CN113005140B - GS expression vector with double expression cassettes and application thereof - Google Patents

GS expression vector with double expression cassettes and application thereof Download PDF

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CN113005140B
CN113005140B CN202010436157.4A CN202010436157A CN113005140B CN 113005140 B CN113005140 B CN 113005140B CN 202010436157 A CN202010436157 A CN 202010436157A CN 113005140 B CN113005140 B CN 113005140B
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CN113005140A (en
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赵丽丽
朱中松
刘忠
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Lunan Pharmaceutical Group Corp
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    • C12N2800/60—Vectors containing traps for, e.g. exons, promoters

Abstract

The invention belongs to the technical field of biology, and particularly relates to construction and application of a glutamine synthetase expression vector in development of a high-expression cell strain. The glutamine synthetase expression vector provided by the invention is provided with a first expression box element, a second expression box element, a glutamine synthetase expression element, an ampicillin beta lactamase hydrolase expression element and a replication initiation site ORI, the vector is named as PGS-2, two proteins can be simultaneously expressed or two subunits of one protein are expressed by using the constructed glutamine synthetase expression vector, and finally, the assembly is carried out, so that the glutamine synthetase expression vector has better operation convenience compared with the prior method, and the protein expression quantity is improved by more than 8 times.

Description

GS expression vector with double expression cassettes and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to construction and application of a glutamine synthetase expression vector in development of a high-expression cell strain.
Background
In 2018, the biotechnology protein medicines account for 9 out of the 10 most popular medicines in the global market-free medicine ranking list, and the antibody medicine Wang Xiumei (Humira) is top in 2012 and sold in 199.36 billions of U.S. gold in 2018. Protein medicines become the dominant force of the medicine market, and have the effect of brightening eyes in diseases such as tumor diseases, vascular diseases, rheumatic diseases, hypertension and the like.
The development of biotechnology is also day-to-day, and protein drugs gradually evolve from cytokines, fc fusion proteins, monoclonal antibodies and nanobodies to bispecific antibodies, and along with the progress of the modification technology of modern culture medium technology, cell strains and vectors, the expression level of cells is gradually increased, and the expression level of antibodies is also from mg/L to g/L.
In the large-scale production of mammalian cells, the most common gene screening amplification systems for amplifying selectable marker genes are the dihydrofolate reductase system (DHFR) and the glutamine synthetase system (GS). The dihydrofolate reductase can be inhibited by folic acid analogue Methotrexate (MTX), and the target gene and the DHFR screening marker gene connected with the target gene are coupled and amplified by thousands of times under the condition that the concentration of MTX is continuously increased, so that the expression quantity of the target gene is increased. The glutamine synthetase system (GS) is a recently developed gene screening amplification system, has obvious superiority compared with the DHFR system, is widely accepted internationally at present, and can amplify GS genes and target genes connected with the GS genes by adding Methionine Sulfonamide (MSX) which is an inhibitor of glutamine synthetase into the system, so that the aim of improving the expression level of the target genes is fulfilled. Compared with the DHFR system, the GS system has better application convenience, the GS system can finish screening of the expression cell strain only by one round of pressurization, multiple rounds of pressurization are not needed, the expression quantity of the protein is improved, and the DHFR system needs multiple rounds of gradual pressurization, and has long time period and complex operation. In addition, glutamine is not required to be added in the GS system during large-scale fermentation culture, ammonia and glutamic acid in cells are utilized to synthesize glutamine, so that damage to cells caused by too high ammonia concentration in a system due to glutamine decomposition is avoided, the cost is reduced, the process control difficulty is reduced, and the cells have better performance in large-scale development.
Currently, the commercially preferred cell lines include the CHOK1SV GS-KO cell line from Lonza, the CHOZN CHO K1 cell line from Merk (original SAFC) and the CHO GS null cell line from Horizon Discovery. Lonza and Merk were GS knocked out using ZFN technology and Horizon Discovery was GS knocked out using rAAV virus technology. Different cell expression systems are respectively introduced into domestic companies to express proteins, but the use cost of the systems is generally millions to tens of millions, and even the sales of medicines after marketing are divided into categories, so that the use of the systems is severely limited. There is an urgent need to develop a cell expression system with high expression level suitable for us. The PXC17.4 vector and the PXC18.4 vector of the Lonza company are combinations of the two vectors, the PXC17.4 vector is used when one protein is expressed, when the two proteins are expressed, the two proteins are cloned into the PXC17.4 vector and the PXC18.4 vector respectively, and an expression system containing two expression cassettes is constructed through enzyme digestion and connection, so that the operation is complex. Patent CN104195173 discloses a glutamine expression vector with double expression cassettes, which also contains two expression cassettes, but the expression quantity in the examples is only 600mg/L, the expression quantity is low, and the application of the glutamine expression vector is severely limited. Aiming at the current demands of an expression system, a GS double-expression-cassette expression vector with higher expression quantity is developed, and the GS double-expression-cassette expression vector with higher expression quantity is successfully used on various proteins.
Disclosure of Invention
The invention discloses a glutamine synthetase expression vector with double expression cassettes and application of the vector in preparation of antibodies, thereby solving the problems of low yield, complex operation, high price and the like of products in the prior art.
A first object of the present invention is to provide a glutamine synthetase expression vector having two protein expression cassettes, which can express two proteins simultaneously, or two subunits of one protein, and finally assembled, and which is named PGS-2.
The glutamine synthetase expression vector comprises a first expression cassette element, a second expression cassette element, a glutamine synthetase expression element, an ampicillin beta lactamase hydrolase expression element and a replication initiation site ORI.
The gene sequence of the glutamine synthetase expression vector is shown in SEQ ID NO:1 is shown in the specification; the gene sequence of the first expression cassette element is shown in SEQ ID NO:2 is shown in the figure; the gene sequence of the second expression cassette element is shown in SEQ ID NO:3 is shown in the figure; the gene sequence of the glutamine synthetase expression element is shown as SEQ ID NO:4 is shown in the figure; the gene sequence of the ampicillin beta lactamase hydrolase expression element is shown in SEQ ID NO:5 is shown in the figure; the sequence of the ORI gene of the replication initiation site is shown in SEQ ID NO: shown at 6.
Preferably, the glutamine synthetase expression vector further comprises a promoter, a terminator and an enzyme cleavage site.
Preferably, the promoter is selected from one or more of hCMV, hEF-1 a, SV 40.
Preferably, the terminator is selected from one or more of SV40-PolyA, TK-PolyA, SV 40.
Preferably, the cleavage site is selected from one or more of HindIII enzyme, ecoRI enzyme, pacI enzyme or NotI enzyme.
More preferably, the first expression cassette element comprises the hCMV promoter and the SV40-PolyA terminator, hindIII and EcoRI cleavage sites.
More preferably, the second expression cassette element comprises the hEF-1. Alpha. Promoter and TK-PolyA terminator, pacI and Not I cleavage sites.
More preferably, the glutamine synthetase expression element comprises an SV40 promoter and an SV40 terminator.
A second object of the present invention is to provide the use of a glutamine synthetase expression vector in the expression of a protein.
The glutamine synthetase expression vector of the invention can be used for expressing cytokines, fusion proteins, antibodies, bispecific antibodies, membrane proteins and the like.
In a preferred embodiment, the amino acid sequence encoded is as set forth in SEQ ID NO:9, cloning the polynucleotide of FGF21-Fc into an expression vector PGS-2, constructing a PGS-2-FGF21-Fc expression vector, and then transfecting the recombinant expression vector into CHO-K1 cells to express target proteins; the p327.7-FGF21-Fc expression vector is constructed according to the method disclosed by the prior art, and then the recombinant expression vector is transfected into CHO-K1 cells to express the target protein. The result shows that the carrier PGS-2 of the invention can greatly improve the expression quantity of FGF21-Fc fusion protein, and compared with the carrier p327.7, the expression quantity is improved by 8.23 times.
In a more preferred embodiment, first, the nucleotide sequence set forth in SEQ id no:9, carrying out double digestion on the PUC57-FGF21-Fc shown in the specification by using Hind III and EcoRI enzymes, recovering a target fragment, connecting the target fragment with a carrier PGS-2 which is subjected to double digestion by using the restriction enzymes Hind III and EcoRI, constructing a recombinant eukaryotic expression carrier PGS-2-FGF21-Fc, converting E.coli DH5a, picking up positive clones after overnight culture, carrying out small plasmid extraction, carrying out agarose gel electrophoresis identification after double digestion by using the restriction enzymes Hind III and EcoRI, sequencing the clones which are identified to be positive, and selecting clones which are sequenced correctly for carrying out large plasmid extraction. Then linearizing with restriction enzyme NruI, recovering plasmid by ethanol precipitation method, and preserving at-20deg.C.
Next, the recombinant expression vector described above transfects chinese hamster ovary cells (CHO-K1 cells) and expresses the protein of interest:
the linearized plasmid PGS-2-FGF21-Fc was transfected into CHO-K1 cells by the liposome method (Freestyle MAX, invitrogen) and the FGF21-Fc fusion protein expression was examined. Plasmid transfection was followed by Freestyle TM Instructions for use of MAX transfection reagent the day before transfection of CHO-K1 cells was followed by 0.5X10 6 Per ml passage, day of transfection counts, cell density was adjusted to 1X 10 6 /ml, culture at 37 ℃. Then, a transfection solution was prepared, and 37.5. Mu.g of linearized plasmid PGS-2-FGF21-Fc was added to OptiPRO, respectively TM SFM media to 1.5ml,37.5ul Freestyle TM MAX transfection reagent 1.45ml OptiPRO was added TM SFM culture medium, respectively mixing, slowly adding the transfection reagent mixed solution into the DNA mixed solution, mixing, standing at room temperature for 10 min, and slowly dripping the transfection mixed solution into 30ml of CHO-K1 cells. 8% CO 2 Culturing at 37℃and 130 rpm. After 48h of transfection, 50 μm MSX was cultivated under pressure. Restoring the cell viability to 90%, and freezing and storing for 4 times; and taking a part of the cells to 0.3X10 6 Inoculating 30 ml/ml, culturing with feed, and collecting supernatant. The expression quantity of the protein measured by an extinction coefficient method reaches 4.2g/L.
