CN108103088B - Optimized gene of recombinant GLP-1 analogue Fc fusion protein and application thereof - Google Patents
Optimized gene of recombinant GLP-1 analogue Fc fusion protein and application thereof Download PDFInfo
- Publication number
- CN108103088B CN108103088B CN201711390118.XA CN201711390118A CN108103088B CN 108103088 B CN108103088 B CN 108103088B CN 201711390118 A CN201711390118 A CN 201711390118A CN 108103088 B CN108103088 B CN 108103088B
- Authority
- CN
- China
- Prior art keywords
- fusion protein
- glp
- gene
- analogue
- recombinant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
- C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
-
- 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
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- 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
- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Reproductive Health (AREA)
- Cell Biology (AREA)
- Endocrinology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of biological pharmacy, and particularly relates to an optimized gene (rGLP-1-Fc for short) of recombinant GLP-1 analogue Fc fusion protein and application thereof. The Fc fusion gene with the human GLP-1 analogue is introduced into a plasmid through optimization, and is transfected into a CHOK1SV GS-KO cell, and a positive CHOK1SV GS-KO cell strain which stably and efficiently expresses the GLP-1 analogue Fc fusion protein is obtained after screening. The optimized gene constructed by the invention has obviously increased secretory expression amount in a positive cell strain, and the secreted fusion protein has higher biological activity, can directly act on a GLP-1 receptor and has higher affinity. Can quickly, efficiently and durably reduce blood sugar and glycosylated hemoglobin, and has the effects of improving the function of islet beta cells, reducing blood fat, losing weight, delaying gastric emptying, increasing satiety and the like.
Description
Technical Field
The invention belongs to the field of biological pharmacy, and particularly relates to an optimized gene (rGLP-1-Fc for short) of recombinant GLP-1 analogue Fc fusion protein and application thereof.
Background
According to the latest data of IDF, the number of diabetics in the whole world is increased to 4.15 hundred million in 2015, the incidence rate of diabetes is 8.8%, and the incidence rate is estimated to be increased to 10.4% in 6.42 hundred million diabetics in the whole world in 2040. The number of Chinese diabetic patients (20-79 years old) in 2015 is increased by 1120 ten thousand to 1.096 hundred million compared with 2013, and the number of diabetic patients in 20-79 years old in 2040 is estimated to be 1.507 hundred million. In most countries, diabetes and its complications are the leading cause of premature death, with 50% or more of diabetics dying due to cardiovascular disease. In 2015, the number of patients who died due to diabetes (20-79 years) was as high as 500 ten thousand, accounting for 14.5% of all deaths, which corresponds to one adult dying from diabetes every 6 seconds. The rapid global increase of the prevalence of diabetes has placed a very heavy burden on social and economic development and has become a major risk factor affecting global development.
Glucagon-like peptide-1 (GLP-1) is mainly produced by intestinal L cells, promotes regeneration of pancreatic beta-cells, secretes insulin and glucose tolerance, activates cAMP and a coupled second messenger pathway, promotes expression and content of beta-cell protein kinase (Akt1 and MAPK), reduces activity of caspase-3 which is a key enzyme of apoptosis, and fundamentally prevents and treats diabetes, so that GLP-1 analogues are hot research objects in diabetes. According to IMS data statistics, the total sale amount of 6 GLP-1 receptor agonist drugs in 2015 is $ 39.12 hundred million, which accounts for 10.37 percent of the total sale amount of 32 hypoglycemic drugs. Analysts expect that the market for GLP-1 analogs will break through $ 60 billion after 5 years.
