operation easily pollutes；And Pichia sp. has strong promoter (AOX) because of it, can carrying out High Density Cultivation, toxigenic capacity is relatively low to take advantage in foreign protein producer face.And carry out pharmaceutical protein currently with yeast as expression system and produce research not relevant at home.
As the production carrier of people's source protein, Pichia sp. modifies the difference of link and mammalian cell after must solving albumen.α-1,6 mannose transferase is present in Pichia sp. endoplasmic reticulum, and effect is to add mannose to Man8GlcNAc2The mannose arm that the α-1,3 of oligonucleotide chain connects forms Man9GlcNAc2Sugar chain structure.Pichia sp. other mannose transferase endogenic, using this sugar chain as substrate, continues to add mannose, forms high mannose type structure.Therefore we must disturb the expression of yeast cells glycosylation-related enzymes, by Pichia sp. α-1 in this patent, 6 mannose transferases (OCH1) gene knockout, the formation of high mannose type sugar chain can be stoped, thus reducing the glycosylation difference of yeast expression albumen and naive albumen, it is ensured that the safety of pharmaceutical protein.
Accompanying drawing explanation
Accompanying drawing is for providing a further understanding of the present invention, and constitutes a part for description, is used for together with embodiments of the present invention explaining the present invention, is not intended that limitation of the present invention.In the accompanying drawings:
Fig. 1 is the structure schematic diagram of OCH1 gene knockout carrier；
Fig. 2 is URA3 gene clone；
Fig. 3 is wild type OCH1 gene clone；
Fig. 4 is OCH1 left arm (5 ' end) and right arm (3 ' end) clone；
Fig. 5 is och1 fusion gene cloning；
Fig. 6 is the structure of CD20 expression plasmid；
Fig. 7 is pPIC9 (C2B8(ScFvHL)2Fc) connection result；
Fig. 8 is Δ och1/CD20 protein expression result.
Detailed description of the invention
Hereinafter the preferred embodiments of the present invention are illustrated, it will be appreciated that preferred embodiment described herein only by the bright and explanation present invention, is not intended to limit the present invention.
Embodiment
A kind of OCH1 deficiency anti-CD 20 tetravalent antibody expresses the construction method of Pichia sp., it is characterised in that comprise the following steps:
One, the structure of positive colony Δ och1 bacterial strain
1), structure is fused into gene Δ OCH1
According to the Pichia sp. OCH1 gene order design two of report in Genebank (E12456) to nucleotide primer OCH1-5F, OCH1-5R, OCH1-3F, OCH1-3R.With X-33 strain gene group for template, the homology arm sequence at clone OCH1 two ends.The product SEQIDNO:10 of OCH1-5F, OCH1-5R clone；The sequence such as SEQIDNO:11 of the product of OCH1-3F, OCH1-3R clone
Fig. 4: OCH1 left arm (5 ' end) and right arm (3 ' end) clone's electrophoresis pattern, 1 is lacked between the homology arm of both sides, the reading frame sequence of about 200bp, merge fragment och1 size and be about 1600bp, sequence is SEQIDNO:12 such as, lacks the open reading frame of OCH1 gene in described fusion gene.
Primer sequence is as follows:
OCH1-5F, sequence is SEQIDNO:1 such as
OCH1-5R, sequence is SEQIDNO:2 such as
OCH1-3F:, sequence is SEQIDNO:3 such as
OCH1-3R: sequence is SEQIDNO:4 such as
2), clone Orotidine-5 '-phosphate decarboxylase gene URA3；
Primer sequence inquiry Pichia sp. URA3 gene Genbank (AF321098), this DNA encoding the protein region of extension increasing sequence (CDS sequence), URA3 gene C DS region sequence sequence such as SEQIDNO:13, it is sized to 838bp fragment.Fig. 2 is the electrophoresis pattern of URA3 gene clone.
U5 sequence such as SEQIDNO:5
U3 sequence such as SEQIDNO:6
3), by 1) in gained fusion gene och1 and 2) in gained URA3 be connected into pichia vector pPICZ α A, build and obtain pPICZ α A-URA3-och1 plasmid；Fig. 1 is the structure figure of OCH1 knockout carrier；
4), by pPICZ α A-URA3-och1 Plastid transformation Pichi strain JC308
Plasmid pPICZ α A-URA3-och1 is after BstEII linearisation, electroporated Pichia sp. JC308, cultivating 3～5 days at 30 DEG C on MD+His+Arg+Ade+Sorbitol flat board, the first time that is extracting Yeast genome checking correct recombinates correct, can carry out second time restructuring.