In another embodiment, the amino acid sequence encoded is as set forth in SEQ ID NO:12 and a PD-1 heavy chain as set forth in SEQ ID NO:15 into an expression vector PGS-2, constructing a PGS-2-PD-1 expression vector, and then transfecting a CHO-K1 cell with the recombinant expression vector to express a target protein; the p327.7-PD-1 expression vector containing the PD-1 light chain and the heavy chain is constructed according to the method disclosed by the prior art, and then the recombinant expression vector is transfected into CHO-K1 cells to express the target protein. The results of the table show that the vector PGS-2 of the invention can greatly improve the expression level of the PD-1 antibody, and compared with the vector p327.7, the vector PGS-2 is improved by 11.82 times.
In another example, the full-length amino acid sequence of SIRPa protein (shown as SEQ ID NO: 16) was submitted to sequence optimization by Nanjing Style Inc. to synthesize the nucleotide sequence. And constructing PGS-2-SIRPa expression plasmid at the first expression cassette, transfecting CHO-K1 cells, screening monoclonal, and carrying out flow detection by using a PE marked SIRPa antibody to obtain a cell strain with high expression of SIRPa membrane protein.
Compared with the prior art, the invention has the following advantages:
the PGS-2 vector is a double expression cassette GS vector, can express two proteins simultaneously or two subunits of one protein, and is finally assembled. Compared with the prior art, the method has better operation convenience, and the protein expression quantity is improved by more than 8 times.
Drawings
FIG. 1 is a structural map of PGS-2 vector;
FIG. 2 is an SDS-PAGE electrophoresis of PD-1 antibody expression, M: low molecular weight protein Marker,1-8: FGF21-Fc fusion protein;
FIG. 3 is an SDS-PAGE electrophoresis of FGF21-FC protein expression, M: low molecular weight protein Marker,1-8: PD-1 antibodies;
FIG. 4 is an SDS-PAGE electrophoresis of CD73-ECD protein expression, M: protein Marker,1: CD73-ECD protein;
FIG. 5 is a flow chart for detecting SIRPa high expressing cell lines, 1: negative control CHO-K1,2: CHO-K1-SIRPa high expression cell strain.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Materials, raw materials, and the like used in the present invention are commercially available without any particular explanation.
EXAMPLE 1 PGS-2 expression vector complete Gene Synthesis and functional elements
The complete sequence of the PGS-2 expression vector was designed and then was synthesized by Shanghai Bioengineering Co. The total length of the sequence is 8866bp, the structural schematic diagram is shown in figure 1, and the total length of the sequence is shown in SEQ ID NO: 1.
The PGS-2 expression vector comprises a first expression cassette element, a second expression cassette element, a glutamine synthetase expression element, an ampicillin β lactamase hydrolase expression element, and a replication initiation site ORI.
The first expression cassette element comprises hCMV promoter and SV40-PolyA terminator, hindIII and EcoRI cleavage sites, and the gene sequence is shown in SEQ ID NO: 2.
The second expression cassette element comprises the hEF-1. Alpha. Promoter and TK-PolyA terminator, pacI and NotI cleavage sites, the gene sequence of which is shown in SEQ ID NO: 3.
The glutamine synthetase expression element comprises an SV40 promoter and an SV40 terminator, and the gene sequence of the glutamine synthetase expression element is shown as SEQ ID NO: 4.
The gene sequence of the ampicillin beta lactamase hydrolase expression element is shown in SEQ ID NO: shown at 5.
The gene sequence of the Origin of replication is shown in SEQ ID NO: shown at 6.
Example 2 high FGF21-Fc protein expression
1. Construction of FGF21-Fc recombinant plasmid
FGF21-Fc protein (amino acid sequence shown as SEQ ID NO: 7) is obtained by optimizing nucleotide sequence by Nanjing Jinsri Co., ltd, adding signal peptide sequence (shown as SEQ ID NO: 8) and Kozak sequence to the nucleotide sequence, adding HindIII cleavage site before the N-terminal, and EcoRI cleavage site after the C-terminal. The optimized nucleotide sequence PUC57-FGF21-Fc of the coded amino acid is shown as SEQ ID NO: shown at 9.
The synthesized PUC57-FGF21-Fc nucleotide sequence is subjected to double digestion by HindIII and EcoRI enzymes, a vector PGS-2 is subjected to double digestion, a target fragment is recovered, a T4 DNA ligase of NEB is connected with the nucleotide sequences of the vector PGS-2 and FGF21-Fc, a connection product is transformed into escherichia coli DH5a, a PGS-2-FGF21-Fc expression strain is constructed, positive clones are selected for small plasmid extraction after overnight culture, agarose gel electrophoresis identification is performed after restriction enzymes HindIII and EcoRI are subjected to double digestion, the clones with positive identification are sent to Shanghai bioengineering limited company for sequencing, and a large number of plasmids are extracted by selecting clones with correct sequencing. Then linearizing with restriction enzyme NruI, recovering plasmid by ethanol precipitation method, and preserving at-20deg.C.
According to the method disclosed in the prior art, the complete sequence synthesis of p327.7 expression vector complete gene is carried out by Shanghai bioengineering limited company, FGF21-Fc protein nucleotide sequence optimization is carried out by Nanjing Jinsri limited company, xhoI and EcoRI restriction sites are added, and an initiation codon ATG, a Kozak sequence and a termination codon TAA are added. The optimized FGF21-Fc protein nucleotide sequence is subjected to double digestion cloning to a p327.7 expression vector through XhoI and EcoRI, escherichia coli DH5a is transformed, a p327.7-FGF21-Fc expression strain is constructed, and plasmids are extracted for identification.
2. FGF21-Fc recombinant plasmid transformed CHO-K1 cell
The linearized PGS-2-FGF21-Fc and p327.7-FGF21-Fc plasmids were transfected into CHO-K1 cells by the liposome method (Freestyle MAX, invitrogen) to examine the expression of FGF21-Fc fusion proteins. Plasmid transfection was followed by Freestyle TM Instructions for use of MAX transfection reagent the day before transfection of CHO-K1 cells was followed by 0.5X10 6 Per ml passage, day of transfection counts, cell density was adjusted to 1X 10 6 /ml, culture at 37 ℃. Then, a transfection solution was prepared, and 37.5. Mu.g of linearized plasmid was added to OptiPRO, respectively TM SFM media to 1.5ml,37.5ul Freestyle TM MAX transfection reagent 1.45ml OptiPRO was added TM SFM culture medium, respectively mixing, slowly adding the transfection reagent mixed solution into the DNA mixed solution, mixing, standing at room temperature for 10 min, and slowly dripping the transfection mixed solution into 30ml of CHO-K1 cells. 8% CO 2 Culturing at 37℃and 130 rpm. After 48h of transfection, 50um MSX was pressure cultured. Restoring the cell viability to 90%, and freezing and storing for 4 times; and taking a part of the cells to 0.3X10 6 30ml of inoculated/ml, fed-batch cultured, and the supernatant collected, and protein expression was measured by SDS-PAGE.
3. Preparation of FGF21-Fc secretion monoclonal cell strain by limiting dilution method
Limiting dilution is carried out on the logarithmic growth phase cells obtained in the step 2, the cell density is 2/ml, 200 mu l/hole is obtained, the cells are inoculated into a 96-well plate, after 7 days, the cells are subjected to microscopic examination and monoclonal, sampling is carried out after 17 days, clones with higher expression level are screened by an HTRF method, the same limiting dilution method is used for limiting dilution for the second round, finally, the clones with higher expression level are obtained, and the cell expression level is measured by freezing and storage of a cell bank and feed culture modes.
4. Purification of proteins
And 3, filtering the cell supernatant obtained in the step 3 and cultured by a 0.4 mu m filter membrane, and purifying by using a Protein A affinity chromatography method. Firstly preparing a protein A affinity column, balancing the column by using PBS, passing the cell culture supernatant which is centrifuged and filtered by a 0.4 mu m filter membrane through the column, washing the cell culture supernatant by using PBS until the OD value is close to zero, eluting the cell culture supernatant by using 50mmol/L glycine-HCL solution with pH of 7.5, collecting eluent in a peak area, dialyzing the eluent for later use, and determining the expression quantity of the protein by using an extinction coefficient method.
Example 3 high expression of PD-1 antibodies
1. PD-1 antibody expression vector construction
The PD-1 antibody has obvious drug effect in tumors, nucleotide sequence optimization is carried out by Nanjing gold Style Limited company, and a PUC-57-PD-1-H plasmid containing a PD-1 heavy chain and a PUC-57-PD-1-L plasmid containing a PD-1 light chain are constructed, specifically: the PD-1 heavy chain amino acid sequence is shown as SEQ ID NO:10, a signal peptide sequence (SEQ ID NO: 11) and a Kozak sequence were added to the nucleotide sequence encoding the amino acid, the stop codon TGA was added to the N-terminus, a HindIII cleavage site was added to the N-terminus, and an EcoRI cleavage site was added to the C-terminus. The nucleotide sequence of the optimized heavy chain amino acid of the PD-1 antibody is shown as SEQ ID NO: shown at 12. The PD-1 antibody light chain amino acid sequence is shown as SEQ ID NO:13, a signal peptide sequence (SEQ ID NO: 14) and a Kozak sequence were added to the nucleotide sequence encoding the amino acid, the stop codon TGA was added before the N-terminus, the PacI cleavage site was added after the C-terminus. The nucleotide sequence of the optimized light chain amino acid of the PD-1 antibody is shown as SEQ ID NO: 15.