At present, a plurality of GLP-1 analogues are used for treating diabetes mellitus on the market, but due to degradation of dipeptidyl peptidase IV and rapid clearance of blood, the half-life period of the medicament is short, the long-acting effect cannot be maintained, and the common strategy is to perform amino acid mutation and Fc fusion on GLP-1. In addition, the DHFR expression system is generally adopted in the industry at present, but the MTX continuous pressurization causes the instability of a foreign gene in a cell genome, which not only causes the lack of stability of a cell strain, but also causes the lower expression level, the GLP-1 fusion protein expression level in the prior art is about 1g/L or less than 1g/L, for example, the GLP-1-IgG2 sigma Fc fusion protein sequence constructed by the people of the Yang-class and the like is inserted into a plasmid, cells are transfected, and finally the GLP-1 fusion protein expression level is 1g/L (Yang, Wande friend, Liu Nei Hui, and the like; the eukaryotic expression of the long-acting GLP-1-IgG2 sigma Fc fusion protein is communicated with the biological activity identification [ J ] biotechnology, 2016,27(2):173 and 177.); in the patent (Saxiong, Guo 39040; lao, Von Jinmeng. a recombinant human GLP-1-Fc fusion protein, CN 104327187A [ P ].2015.), the expression level of GLP-1-Fc fusion protein is also about 1 g/L; in the patent (Huangyanshan, Yangxian, Xuzheng, etc. GLP-1 analogue fusion protein and the preparation method and the application thereof, CN 103408669B [ P ] 2016), the yield of the GLP-1-Fc fusion protein is only 10 mug/mL. On the other hand, the selection and optimization of the signal peptide are also a main factor for the efficient expression of the fusion protein. In general, the construction and optimization of a target gene, the selection of a suitable expression system, and the screening of stable cell lines are of great significance for drug development.
Disclosure of Invention
The invention aims to solve the technical problems and provides a high-efficiency expression method of GLP-1 analogue Fc fusion protein, which comprises the selection and optimization of signal peptide genes in the fusion protein, the optimization of fusion protein genes, the construction of cell strains, monoclonal screening, the expression of GLP-1 analogue Fc fusion protein, purification and biological activity detection, so that the expression quantity of the GLP-1 analogue Fc fusion protein is obviously improved compared with the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
a recombinant fusion protein gene, the recombinant fusion protein gene has a gene sequence with A-B structure, wherein A is a signal peptide gene, and B is a gene for encoding GLP-1 analogue Fc fusion protein; in the fusion protein gene, the nucleotide sequence of the signal peptide A is shown in SEQ ID NO. 1.
Further, the nucleotide sequence of the gene B for encoding the GLP-1 analogue Fc fusion protein is shown as SEQ ID NO. 2.
Furthermore, the amino acid sequence of the recombinant fusion protein gene is shown in SEQ ID NO. 3.
The invention provides a recombinant expression vector containing the SEQ ID NO.1 and SEQ ID NO.2 sequences.
Preferably, the recombinant expression vector is pXC17.4.
The invention provides a host cell containing the recombinant expression vector.
Preferably, the host cell is a CHOK1SV GS-KO cell.
The invention also provides a method for producing the GLP-1 analogue Fc fusion protein, which comprises the following steps: recombining a signal peptide gene shown in SEQ ID NO.1 and a gene of an Fc fusion protein which is shown in SEQ ID NO.2 and used for coding a GLP-1 analogue to obtain an optimized recombinant fusion protein gene; inserting the recombinant fusion protein gene into an expression vector to obtain a recombinant expression vector; carrying out PvuI linearization on the recombinant expression vector, transforming host cells, and screening to obtain a positive host strain; culturing the positive host strain, inducing the expression of the GLP-1 analogue Fc fusion protein, and recovering and purifying the expressed GLP-1 analogue Fc fusion protein. In the above method, the recombinant expression vector is preferably pXC17.4, and the host cell is preferably CHOK1SV GS-KO cell.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention has the following beneficial effects:
obtaining a signal peptide gene sequence A and a GLP-1 analog Fc fusion protein coding gene sequence B through screening and optimization, then constructing a GLP-1 analog Fc fusion protein recombinant gene fragment, then introducing the recombinant gene fragment into a vector pXC17.4, transfecting to CHOK1SV GS-KO cells, screening to obtain a positive host, and obtaining the rGLP-1-Fc with the expression quantity reaching 3.4 g/L. The secretory fusion protein can obviously enhance the affinity of the dimeric GLP-1 receptor due to a dimeric structure formed by Fc, prolong the half-life period of the GLP-1 in vivo and enhance the efficacy. The problems of instability of cell strains, low expression level and the like are overcome, and the produced rGLP-1-Fc has high biological activity and important application value for medical drug development.