There is a recombinant bacterial strain in picking, in inoculation YPD culture medium, 200rpm shake-flask culture 10 hours at 26 DEG C, bacterium solution is coated in MD+His+Ura+Arg+His+Sorbitol+5FOA flat board, cultivates 5 days at 26 DEG C.Single bacterium colony two pieces of YPD flat boards of dibbling respectively will be grown on flat board, respectively 26 DEG C with 38 DEG C of cultivation, can grow at the former and can not grow bacterium colony the latter is possible OCH1 defect strain.
The clone of the genome checking positive is denoted as Δ och1, and checking primer is as follows:
P1: sequence is SEQIDNO:7 such as
P2: sequence is SEQIDNO:8 such as
Through genome screening and phenotypic screen, obtain positive colony Δ och1 bacterial strain；
Two, the structure of anti-CD 20 tetravalent antibody expression strain
Fig. 6 is the structure collection of illustrative plates of CD20 expression plasmid.
1), human anti cd 20 monoclonal antibody sequence information, design C disclosed in United States Patent (USP) 6,399,0612B8Single-chain antibody ScFv gene and people's IgG antibody1The fusion gene C of Fc2B8(ScFvHL)2Fc sequence, Fig. 7 is fusion gene C2B8(ScFvHL)2The electrophoresis pattern of Fc.C2B8(ScFvHL)2The sequence of Fc such as SEQIDNO:9, C2B8(ScFvHL)2The amino acid whose sequence such as SEQIDNO:14 of Fc translation
Clone CD20 monoclonal antibody C2B8Single-chain antibody ScFv gene and people's IgG antibody 1Fc gene；By two C2B8After single-chain antibody ScFv gene tandem and with people's IgG antibody1Fc gene connects, and obtains fusion gene C2B8(ScFvHL)2Fc；
With reference to the domestic patent CN101544694A anti-CD 20 tetravalent antibody data announced and sequence, entrust the correlated series C of Shanghai Sheng Gong biological engineering company limited full genome synthesis anti-humen CD 20 tetravalent antibody2B8(ScFvHL)2Fc, sequence details are with reference to nucleotides sequence list:
2), by step 1) in the fusion gene cloning that obtains to yeast expression vector pPIC9, constitute recombinant expression carrier；
3), by step 2) in the recombinant expression carrier that obtains convert Pichia yeast engineering JC308/ Δ OCH1pPIC9 (C2B8(ScFvHL)2Fc) plasmid is after StuI linearisation, electroporated Δ och1.Genome is verified as the clone of the positive and is denoted as Δ och1/CD20.
4) abduction delivering of anti-CD 20 tetravalent antibody
Picking positive strain Δ och1/CD20, in YPD30 DEG C of overnight incubation, is inoculated in the BMMY culture medium of 100mL with 1% inoculum concentration, is cultured to OD600=6.0, collect thalline, be inoculated in BMGY culture medium and cultivate, added methanol every 24 hours and carry out abduction delivering to final concentration of 1%.30 DEG C of methanol induction fermentation receive cell after 60 hours, with (4 DEG C) protein extract (5% glycerol of pre-cooling, 1mMPMSF and 50mM phosphate buffer, pH7.4) cell is washed and resuspended, adding bead turbula shaker to vibrate crack protein, 10000 × g is centrifugal to be taken supernatant for 30 minutes and carries out SDS-PAGE detection.Fig. 8 is Δ och1/CD20 protein expression result.