The PGS-2 expression vector and the synthesized PUC-57-PD-1-H vector containing heavy chain nucleotide sequences are subjected to double enzyme digestion by HindIII and EcoRI enzymes respectively, target fragments are recovered, the heavy chain nucleotide sequences of the vector PGS-2 and PD-1 are connected by using T4 DNA ligase of NEB, and the connection products are transformed into escherichia coli DH5a to construct the PGS-2-PD-1-H expression strain. After identification, PGS-2-PD-1-H is subjected to double digestion by PacI and NotI enzymes, meanwhile, a PUC-57-PD-1-L vector containing a light chain nucleotide sequence is subjected to double digestion by PacI and NotI enzymes, a target fragment is recovered, the light chain nucleotide sequences of the vector PGS-2-PD-1-H and PD-1 are connected by using T4 DNA ligase of NEB, and the connection product is transformed into E.coli DH5a to construct a PGS-2-PD-1 expression strain containing a PD-1 light chain and a PD-1 heavy chain, so that the identification result is correct.
According to the method disclosed in the prior art, the complete sequence synthesis of p327.7 expression vector complete gene is carried out by Shanghai bioengineering limited company, the nucleotide sequence optimization of PD-1 antibody is carried out by Nanjing Jinsri limited company, xhoI and EcoRI enzyme cutting sites are added to PD-1L chain coding gene, xbaI and SaII enzyme cutting sites, and start codon ATG, kozak sequence and stop codon TAA are added to PD-1H chain coding gene. The optimized PD-1L chain nucleotide sequence is subjected to double digestion cloning to a p327.7 expression vector through XhoI and EcoRI, a p327.7-PD-1-L expression vector is constructed, and after identification, the PD-1H chain nucleotide sequence is subjected to double digestion cloning to a p327.7-PD-1-L expression vector through XbaI and SaII. Then, E.coli DH5a is transformed to construct p327.7-PD-1 expression strain, and the identification result is correct.
2. Transformation of CHO-K1 cells with PD-1 recombinant plasmids
The correct PGS-2-PD-1, p327.7-PD-1 expression strains were identified, plasmid extraction was performed using OMEGA plasmid bulk extraction kit, linearization was performed using pvuI, and CHO-K1 cells were transfected with linearized PGS-2-PD-1, p327.7-PD-1 plasmids using the liposome method (Freestyle MAX, invitrogen). Plasmid transfection was followed by Freestyle TM Instructions for use of MAX transfection reagent the day before transfection of CHO-K1 cells was followed by 0.5X10 6 Per ml passage, day of transfection counts, cell density was adjusted to 1X 10 6 /ml, culture at 37 ℃. Then, a transfection solution was prepared, and 37.5. Mu.g of linearized plasmid was added to OptiPROT, respectivelyM SFM Medium to 1.5ml, 37.5. Mu.l Freshtyle TM MAX transfection reagent 1.45ml OptiPRO was added TM SFM culture medium, respectively mixing, slowly adding the transfection reagent mixed solution into the DNA mixed solution, mixing, standing at room temperature for 10 min, and slowly dripping the transfection mixed solution into 30ml of CHO-K1 cells. 8% CO 2 Culturing at 37℃and 130 rpm. After 48h of transfection, 50 μm MSX was cultivated under pressure. Restoring the cell viability to 90%, and freezing and storing for 4 times; and taking a part of the cells to 0.3X10 6 30ml of inoculated/ml, fed-batch cultured, and the supernatant collected, and protein expression was measured by SDS-PAGE.
3. Preparation of PD-1 antibody monoclonal cell strain by limiting dilution method
Limiting dilution is carried out on the logarithmic growth phase cells obtained in the step 2, the cell density is 2/ml, 200 mu l/hole is obtained, the cells are inoculated into a 96-well plate, after 7 days, the cells are subjected to microscopic examination and monoclonal, sampling is carried out after 17 days, clones with higher expression level are screened by an HTRF method, the same limiting dilution method is used for limiting dilution for the second round, finally, the clones with higher expression level are obtained, and the cell expression level is measured by freezing and storage of a cell bank and feed culture modes.
4. Purification of PD-1 antibodies
The CHO-K1-PD-1 cells obtained in the above step 3 were cultured at 0.3X10 6 Inoculating 30ml of Gibco Feed C culture medium for the fourth day, measuring sugar and supplementing sugar every other day to make sugar content not less than 4g/L, culturing until activity is less than 90%, centrifuging for 10 min after 4500 rpm, and collecting sample for protein purification. The cell supernatant cultured with the feed was filtered through a 0.4um filter, and then purified by Protein A affinity chromatography. Firstly preparing a protein A affinity column, balancing the column by using PBS, filtering a cell culture supernatant filtered by a 0.4um filter membrane, washing the cell culture supernatant by using PBS until the OD value is close to zero, eluting by using a glycine-HCL solution with the pH of 7.5 at the concentration of 50mmol/L, collecting an eluent in a peak area, dialyzing for later use, and determining the expression quantity of the protein by using an extinction coefficient method.
Example 4 expression of CD73-ECD protein
1. CD73-ECD high expression vector construction
Human CD73 protein (UNIPOT: P21589) is extracellular anchoring protein, amino acid 1-36 is signal peptide, amino acid 27-549 is extracellular region, 550-574 is anchoring region. The nucleotide sequence of the CD73 extracellular region protein sequence is added with a 6X HIS label, and is optimized by Nanjing Jinsri Co., ltd, and the nucleotide sequence is named as CD73-ECD, and the amino acid sequence is shown as SEQ ID NO:16 and constructing a PUC-57-CD73-ECD plasmid containing the CD73-ECD sequence. Specific: a HindIII restriction site and a Kozak sequence are added to the N end of the CD73-ECD sequence, and a stop codon TGA and an EcoRI restriction site are added to the C end. The optimized nucleotide sequence of the coding SIRPa amino acid is shown as SEQ ID NO: shown at 17.
Double-enzyme cutting the synthesized PUC5-57-CD73-ECD vector by using Hind III and EcoRI enzymes, recovering a target fragment, connecting the target fragment with a PGS-2 expression vector which is also subjected to double-enzyme cutting by using restriction enzymes Hind III and EcoRI by using T4 DNA ligase of NEB, constructing a PGS-2-CD73-ECD expression vector, transforming E.coli DH5a, constructing a PGS-2-CD73-ECD expression strain, and extracting plasmids for identification.
According to the method disclosed in the prior art, the complete sequence synthesis of p327.7 expression vector complete gene is carried out by Shanghai bioengineering limited company, CD73-ECD protein nucleotide sequence optimization is carried out by Nanjing Jinsri limited company, xhoI and EcoRI enzyme cutting sites are added, and an initiation codon ATG, a Kozak sequence and a termination codon TAA are added. The optimized CD73-ECD protein nucleotide sequence is cloned to a p327.7 expression vector through double enzyme digestion of XhoI and EcoRI, escherichia coli DH5a is transformed, a p327.7-CD73-ECD expression strain is constructed, and plasmid identification is extracted.
2. Transformation of CHO-K1 cells with PGS-2-CD73-ECD plasmid
Plasmid extraction was performed using OMEGA plasmid Mass extraction kit to identify the correct PGS-2-CD73-ECD expression strain, p327.7-CD73-ECD expression strain, linearization was performed using PvuI, and the linearized plasmid was transfected into CHO-K1 cells using the liposome method (Freestyle MAX, invitrogen). Plasmid transfection was followed by Freestyle TM Instructions for use of MAX transfection reagent the day before transfection of CHO-K1 cells was followed by 0.5X10 6 Per ml passage, day of transfection counts, cell density was adjusted to 1X 10 6 /ml, culture at 37 ℃. Then, a transfection solution was prepared, and 37.5. Mu.g of linearized plasmid was added to OptiPRO, respectively TM SFMCulture medium to 1.5ml, 37.5. Mu.l Freestyle TM MAX transfection reagent 1.45ml OptiPRO was added TM SFM culture medium, mixing, slowly adding transfection reagent mixed solution into DNA mixed solution, mixing, standing at room temperature for 10 min, and slowly dripping transfection mixed solution into 30ml CHO-K1 cells. 8% CO 2 Culturing at 37℃and 130 rpm. After 48h of transfection, 50um MSX was pressure cultured. Cell viability was restored to 90% and 4 frozen.
3. Preparation of CD73-ECD protein monoclonal cell strain by limiting dilution method
Limiting dilution is carried out on the logarithmic growth phase cells obtained in the step 2, the cell density is 2/ml, 200 mu l/hole is diluted, the cells are inoculated into a 96-well plate, after 7 days, a microscope is adopted for microscopic examination of single clone, sampling is carried out after 17 days, the FORTEBIO method is adopted for screening clones with higher expression level, the same limiting dilution method is adopted for carrying out the second round of limiting dilution, finally, the clones with higher expression level are obtained, and the cell expression level is measured by the way of freezing and storing cell libraries and feeding culture.