Drawings
FIG. 1 is a schematic diagram of recombinant expression plasmid pXC17.4-rGLP-1-Fc.
FIG. 2 is a schematic diagram of the imaging of Clone Select Image after incubation in 96-well plates of a stable transfer library.
FIG. 3 is a schematic of the imaging of ClonePix2 after cloning of the semi-solid medium.
FIG. 4ClonePix2 graphic representation of Clone Select Image after 96 well culture of selected clones.
FIG. 5 is a graph showing the growth tendency of cells during a single-clone fed-batch fermentation.
FIG. 6 is a schematic diagram showing the change in expression level of cells during a fermentation process with a monoclonal feed.
FIG. 7 is a schematic diagram showing the change of glucose and glutamic acid in cells during a fed-batch fermentation process of a single clone.
FIG. 8 is a diagram showing the results of the measurement of the biological activity of the GLP-1 analog Fc fusion protein.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the invention is not limited thereto.
Description of the drawings: vectors pXC17.4 and CHOK1SV GS-KO cells were purchased from lonza, Switzerland; pool spread semisolid medium purchased from Jackson Immunoresearch Laboratories, Inc.; control Trulicity was purchased from Gift Inc.
Example 1 construction of recombinant expression plasmid pXC17.4-rGLP-1-Fc
1. Total gene synthesis of recombinant GLP-1 analogue Fc fusion protein gene (rGLP-1-Fc)
The Signal peptide A (SEQ ID NO: 1, 19aa) was obtained by screening and optimization based on the Signal peptide provided in the literature (Haryadi R, Ho S, Kok Y J, et al. optimization of Heavy Chain and Light Chain Signal Peptides for High Level Expression of Therapeutic Antibodies in CHO Cells [ J ]. Plos One,2015,10(2): e0116878.), and two amino acid sequences were combined together according to the amino acid sequence of GLP-1 analogs published by patents (A.M.Vickers, R.L.millet sequence, W.Gelsel.GLP-1 analog fusion protein, CN 1802386B [ P ].2010.), optimization strategy of Chinese hamster ovary Cells (CHO Cells), sequence was optimized by adding sequence of HindIII and HindIk at the 5' end of the sequence, sequence of double-ending with Koyak codon addition, obtaining a GLP-1 analogue Fc fusion protein gene B (SEQ ID NO: 2), and carrying out whole-gene synthesis on the optimized signal peptide A gene and the optimized GLP-1 analogue Fc fusion protein gene B to obtain a recombinant fusion protein gene rGLP-1-Fc.
2. Construction of recombinant expression plasmid pXC17.4-rGLP-1-Fc
The recombinant fusion protein gene rGLP-1-Fc is subjected to double enzyme digestion by HindIII and EcoRII, the nucleotide sequence C (SEQ ID NO.4, 903bp) of the rGLP-1-Fc fragment is recovered, the nucleotide sequence C is connected with the nucleotide sequence D (SEQ ID NO.5, 6893bp) of the vector fragment recovered after double enzyme digestion by HindIII and EcoRI of pXC17.4, and the final recombinant expression plasmid pXC17.4-rGLP-1-Fc (7796bp) is obtained by screening, and the result is shown in figure 1. Primers are designed at two ends of the rGLP-1-Fc gene fragment for sequencing, and the result shows that the cloned gene fragment is consistent with theory.