Later stage fermentation adopts 2L water jacket rustless steel fermentation tank (band oxygen supply device, pH value microprocessor controller, dissolved oxygen, stirring, temperature and nutrient fodder, with electronics foam control), the fermentation basal salt contained in the 1L culture medium of fermentation tank and 4% glycerol, these are all through 30 minutes, 122 DEG C of sterilizations, afterwards the aseptic initial inoculation cell (BMGY cultivation) adding 2.4ml/L trace salt (PTM1) and 100mL.Stirring maintain 800rpm, temperature at 30 DEG C, oxygen 30%, pH5.0, ammonium hydroxide 25%.First glycerol balance period continues 24 hours until glycerol depletion, and then the glycerol supply phase is continuously added into 50% (w/v) glycerol and 12mL/LPTM1, trace salt speed be that 18ml/h/l is until glycerol supply concentration reaches 100 200g/L.The methanol supply phase when starting 12mL/LPTM1 supply 18 hours with the speed of 2g/h/L, 4g/h/L, 3 hours or 6g/h/L, 3 hours, final 6 7g/h/L continue 5 to 6 days until fermentation ends.Collect cell: 4 DEG C, centrifugal 10 minutes of 2500 × g, extraction purification destination protein.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, it is not limited to the present invention, although the present invention being described in detail with reference to previous embodiment, for a person skilled in the art, technical scheme described in foregoing embodiments still can be modified by it, or wherein portion of techniques feature carries out equivalent replacement.All within the spirit and principles in the present invention, any amendment of making, equivalent replacement, improvement etc., should be included within protection scope of the present invention.
Claims (6)
1. the construction method of an OCH1 deficiency anti-CD 20 tetravalent antibody expression Pichia sp., it is characterised in that comprise the following steps:
One, the structure of recombinant bacterial strain △ och1
1), structure is fused into gene och1
With pichia pastoris X-33 genome for template, two sections of homologous sequences of upstream and downstream of clone's OCH1 gene, two sections of homologous sequences of upstream and downstream of OCH1 gene are fused into gene och1, described fusion gene lacks the open reading frame of OCH1 gene；
2), clone Orotidine-5 '-phosphate decarboxylase gene URA3；
3), by 1) in gained be fused into gene och1 and 2) in gained URA3 import pichia vector pPICZ α A, build and obtain pPICZ α A-URA3-och1 plasmid；
4), by pPICZ α A-URA3-och1 Plastid transformation Pichi strain JC308, through genome screening and phenotypic screen, positive colony △ och1 bacterial strain is obtained；
Two, the structure of anti-CD 20 tetravalent antibody expression strain
5), C is built2B8Single-chain antibody ScFv gene and people's IgG antibody1The fusion gene of Fc
Clone CD20 monoclonal antibody C2B8Single-chain antibody ScFv gene and people's IgG antibody1Fc gene；By two C2B8After single-chain antibody ScFv gene tandem and with people's IgG antibody1Fc gene connects, and obtains fusion gene；
6), by step 1) in the fusion gene cloning that obtains to yeast expression vector pPIC9, constitute recombinant expression carrier；
7), by step 2) in the recombinant expression carrier that obtains convert Pichia yeast engineering △ och1, screening positive clone.
2. a kind of OCH1 deficiency anti-CD 20 tetravalent antibody as claimed in claim 1 expresses the construction method of Pichia sp., it is characterised in that the method for described screening positive clone △ och1 bacterial strain is:
Plasmid pPICZ α A-URA3-och1 is after BstEII linearisation, electroporated Pichia sp. JC308, cultivating 3～5 days at 30 DEG C on MD+His+Arg+Ade+Sorbitol flat board, the first time that is extracting Yeast genome checking correct recombinates correct, can carry out second time restructuring.
There is a recombinant bacterial strain in picking, in inoculation YPD culture medium, 200rpm shake-flask culture 10 hours at 26 DEG C, bacterium solution is coated in MD+His+Ura+Arg+His+Sorbitol+5FOA flat board, cultivates 5 days at 26 DEG C.Single bacterium colony two pieces of YPD flat boards of dibbling respectively will be grown on flat board, respectively 26 DEG C with 38 DEG C of cultivation, can grow at the former and can not grow bacterium colony the latter is possible positive colony △ och1 bacterial strain.
3. merging a fragment och1, sequence is such as shown in SEQIDNO:12.
4. a C2B8Single-chain antibody ScFv gene and people's IgG antibody1The aminoacid of the fusion gene translation of Fc, sequence is such as shown in SEQIDNO:14.
5. the Pichi strain that the construction method described in claim 1 builds.
6. the application in fermentation production of protein of the bacterial strain described in claim 5.
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