4. Purification of CD73-ECD proteins
The CHO-K1-CD73-ECD protein cells obtained in the above step 3 were cultured at 0.3X10 6 Inoculating 30ml of Gibco Feed C culture medium for the fourth day, measuring sugar and supplementing sugar every other day to make sugar content not less than 4g/L, culturing until activity is less than 90%, centrifuging for 10 min after 4500 rpm, and collecting sample for protein purification. The cell supernatant cultured with the feed was filtered through a 0.4um filter and purified by NI affinity chromatography. Firstly preparing an NI affinity column, balancing the column by using 20mm imidazole-PBS, passing the cell culture supernatant filtered by a 0.4um filter membrane through the column, washing the cell culture supernatant by using 20mm imidazole-PBS until the OD value is close to zero, eluting by using 500mm imidazole PBS, collecting the eluent in a peak area, and measuring the expression quantity of the protein by using an ultrafiltration liquid exchange and extinction coefficient method. The results of measuring the expression level of each protein are shown in Table 1.
TABLE 1 expression level of proteins
As can be seen from Table 1, the PGS-2 vector of the present invention has an increased protein expression level by 8-fold or more as compared with the p327.7 vector.
EXAMPLE 5 SDS-PAGE to identify protein expression
SDS-PAGE reduction electrophoresis detects protein expression of cell culture supernatants. Electrophoresis is carried out according to the method of the third appendix of Chinese pharmacopoeia, the gray scale of the electrophoresis chart is scanned, the molecular weight and the expression quantity are identified, and the results are shown in figures 2, 3 and 4.
EXAMPLE 6 construction of SIRPa high expressing cell lines
1. SIRPa high expression vector construction
Human SIRPa protein (UNIPAT (P78324), SEQ ID NO:18 in the present invention) is a primary transmembrane protein, amino acids 1 to 30 are signal peptides, amino acids 31 to 373 are extracellular regions, amino acids 374 to 394 are transmembrane regions, and amino acids 395 to 504 are cytoplasmic regions. The SIRPa protein sequence was nucleotide sequence optimized by Nanjing gold Style, inc., and a PUC-57-SIRPa plasmid containing the SIRPa sequence was constructed. Specific: a HindIII enzyme cutting site and a Kozak sequence are added to the N end of the SIRPa sequence, and a termination codon TGA and an EcoRI enzyme cutting site are added to the C end. The optimized nucleotide sequence of the coding SIRPa amino acid is shown as SEQ ID NO: 19.
Double-enzyme cutting the synthesized PUC5-57-SIRPa vector containing heavy chain nucleotide sequence by HindIII and EcoRI enzymes, recovering target fragments, connecting the target fragments with a PGS-2 expression vector which is also subjected to double-enzyme cutting by restriction enzymes HindIII and EcoRI by using T4 DNA ligase of NEB, constructing a PGS-2-SIRPa expression vector, transforming E.coli DH5a, constructing a PGS-2-SIRPa expression strain, and extracting plasmids for identification.
2. Transformation of CHO-K1 cells with PGS-2-SIRPa plasmid
The correct PGS-2-SIRPa expression strain was identified, plasmid extraction was performed using OMEGA plasmid mass extraction kit, linearization was performed using PvuI, and CHO-K1 cells were transfected with the linearized PGS-2-SIRPa plasmid using the liposome method (Freestyle MAX, invitrogen). Plasmid transfection was followed by Freestyle TM Instructions for use of MAX transfection reagent the day before transfection of CHO-K1 cells was followed by 0.5X10 6 Per ml passage, day of transfection count, cell conditioningDensity of 1X 10 6 /ml, culture at 37 ℃. Then, a transfection solution was prepared, and 37.5. Mu.g of linearized plasmid was added to OptiPRO, respectively TM SFM medium to 1.5ml, 37.5. Mu.l Freshtyle TM MAX transfection reagent 1.45ml OptiPRO was added TM SFM culture medium, mixing, slowly adding transfection reagent mixed solution into DNA mixed solution, mixing, standing at room temperature for 10 min, and slowly dripping transfection mixed solution into 30ml CHO-K1 cells. 8% CO 2 Culturing at 37℃and 130 rpm. After 48h of transfection, 50um MSX was pressure cultured. Cell viability was restored to 90% and 4 frozen.
3. Preparation of SIRPa protein high-expression monoclonal cell strain by limiting dilution
Limiting dilution is carried out on the logarithmic phase cells obtained in the step 2, the limiting dilution is carried out until the cell density is 2/ml, 200 mu l/hole is obtained, the cells are inoculated into a 96-well plate, after 7 days, the monoclonal cells are detected under a microscope, after 17 days, the monoclonal cells are taken for expansion culture, and after the cells are amplified to a certain concentration, the flow detection is carried out.
4. Flow detection SIRPa high expression cell strain
The control negative cells adopt CHO-K1 empty cells, the detection cells are SIRPa expression cell strains of monoclonal culture, PBS is used for resuspending the cells, and the cell concentration is regulated to ensure that the number of cells in each hole is 2 multiplied by 10 6 /ml. 100ul of 96-well plates were plated, 10ul of PE-SIRPa antibody was added to each well, incubated for 1 hour at 4 ℃, spun at 2000 rpm for 3 minutes, washed twice with PBS, resuspended with 200 ul PBS, and detected by flow cytometry. The detection result is shown in FIG. 5, and the result shows that PGS-2 can be used as an expression vector for high expression of membrane protein, and can be used for constructing a cell strain with high expression of membrane protein.
Sequence listing
<110> Lunan pharmaceutical group Co., ltd
<120> GS expression vector with double expression cassettes and application thereof
<160> 19
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8831
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ggatcccccg ggggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc 60
gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta 120
aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg 180
tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca 240
caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc 300
gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt 360
ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga 420
gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc 480
tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg 540
ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg 600
gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg 660
cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct 720
gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc 780
cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc 840
tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa 900
agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc 960
aggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg 1020
ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg 1080
cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc 1140
aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg atctagactc 1200
gagtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga 1260
ccgatccagc ctccgcggcc gggaacggtg cattggaacg cggattcccc gtgccaagag 1320
tgacgtaagt accgcctata gagtctatag gcccaccccc ttggcttctt atgcatgcta 1380
tactgttttt ggcttggggt ctatacaccc ccgcttcctc atgttatagg tgatggtata 1440
gcttagccta taggtgtggg ttattgacca ttattgacca ctcccctatt ggtgacgata 1500
ctttccatta ctaatccata acatggctct ttgccacaac tctctttatt ggctatatgc 1560
caatacactg tccttcagag actgacacgg actctgtatt tttacaggat ggggtctcat 1620
ttattattta caaattcaca tatacaacac caccgtcccc agtgcccgca gtttttatta 1680
aacataacgt gggatctcca cgcgaatctc gggtacgtgt tccggacatg ggctcttctc 1740
cggtagcggc ggagcttcta catccgagcc ctgctcccat gcctccagcg actcatggtc 1800
gctcggcagc tccttgctcc taacagtgga ggccagactt aggcacagca cgatgcccac 1860
caccaccagt gtgccgcaca aggccgtggc ggtagggtat gtgtctgaaa atgagctcgg 1920
ggagcgggct tgcaccgctg acgcatttgg aagacttaag gcagcggcag aagaagatgc 1980
aggcagctga gttgttgtgt tctgataaga gtcagaggta actcccgttg cggtgctgtt 2040
aacggtggag ggcagtgtag tctgagcagt actcgttgct gccgcgcgcg ccaccagaca 2100
taatagctga cagactaaca gactgttcct ttccatgggt cttttctgca gtcaccgtcc 2160
ttgacacgtt aattaagcgg ccgcgaatcg atgtttgtgg tggccgctgc cacaggcgtg 2220
cagtctgagg aggagctgca ggtcatccag cctgataagt ccgtgctggt ggctgctgga 2280
gagaccgcta cactgaggtg caccgccaca tccctgatcc cagtgggacc tatccagtgg 2340
ttcaggggag ctggaccagg ccgggagctg atctacaatc agaaggaggg ccacttccct 2400
cgggtgacca cagtgagcga cctgaccaag aggaacaata tggatttttc tatccggatc 2460
ggcagtcgac ctcgggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 2520
catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 2580
caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 2640
ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 2700
aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 2760
gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 2820
cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 2880
ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 2940
tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 3000
tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 3060
cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 3120
tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 3180
tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 3240
tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 3300
cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 3360
ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 3420
tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 3480
gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 3540
agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 3600
atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 3660
tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 3720
gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 3780
agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 3840
cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 3900
ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 3960
ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 4020
actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 4080
ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 4140
atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 4200
caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 4260
attatcatga cattaaccta taaaaatagg cgtatcacga ggccctgatg gctctttgcg 4320
gcacccatcg ttcgtaatgt tccgtggcac cgaggacaac cctcaagaga aaatgtaatc 4380
acactggctc accttcgggt gggcctttct gcgtttataa ggagacactt tatgtttaag 4440
aaggttggta aattccttgc ggctttggca gccaagctag atccggctgt ggaatgtgtg 4500
tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca 4560
tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 4620
gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc 4680
gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat 4740
ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt 4800
ttttggaggc ctaggctttt gcaaaaagct agcttggggc caccgctcag agcaccttcc 4860
accatggcca cctcagcaag ttcccacttg aacaaaaaca tcaagcaaat gtacttgtgc 4920
ctgccccagg gtgagaaagt ccaagccatg tatatctggg ttgatggtac tggagaagga 4980
ctgcgctgca aaacccgcac cctggactgt gagcccaagt gtgtagaaga gttacctgag 5040
tggaattttg atggctctag tacctttcag tctgagggct ccaacagtga catgtatctc 5100
agccctgttg ccatgtttcg ggaccccttc cgcagagatc ccaacaagct ggtgttctgt 5160
gaagttttca agtacaaccg gaagcctgca gagaccaatt taaggcactc gtgtaaacgg 5220
ataatggaca tggtgagcaa ccagcacccc tggtttggaa tggaacagga gtatactctg 5280
atgggaacag atgggcaccc ttttggttgg ccttccaatg gctttcctgg gccccaaggt 5340
ccgtattact gtggtgtggg cgcagacaaa gcctatggca gggatatcgt ggaggctcac 5400
taccgcgcct gcttgtatgc tggggtcaag attacaggaa caaatgctga ggtcatgcct 5460
gcccagtggg agttccaaat aggaccctgt gaaggaatcc gcatgggaga tcatctctgg 5520
gtggcccgtt tcatcttgca tcgagtatgt gaagactttg gggtaatagc aacctttgac 5580
cccaagccca ttcctgggaa ctggaatggt gcaggctgcc ataccaactt tagcaccaag 5640
gccatgcggg aggagaatgg tctgaagcac atcgaggagg ccatcgagaa actaagcaag 5700
cggcaccggt accacattcg agcctacgat cccaaggggg gcctggacaa tgcccgtcgt 5760
ctgactgggt tccacgaaac gtccaacatc aacgactttt ctgctggtgt cgccaatcgc 5820
agtgccagca tccgcattcc ccggactgtc ggccaggaga agaaaggtta ctttgaagac 5880
cgccgcccct ctgccaattg tgaccccttt gcagtgacag aagccatcgt ccgcacatgc 5940
cttctcaatg agactggcga cgagcccttc caatacaaaa actaattaga ctttgagtga 6000
tcttgagcct ttcctagttc atcccacccc gccccagaga gatctttgtg aaggaacctt 6060
acttctgtgg tgtgacataa ttggacaaac tacctacaga gatttaaagc tctaaggtaa 6120
atataaaatt tttaagtgta taatgtgtta aactactgat tctaattgtt tgtgtatttt 6180
agattccaac ctatggaact gatgaatggg agcagtggtg gaatgccttt aatgaggaaa 6240
acctgttttg ctcagaagaa atgccatcta gtgatgatga ggctactgct gactctcaac 6300
attctactcc tccaaaaaag aagagaaagg tagaagaccc caaggacttt ccttcagaat 6360
tgctaagttt tttgagtcat gctgtgttta gtaatagaac tcttgcttgc tttgctattt 6420
acaccacaaa ggaaaaagct gcactgctat acaagaaaat tatggaaaaa tattctgtaa 6480
cctttataag taggcataac agttataatc ataacatact gttttttctt actccacaca 6540
ggcatagagt gtctgctatt aataactatg ctcaaaaatt gtgtaccttt agctttttaa 6600
tttgtaaagg ggttaataag gaatatttga tgtatagtgc cttgactaga gatcataatc 6660
agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg 6720
aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat 6780
ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat 6840
tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat ctctagcttc 6900
gtgtcaagga cggtgagggt tgacattgat tattgactag ttattaatag taatcaatta 6960
cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 7020
gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 7080
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 7140
ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 7200
atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 7260
cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 7320
acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 7380
acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 7440
actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 7500
gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc 7560
atagaagaca ccgggaccga tccagcctcc ggactctatc agatcgcctg gagacgccat 7620
ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg cggccgggaa 7680
cggtgcattg gaacgcggat tccccgtgcc aagagtgacg taagtaccgc ctatagagtc 7740
tataggccca cccccttggc ttcttatgca tgctatactg tttttggctt ggggtctata 7800
cacccccgct tcctcatgtt ataggtgatg gtatagctta gcctataggt gtgggttatt 7860
gaccattatt gaccactccc ctattggtga cgatactttc cattactaat ccataacatg 7920
gctctttgcc acaactctct ttattggcta tatgccaata cactgtcctt cagagactga 7980
cacggactct gtatttttac aggatggggt ctcatttatt atttacaaat tcacatatac 8040
aacaccaccg tccccagtgc ccgcagtttt tattaaacat aacgtgggat ctccacgcga 8100
atctcgggta cgtgttccgg acatgggctc ttctccggta gcggcggagc ttctacatcc 8160
gagccctgct cccatgcctc cagcgactca tggtcgctcg gcagctcctt gctcctaaca 8220
gtggaggcca gacttaggca cagcacgatg cccaccacca ccagtgtgcc gcacaaggcc 8280
gtggcggtag ggtatgtgtc tgaaaatgag ctcggggagc gggcttgcac cgctgacgca 8340
tttggaagac ttaaggcagc ggcagaagaa gatgcaggca gctgagttgt tgtgttctga 8400
taagagtcag aggtaactcc cgttgcggtg ctgttaacgg tggagggcag tgtagtctga 8460
gcagtactcg ttgctgccgc gcgcgccacc agacataata gctgacagac taacagactg 8520
ttcctttcca tgggtctttt ctgcagtcac cgtccttgac acgaagctta ggccgcgaat 8580
tcatcgatat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc 8640
tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt 8700
ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 8760
catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 8820
tctggcgcgc c 8831
<210> 2
<211> 1907
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60
gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120
ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180
ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240
atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300
cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360
tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420
agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480
tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540
aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc 600
gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc 660
gatccagcct ccggactcta tcagatcgcc tggagacgcc atccacgctg ttttgacctc 720
catagaagac accgggaccg atccagcctc cgcggccggg aacggtgcat tggaacgcgg 780
attccccgtg ccaagagtga cgtaagtacc gcctatagag tctataggcc cacccccttg 840
gcttcttatg catgctatac tgtttttggc ttggggtcta tacacccccg cttcctcatg 900
ttataggtga tggtatagct tagcctatag gtgtgggtta ttgaccatta ttgaccactc 960
ccctattggt gacgatactt tccattacta atccataaca tggctctttg ccacaactct 1020
ctttattggc tatatgccaa tacactgtcc ttcagagact gacacggact ctgtattttt 1080
acaggatggg gtctcattta ttatttacaa attcacatat acaacaccac cgtccccagt 1140
gcccgcagtt tttattaaac ataacgtggg atctccacgc gaatctcggg tacgtgttcc 1200
ggacatgggc tcttctccgg tagcggcgga gcttctacat ccgagccctg ctcccatgcc 1260
tccagcgact catggtcgct cggcagctcc ttgctcctaa cagtggaggc cagacttagg 1320
cacagcacga tgcccaccac caccagtgtg ccgcacaagg ccgtggcggt agggtatgtg 1380
tctgaaaatg agctcgggga gcgggcttgc accgctgacg catttggaag acttaaggca 1440
gcggcagaag aagatgcagg cagctgagtt gttgtgttct gataagagtc agaggtaact 1500
cccgttgcgg tgctgttaac ggtggagggc agtgtagtct gagcagtact cgttgctgcc 1560
gcgcgcgcca ccagacataa tagctgacag actaacagac tgttcctttc catgggtctt 1620
ttctgcagtc accgtccttg acacgaagct taggccgcga attcatcgat atcataatca 1680
gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga 1740
acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg 1800
gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt 1860
ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgg 1907
<210> 3
<211> 2452
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60
ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt 120
gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180
gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc 240
gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 300
acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg 360
gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg 420
cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct 480
ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg 540
caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc 600
gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga 660
gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct 720
ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca 780
gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg 840
acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg 900
tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat 960
tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg 1020
gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa 1080
ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca 1140
gtggttcaaa gtttttttct tccatttcag gtgtcgtgat ctagactcga gtcagatcgc 1200
ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct 1260
ccgcggccgg gaacggtgca ttggaacgcg gattccccgt gccaagagtg acgtaagtac 1320
cgcctataga gtctataggc ccaccccctt ggcttcttat gcatgctata ctgtttttgg 1380
cttggggtct atacaccccc gcttcctcat gttataggtg atggtatagc ttagcctata 1440
ggtgtgggtt attgaccatt attgaccact cccctattgg tgacgatact ttccattact 1500
aatccataac atggctcttt gccacaactc tctttattgg ctatatgcca atacactgtc 1560
cttcagagac tgacacggac tctgtatttt tacaggatgg ggtctcattt attatttaca 1620
aattcacata tacaacacca ccgtccccag tgcccgcagt ttttattaaa cataacgtgg 1680
gatctccacg cgaatctcgg gtacgtgttc cggacatggg ctcttctccg gtagcggcgg 1740
agcttctaca tccgagccct gctcccatgc ctccagcgac tcatggtcgc tcggcagctc 1800
cttgctccta acagtggagg ccagacttag gcacagcacg atgcccacca ccaccagtgt 1860
gccgcacaag gccgtggcgg tagggtatgt gtctgaaaat gagctcgggg agcgggcttg 1920
caccgctgac gcatttggaa gacttaaggc agcggcagaa gaagatgcag gcagctgagt 1980
tgttgtgttc tgataagagt cagaggtaac tcccgttgcg gtgctgttaa cggtggaggg 2040
cagtgtagtc tgagcagtac tcgttgctgc cgcgcgcgcc accagacata atagctgaca 2100
gactaacaga ctgttccttt ccatgggtct tttctgcagt caccgtcctt gacacgttaa 2160
ttaagcggcc gcgaatcgat gtttgtggtg gccgctgcca caggcgtgca gtctgaggag 2220
gagctgcagg tcatccagcc tgataagtcc gtgctggtgg ctgctggaga gaccgctaca 2280
ctgaggtgca ccgccacatc cctgatccca gtgggaccta tccagtggtt caggggagct 2340
ggaccaggcc gggagctgat ctacaatcag aaggagggcc acttccctcg ggtgaccaca 2400
gtgagcgacc tgaccaagag gaacaatatg gatttttcta tccggatcgg ca 2452
<210> 4
<211> 2405
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt 60
atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca 120
gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta 180
actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 240
ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag 300
tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctagcttg gggccaccgc 360
tcagagcacc ttccaccatg gccacctcag caagttccca cttgaacaaa aacatcaagc 420
aaatgtactt gtgcctgccc cagggtgaga aagtccaagc catgtatatc tgggttgatg 480
gtactggaga aggactgcgc tgcaaaaccc gcaccctgga ctgtgagccc aagtgtgtag 540
aagagttacc tgagtggaat tttgatggct ctagtacctt tcagtctgag ggctccaaca 600
gtgacatgta tctcagccct gttgccatgt ttcgggaccc cttccgcaga gatcccaaca 660
agctggtgtt ctgtgaagtt ttcaagtaca accggaagcc tgcagagacc aatttaaggc 720
actcgtgtaa acggataatg gacatggtga gcaaccagca cccctggttt ggaatggaac 780
aggagtatac tctgatggga acagatgggc acccttttgg ttggccttcc aatggctttc 840
ctgggcccca aggtccgtat tactgtggtg tgggcgcaga caaagcctat ggcagggata 900
tcgtggaggc tcactaccgc gcctgcttgt atgctggggt caagattaca ggaacaaatg 960
ctgaggtcat gcctgcccag tgggagttcc aaataggacc ctgtgaagga atccgcatgg 1020
gagatcatct ctgggtggcc cgtttcatct tgcatcgagt atgtgaagac tttggggtaa 1080
tagcaacctt tgaccccaag cccattcctg ggaactggaa tggtgcaggc tgccatacca 1140
actttagcac caaggccatg cgggaggaga atggtctgaa gcacatcgag gaggccatcg 1200
agaaactaag caagcggcac cggtaccaca ttcgagccta cgatcccaag gggggcctgg 1260
acaatgcccg tcgtctgact gggttccacg aaacgtccaa catcaacgac ttttctgctg 1320
gtgtcgccaa tcgcagtgcc agcatccgca ttccccggac tgtcggccag gagaagaaag 1380
gttactttga agaccgccgc ccctctgcca attgtgaccc ctttgcagtg acagaagcca 1440
tcgtccgcac atgccttctc aatgagactg gcgacgagcc cttccaatac aaaaactaat 1500
tagactttga gtgatcttga gcctttccta gttcatccca ccccgcccca gagagatctt 1560
tgtgaaggaa ccttacttct gtggtgtgac ataattggac aaactaccta cagagattta 1620
aagctctaag gtaaatataa aatttttaag tgtataatgt gttaaactac tgattctaat 1680
tgtttgtgta ttttagattc caacctatgg aactgatgaa tgggagcagt ggtggaatgc 1740
ctttaatgag gaaaacctgt tttgctcaga agaaatgcca tctagtgatg atgaggctac 1800
tgctgactct caacattcta ctcctccaaa aaagaagaga aaggtagaag accccaagga 1860
ctttccttca gaattgctaa gttttttgag tcatgctgtg tttagtaata gaactcttgc 1920
ttgctttgct atttacacca caaaggaaaa agctgcactg ctatacaaga aaattatgga 1980
aaaatattct gtaaccttta taagtaggca taacagttat aatcataaca tactgttttt 2040
tcttactcca cacaggcata gagtgtctgc tattaataac tatgctcaaa aattgtgtac 2100
ctttagcttt ttaatttgta aaggggttaa taaggaatat ttgatgtata gtgccttgac 2160
tagagatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc 2220
cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta 2280
ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 2340
ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 2400
ggatc 2405
<210> 5
<211> 861
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 60
agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 120
cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 180
ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 240
gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 300
cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 360
cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 420
ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 480
catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 540
tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 600
ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 660
catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 720
cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 780
cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 840
acggaaatgt tgaatactca t 861
<210> 6
<211> 615
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 60
ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 120
aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 180
tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 240
gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 300
cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 360
ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 420
cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 480
gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 540
cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 600
tcaagaagat ccttt 615
<210> 7
<211> 459
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 7
His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val
1 5 10 15
Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His
20 25 30
Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser
35 40 45
Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
50 55 60
Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly
65 70 75 80
Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg
85 90 95
Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His
100 105 110
Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro
115 120 125
Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro
130 135 140
Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val
145 150 155 160
Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser
165 170 175
Pro Ser Tyr Ala Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
180 185 190
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
195 200 205
Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg
210 215 220
Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Val Glu Cys Pro
225 230 235 240
Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro
245 250 255
Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr
260 265 270
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn
275 280 285
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
290 295 300
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
305 310 315 320
Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
325 330 335
Asn Lys Gly Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Thr Lys
340 345 350
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
355 360 365
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
370 375 380
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
385 390 395 400
Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe
405 410 415
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
420 425 430
Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr
435 440 445
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 8
<211> 19
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 8
Met Arg Ser Leu Gly Ala Leu Leu Leu Leu Leu Ser Ala Cys Leu Ala
1 5 10 15
Val Ser Ala
<210> 9
<211> 1456
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
aagcttgccg ccaccatgag gagcctcggg gccctgctct tgctgctgag cgcctgcctg 60
gcggtgagcg ctcaccctat tcccgatagc tcccccctcc tgcagttcgg aggccaggtg 120
aggcagcggt acctgtacac cgacgacgct cagcagaccg aagctcacct ggagatcagg 180
gaggatggaa ccgtcggcgg agctgctgac cagtcccccg agagcctgct gcagctgaag 240
gccctgaagc ccggagtcat ccagatcctg ggcgtgaaga cctcccggtt tctgtgtcag 300
cggcccgatg gagccctgta cggctccctg cattttgacc ccgaggcctg tagcttcagg 360
gagctgctgc tggaagacgg ctacaacgtg taccagagcg aagctcacgg actgcccctg 420
cacctgcctg gcaacaaatc ccctcacagg gaccccgctc ccaggggacc tgccaggttc 480
ctgcctctgc ccggactgcc tcctgctcct cccgaacctc ctggcatcct cgctcctcag 540
ccccctgatg tcggcagcag cgaccctctg tccatggtcg gccccagcca aggcaggagc 600
ccttcctacg cttccggatc cggtggcggt ggctccggtg gaggcggaag cggcggtgga 660
ggatcaggcg gtggaggtag cggcggaggc ggtagctcca gctctagtaa agctccccct 720
ccttccctgc cctcaccctc aagactgcct ggaccttccg acactcccat cctgccacag 780
gtggagtgcc ctccatgtcc agcaccccct gtcgcaggtc catctgtgtt cctgtttcca 840
cccaagccta aagaccagct gatgatctcc cgcaccccag aagtcacctg tgtggtcgtg 900
gatgtgagcc atgaagaccc cgaggtccag ttcaattggt acgtggatgg cgtcgaggtg 960
cacaacgcta agacaaaacc tagagaagag cagttcaact ctacctttcg cgtcgtgagt 1020
gtgctgacag tcgtgcacca ggactggctg aatggcaagg agtataagtg caaagtgagc 1080
aacaaaggac tgcctgcctc aatcgaaaag actatttcca agaccaaagg acagccaaga 1140
gagccccagg tgtacaccct gcctccaagc cgcgaagaga tgactaaaaa tcaggtctct 1200
ctgacctgtc tggtgaaggg gttttatcct agtgatatcg ccgtggaatg ggagtcaaac 1260
ggtcagccag agaacaatta caagaccaca ccccctatgc tggacagcga tgggtctttc 1320
tttctgtata gcaaactgac agtggacaag tctcggtggc agcagggtaa cgtcttctct 1380
tgcagtgtgc tgcacgaagc actgcacaat cattacaccc agaagtcact gtcactgagc 1440
ccaggaaaat gaattc 1456
<210> 10
<211> 448
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Arg Gln Lys Asp Thr Ser Trp Phe Val His Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 11
<211> 19
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser
<210> 12
<211> 1423
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
aagcttgccg ccaccatggg ctggtcctgc atcatcctgt ttctggtcgc taccgccaca 60
ggcgtccact ccgaggtgca gctggtggag agcggaggag gactggtgaa gccaggagga 120
tctctgaggc tgagctgtgc tgcctctggc ttcacctttt cctcttacgg catgagctgg 180
gtgagacaga cacccgagaa gcgcctggaa tgggtcgcta ccatctctgg cggcggcaga 240
gacacatact atcctgattc cgtgaagggc agattcacca tcagccgcga taacgccaaa 300
aataatctgt atctgcaaat gtcttccctg aggtctgagg acaccgctct gtactattgc 360
gcccggcaga aggatacatc ttggttcgtg cactggggac agggcaccct ggtgaccgtg 420
tcctccgctt ccaccaaggg cccttccgtg ttccctctcg ctcccagctc caagagcacc 480
agcggaggaa cagctgccct gggatgcctg gtgaaggact acttccccga gcctgtcacc 540
gtgtcctgga acagcggagc cctgacatcc ggcgtgcaca cctttcccgc tgtgctccag 600
tccagcggcc tgtacagcct ctcctccgtg gtgaccgtgc cttcctccag cctcggcacc 660
caaacctaca tctgtaacgt gaaccacaag cccagcaaca ccaaagtgga caagaaggtg 720
gagcccaaat cctgcgacaa gacacacaca tgccctcctt gtcccgctcc cgagctgctc 780
ggaggaccta gcgtgtttct gttccccccc aaacccaagg atcaactcat gatcagccgg 840
acccccgaag tcacatgcgt cgtggtggac gtgtcccacg aggaccccga ggtgaagttc 900
aactggtacg tggacggcgt ggaggtgcac aacgccaaga ccaaacctcg ggaggagcag 960
tacgccagca cctaccgggt cgtctccgtg ctcaccgtgc tgcaccagga ctggctcaac 1020
ggcaaggagt ataagtgcaa ggtgtccaat aaggccctgc ccgcccccat tgagaagacc 1080
attagcaagg ccaagggcca acccagggag cctcaggtgt ataccctgcc ccccagcagg 1140
gaagagatga ccaagaacca ggtgtccctc acatgcctcg tcaagggatt ctaccctagc 1200
gacatcgccg tcgaatggga gtccaacgga cagcccgaga acaactataa gaccacaccc 1260
cccgtgctcg attccgacgg atccttcttt ctctactcca agctcaccgt ggacaagagc 1320
cggtggcagc aaggcaacgt gttctcctgc agcgtgctgc acgaagccct gcacaaccac 1380
tacacccaga agagcctcag cctgagcccc ggaaagtgaa ttc 1423
<210> 13
<211> 218
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 13
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ala Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asp Tyr
20 25 30
Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Val Ala Ser Asn Gln Gly Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys
85 90 95
Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 14
<211> 19
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 14
Met Arg Ser Leu Gly Ala Leu Leu Leu Leu Leu Ser Ala Cys Leu Ala
1 5 10 15
Val Ser Ala
<210> 15
<211> 739
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
ttaattaagc cgccaccatg aggagcctcg gggccctgct cttgctgctg agcgcctgcc 60
tggcggtgag cgctgaaatc gtgctgaccc agtctccagc tacactggcc gtgtcccctg 120
gagagagggc taccatcagc tgtcgggcct ctgagtctgt cgacgattac ggcatctctt 180
tcatgaattg gttccagcag aaacctggcc agccccctaa actgctgatc tatgtcgctt 240
ccaatcaggg aagcggagtg cctgctagat tcagcggatc tggatctgga accgacttca 300
ccctgaacat ccatccaatg gaggaggacg ataccgccat gtacttctgt cagcagtcta 360
aagaagtccc ttggaccttt ggcggcggca caaagctgga gatcaagcgg accgtggccg 420
ccccctccgt gttcatcttt ccccccagcg acgagcagct caaatccggc acagccagcg 480
tggtgtgcct gctgaacaac ttctacccca gggaggccaa ggtccagtgg aaggtggaca 540
acgctctgca gtccggcaac tcccaggagt ccgtgaccga gcaggactcc aaggattcca 600
cctatagcct gtccagcacc ctcaccctca gcaaagccga ctacgagaag cacaaggtct 660
acgcctgcga agtgacacac cagggcctga gctcccccgt gacaaagtcc ttcaacaggg 720
gcgagtgctg agcggccgc 739
<210> 16
<211> 557
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 16
Met Cys Pro Arg Ala Ala Arg Ala Pro Ala Thr Leu Leu Leu Ala Leu
1 5 10 15
Gly Ala Val Leu Trp Pro Ala Ala Gly Ala Trp Glu Leu Thr Ile Leu
20 25 30
His Thr Asn Asp Val His Ser Arg Leu Glu Gln Thr Ser Glu Asp Ser
35 40 45
Ser Lys Cys Val Asn Ala Ser Arg Cys Met Gly Gly Val Ala Arg Leu
50 55 60
Phe Thr Lys Val Gln Gln Ile Arg Arg Ala Glu Pro Asn Val Leu Leu
65 70 75 80
Leu Asp Ala Gly Asp Gln Tyr Gln Gly Thr Ile Trp Phe Thr Val Tyr
85 90 95
Lys Gly Ala Glu Val Ala His Phe Met Asn Ala Leu Arg Tyr Asp Ala
100 105 110
Met Ala Leu Gly Asn His Glu Phe Asp Asn Gly Val Glu Gly Leu Ile
115 120 125
Glu Pro Leu Leu Lys Glu Ala Lys Phe Pro Ile Leu Ser Ala Asn Ile
130 135 140
Lys Ala Lys Gly Pro Leu Ala Ser Gln Ile Ser Gly Leu Tyr Leu Pro
145 150 155 160
Tyr Lys Val Leu Pro Val Gly Asp Glu Val Val Gly Ile Val Gly Tyr
165 170 175
Thr Ser Lys Glu Thr Pro Phe Leu Ser Asn Pro Gly Thr Asn Leu Val
180 185 190
Phe Glu Asp Glu Ile Thr Ala Leu Gln Pro Glu Val Asp Lys Leu Lys
195 200 205
Thr Leu Asn Val Asn Lys Ile Ile Ala Leu Gly His Ser Gly Phe Glu
210 215 220
Met Asp Lys Leu Ile Ala Gln Lys Val Arg Gly Val Asp Val Val Val
225 230 235 240
Gly Gly His Ser Asn Thr Phe Leu Tyr Thr Gly Asn Pro Pro Ser Lys
245 250 255
Glu Val Pro Ala Gly Lys Tyr Pro Phe Ile Val Thr Ser Asp Asp Gly
260 265 270
Arg Lys Val Pro Val Val Gln Ala Tyr Ala Phe Gly Lys Tyr Leu Gly
275 280 285
Tyr Leu Lys Ile Glu Phe Asp Glu Arg Gly Asn Val Ile Ser Ser His
290 295 300
Gly Asn Pro Ile Leu Leu Asn Ser Ser Ile Pro Glu Asp Pro Ser Ile
305 310 315 320
Lys Ala Asp Ile Asn Lys Trp Arg Ile Lys Leu Asp Asn Tyr Ser Thr
325 330 335
Gln Glu Leu Gly Lys Thr Ile Val Tyr Leu Asp Gly Ser Ser Gln Ser
340 345 350
Cys Arg Phe Arg Glu Cys Asn Met Gly Asn Leu Ile Cys Asp Ala Met
355 360 365
Ile Asn Asn Asn Leu Arg His Thr Asp Glu Met Phe Trp Asn His Val
370 375 380
Ser Met Cys Ile Leu Asn Gly Gly Gly Ile Arg Ser Pro Ile Asp Glu
385 390 395 400
Arg Asn Asn Gly Thr Ile Thr Trp Glu Asn Leu Ala Ala Val Leu Pro
405 410 415
Phe Gly Gly Thr Phe Asp Leu Val Gln Leu Lys Gly Ser Thr Leu Lys
420 425 430
Lys Ala Phe Glu His Ser Val His Arg Tyr Gly Gln Ser Thr Gly Glu
435 440 445
Phe Leu Gln Val Gly Gly Ile His Val Val Tyr Asp Leu Ser Arg Lys
450 455 460
Pro Gly Asp Arg Val Val Lys Leu Asp Val Leu Cys Thr Lys Cys Arg
465 470 475 480
Val Pro Ser Tyr Asp Pro Leu Lys Met Asp Glu Val Tyr Lys Val Ile
485 490 495
Leu Pro Asn Phe Leu Ala Asn Gly Gly Asp Gly Phe Gln Met Ile Lys
500 505 510
Asp Glu Leu Leu Arg His Asp Ser Gly Asp Gln Asp Ile Asn Val Val
515 520 525
Ser Thr Tyr Ile Ser Lys Met Lys Val Ile Tyr Pro Ala Val Glu Gly
530 535 540
Arg Ile Lys Phe Ser His His His His His His His His
545 550 555
<210> 17
<211> 1695
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
aagcttgccg ccaccatgtg ccccagggct gctagggctc ctgctaccct gctgctggcc 60
ctgggcgctg tgctgtggcc agctgctggc gcctgggagc tgaccatcct gcacacaaat 120
gacgtgcata gcaggctgga gcagacctct gaggattcca gcaagtgcgt gaacgcctcc 180
agatgtatgg gcggcgtggc tcgcctgttc acaaaggtgc agcagatcag gcgggccgag 240
cctaatgtgc tgctgctgga cgctggcgat cagtaccagg gcaccatctg gttcacagtg 300
tataagggcg ccgaggtggc tcactttatg aacgccctga gatacgacgc catggctctg 360
ggcaatcatg agttcgataa cggcgtggag ggcctgatcg agccactgct gaaggaggcc 420
aagtttccca tcctgagcgc caacatcaag gctaagggcc ctctggctag ccagatctct 480
ggcctgtacc tgccctataa ggtgctgcct gtgggcgacg aggtggtggg aatcgtgggc 540
tatacctcca aggagacacc attcctgagc aatcccggca ccaacctggt gtttgaggac 600
gagatcacag ccctgcagcc agaggtggat aagctgaaga ccctgaatgt gaacaagatc 660
atcgccctgg gccactctgg ctttgagatg gacaagctga tcgctcagaa ggtgcgggga 720
gtggatgtgg tggtgggagg ccatagcaat accttcctgt acacaggcaa ccccccttct 780
aaggaggtgc ctgctggcaa gtatcccttc atcgtgacat ccgacgatgg caggaaggtg 840
cccgtggtgc aggcctacgc tttcggcaag tacctgggct atctgaagat cgagtttgac 900
gagcggggca atgtgatctc ttcccacggc aatcctatcc tgctgaacag ctctatccca 960
gaggacccct ctatcaaggc cgatatcaat aagtggagaa tcaagctgga caactactcc 1020
acccaggagc tgggcaagac aatcgtgtat ctggatggct ccagccagag ctgcagattc 1080
cgcgagtgta atatgggcaa cctgatctgc gacgctatga tcaacaataa cctgcgccac 1140
accgatgaga tgttttggaa tcacgtgtcc atgtgcatcc tgaacggcgg cggcatcagg 1200
agccctatcg acgagcggaa taacggcacc atcacatggg agaacctggc tgctgtgctg 1260
ccattcggcg gcacctttga tctggtgcag ctgaagggct ctacactgaa gaaggccttc 1320
gagcactctg tgcatagata cggccagtcc accggcgagt ttctgcaagt gggcggcatc 1380
cacgtggtgt atgacctgag cagaaagcca ggcgaccgcg tggtgaagct ggatgtgctg 1440
tgcacaaagt gtcgggtgcc ttcttacgac ccactgaaga tggatgaggt gtataaagtg 1500
atcctgccca atttcctggc taacggcggc gatggctttc agatgatcaa ggacgagctg 1560
ctgagacatg attccggcga ccaggatatc aacgtggtgt ctacctacat ctccaagatg 1620
aaagtgatct acccagccgt ggagggccgc atcaagttct cccatcacca ccatcaccat 1680
caccattgag aattc 1695
<210> 18
<211> 504
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 18
Met Glu Pro Ala Gly Pro Ala Pro Gly Arg Leu Gly Pro Leu Leu Cys
1 5 10 15
Leu Leu Leu Ala Ala Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu
20 25 30
Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly
35 40 45
Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro Val Gly
50 55 60
Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr
65 70 75 80
Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu
85 90 95
Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn Ile Thr
100 105 110
Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser
115 120 125
Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val
130 135 140
Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala
145 150 155 160
Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser
165 170 175
Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser
180 185 190
Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser
195 200 205
Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser
210 215 220
Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu
225 230 235 240
Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu
245 250 255
Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr
260 265 270
Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu
275 280 285
Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu
290 295 300
Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val
305 310 315 320
Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp
325 330 335
Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala His
340 345 350
Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser Asn
355 360 365
Glu Arg Asn Ile Tyr Ile Val Val Gly Val Val Cys Thr Leu Leu Val
370 375 380
Ala Leu Leu Met Ala Ala Leu Tyr Leu Val Arg Ile Arg Gln Lys Lys
385 390 395 400
Ala Gln Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro Glu Lys Asn
405 410 415
Ala Arg Glu Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala Asp Leu
420 425 430
Asn Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn
435 440 445
Asn His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro Ala Ser
450 455 460
Glu Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu Asn Arg
465 470 475 480
Thr Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro Ser Phe Ser Glu Tyr
485 490 495
Ala Ser Val Gln Val Pro Arg Lys
500
<210> 19
<211> 1536
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
aagcttgccg ccaccatgga gccagctgga ccagctccag gcaggctggg ccctctgctg 60
tgcctgctgc tggctgcttc ctgtgcttgg agcggagtgg ctggagagga ggagctgcag 120
gtcatccagc ccgacaagtc tgtgctggtg gctgctggag agaccgccac actgagatgc 180
accgctacat ccctgatccc cgtgggccct atccagtggt tcagaggagc tggacctggc 240
cgcgagctga tctacaacca gaaggagggc cacttcccac gcgtgaccac agtgagcgac 300
ctgacaaaga gaaacaacat ggatttttct atccgcatcg gcaatatcac accagccgac 360
gctggcacct actattgcgt gaagttccgg aagggcagcc ccgacgatgt ggagtttaag 420
agcggagctg gaaccgagct gtctgtgagg gctaagccat ccgctccagt ggtgagcgga 480
cctgctgcta gggctacccc acagcacaca gtgagcttca cctgtgagtc tcatggcttt 540
tccccaaggg atatcacact gaagtggttc aagaacggca atgagctgtc tgactttcag 600
accaacgtgg atcccgtggg cgagtccgtg tcctatagca tccattccac agctaaggtg 660
gtgctgaccc gggaggacgt gcacagccag gtcatctgcg aggtggctca tgtgaccctg 720
cagggcgatc ctctgagagg cacagctaat ctgtctgaga ccatccgcgt gccccctaca 780
ctggaggtga cccagcagcc tgtgagagcc gagaaccaag tgaatgtgac atgtcaggtg 840
aggaagtttt acccacagcg gctgcagctg acctggctgg agaacggcaa cgtgtccagg 900
accgagacag cttccaccgt gacagagaac aaggatggca catataattg gatgtcctgg 960
ctgctggtga acgtgagcgc ccaccgggac gatgtgaagc tgacctgcca ggtggagcat 1020
gacggacagc cagccgtgtc taagtcccac gatctgaagg tgtctgctca tcctaaggag 1080
cagggctcca acacagccgc tgagaatacc ggctctaacg agcgcaatat ctacatcgtg 1140
gtgggagtgg tgtgcaccct gctggtggcc ctgctgatgg ccgctctgta tctggtgagg 1200
atccggcaga agaaggctca gggctccacc tccagcacaa ggctgcacga gccagagaag 1260
aacgcccggg agatcaccca ggacacaaac gatatcacct acgccgacct gaatctgcca 1320
aagggcaaga agcctgctcc acaggccgct gagcccaaca atcacacaga gtacgccagc 1380
atccagacct ctccccagcc tgcctccgag gataccctga catatgctga cctggatatg 1440
gtgcatctga atagaacccc aaagcagcca gctccaaagc ctgagccctc cttctccgag 1500
tatgctagcg tgcaggtgcc tcgcaagtga gaattc 1536

Claims (9)

1. A glutamine synthetase expression vector, characterized in that the vector comprises a first expression cassette element, a second expression cassette element, a glutamine synthetase expression element, an ampicillin β lactamase hydrolase expression element, a replication initiation site ORI, and the vector has a sequence as set forth in SEQ id no: 1.
2. The glutamine synthetase expression vector of claim 1, wherein the first expression cassette element comprises hCMV promoter and SV40-PolyA terminator, hindiii and ecori cleavage sites, and the first expression cassette element has a sequence as set forth in SEQ id no: 2.
3. The glutamine synthetase expression vector of claim 1, wherein the second expression cassette element comprises a hEF-1 alpha promoter and TK-poly a terminator, paci and Not i cleavage sites, and the second expression cassette element sequence is set forth in SEQ id no: 3.
4. The glutamine synthetase expression vector of claim 1, wherein the glutamine synthetase expression element comprises an SV40 promoter and an SV40 terminator, and the sequence of the glutamine synthetase expression element is as shown in SEQ id no: 4.
5. The glutamine synthetase expression vector according to any of claims 1 to 4, characterized in that the vector can express two proteins simultaneously or two subunits of one protein and finally assembled, the vector being named PGS-2.
6. Use of the glutamine synthetase expression vector of any one of claims 1-4 for expressing 1-2 proteins.
7. Use of the glutamine synthetase expression vector of any one of claims 1-4 for expressing a cytokine, an antibody, a membrane protein.
8. Use of the glutamine synthetase expression vector of any one of claims 1-4 for expressing a fusion protein.
9. Use of the glutamine synthetase expression vector of any one of claims 1-4 for expressing a bispecific antibody.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1668749A (en) * 2002-07-18 2005-09-14 英国龙沙生物医药股份有限公司 Method of expressing recombinant protein in CHO cells
CN104195173A (en) * 2014-09-02 2014-12-10 北京比洋生物技术有限公司 Glutamine synthetase expression vector with two expression cassettes
CN104946687A (en) * 2015-06-09 2015-09-30 北京东方百泰生物科技有限公司 Mammal dual gene highly efficient screening expression vectors and construction method thereof
CN108085337A (en) * 2017-12-28 2018-05-29 未名生物医药有限公司 A kind of screening technique of GS expression systems cell line
CN108103088A (en) * 2017-12-21 2018-06-01 广东东阳光药业有限公司 Recombinate optimization gene and its application of GLP-1 analog Fc fusion proteins
CN110023500A (en) * 2016-11-16 2019-07-16 新加坡科技研究局 The attenuation glutamine synthelase alternatively marked

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101494072B1 (en) * 2012-03-12 2015-02-17 한화케미칼 주식회사 An expression vector comprising a polynucleotide encoding a modified glutamine synthetase and a method for preparing a target protein employing the same

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1668749A (en) * 2002-07-18 2005-09-14 英国龙沙生物医药股份有限公司 Method of expressing recombinant protein in CHO cells
CN104195173A (en) * 2014-09-02 2014-12-10 北京比洋生物技术有限公司 Glutamine synthetase expression vector with two expression cassettes
CN104946687A (en) * 2015-06-09 2015-09-30 北京东方百泰生物科技有限公司 Mammal dual gene highly efficient screening expression vectors and construction method thereof
CN110023500A (en) * 2016-11-16 2019-07-16 新加坡科技研究局 The attenuation glutamine synthelase alternatively marked
CN108103088A (en) * 2017-12-21 2018-06-01 广东东阳光药业有限公司 Recombinate optimization gene and its application of GLP-1 analog Fc fusion proteins
CN108085337A (en) * 2017-12-28 2018-05-29 未名生物医药有限公司 A kind of screening technique of GS expression systems cell line

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Fujii,W. et al.ACCESSION NO.AB781744.1,Cloning vector pKK-platform DNA, complete sequence.《GenBank》.2013,FEATURES,ORIGIN. *
Optimization of transcriptional regulatory elements for constructing plasmid vectors;Xu ZL et al.;《Gene》;20010711;第272卷(第1-2期);第149-156页 *
Regulation of eukaryotic gene expression by the untranslated gene regions and other non-coding elements;Lucy W Barrett et al.;《Cell Mol Life Sci.》;20121130;第69卷(第21期);第3613-3634页 *
基于GS筛选系统的动物细胞高效表达载体的构建及应用;高招刚;《中国优秀硕士学位论文全文数据库基础科学辑》;20140815(第08期);A006-19 *

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