Example 2 Stable cell Pool Screen
Electrotransformation of pXC17.4-rGLP-1-Fc
The plasmid pXC17.4-rGLP-1-Fc is linearized by utilizing the PvuI enzyme cutting site, and the linearized plasmid pXC17.4-rGLP-1-Fc (the concentration is 0.4ug/uL) is obtained through purification, filter sterilization and sterile detection. Separately, 2X 10 was prepared7Exponential growth phase of cells CHOK1SV GS-KO cells, added to two electroporation cups, each electrode cup containing 1X 107cells, 0.7mL in volume, each added 100. mu.L of linearized DNA at a concentration of 0.4ug/uL to a cuvette and the linearized plasmid pXC17.4-rGLP-1-Fc was electroporated into CHOK1SV GS-KO recipient cells according to the preset program (pulse 300V, 900. mu.F, resistance ∞, exponential wave, 4mm gap).
2. Transfected cells plated in 96-well plates and Pool selection
Resuspending the cells after electric shock in a 1L conical flask containing 200mL of CD CHO AGT culture medium, gently mixing, pouring into a sample adding groove, and sucking cell sap to 96 degrees by using a row gunIn a well plate, 50. mu.L per well (about 5000cells per well) was placed in a constant temperature CO2In an incubator, 37 ℃ and 10% CO2Culturing; after 1 day of incubation, 150. mu.L of CD CHO AGT (containing 33.3. mu. mol. L) was added to each well-1MSX) Medium was pressurized, one Pool per well, placed at 37 ℃ in 10% CO2And (5) standing and culturing. After culturing for about 20 days, the 96-well plate was scanned with a Clone Select Image imager to obtain the confluency of each plate (see FIG. 2). Selecting a well with the confluency of more than or equal to 55% and less than 70%, sucking 120 mu L of cell supernatant, measuring the expression quantity through Fortebio, and simultaneously supplementing 180 mu L of preheated CD CHO AGT culture medium into each well. Selecting 24 Pool rotating 24 pore plates with expression quantity more than or equal to 13ug/mL and relative expression quantity more than or equal to 22 for expanding culture.
Amplification culture and batch culture screening of Pool
After culturing for 3-4 days in a 24-well plate, transferring all the Pool to a 6-well plate for continuous amplification culture, after culturing for 3-4 days, all 24 Pool participate in batch culture of the 6-well plate, after culturing for 7 days, Fortebio measures the expression quantity, and simultaneously calculates SPR (unit pg. d)-1·cell-1And abbreviated as pcd), selecting 8 Pools with the expression quantity (more than or equal to 80ug/mL) or the SPR value (more than or equal to 9pcd) to respectively transfer to a 6-well plate for continuous culture.
EXAMPLE 3 monoclonal ClonePix2 screening
Semi-solid Medium was spread over Pool
1) Culture formula of Pool semisolid culture medium
TABLE 1 semi-solid Medium formulation for Pool cloning of cells
Blowing and sucking evenly at room temperature, and waiting for the bubbles to disappear slowly.
2) Pool spread semisolid culture medium
According to 150cells/2mL semisolid culture medium per well after plating, the cell sap with the required cell amount is added to the semisolid culture medium. The semi-solid medium is gently mixed with the cells by a pipette and left standing at room temperature for a few minutes to allow the large air bubbles to disappear or break. 2mL of the mixed cell sap was pipetted slowlySlowly adding the mixture into each small hole in the six-hole plate, and slightly breaking the bubbles when the bubbles are mixed. After being split and spread, the plates were covered with a lid and kept at 37 ℃ with 5% CO2And (5) standing and culturing.
Selection of monoclonals by ClonePix2
After approximately 10 days of culture in the semi-solid 6-well plate, the colony outgrowth was observed under a microscope, at which time the colony cell mass was imaged with ClonePix2 (see FIG. 3), and based on the threshold values set by fluorescence intensity, cell size, and cell shape imaging, the dominant single colony was picked up with the needle of ClonePix2 into the target 96-well plate (413 colonies in total) and placed in culture.
Example 4 monoclonal well plate screening
1.96 well plate monoclonal screening
The single Clone was cultured in a 96-well plate for about 5 to 6 days, the confluency of the 96-well plate was scanned with a Clone Select Image imager (see FIG. 4), 120. mu.L of cell supernatant was aspirated from a well having a confluency of 35% or more, the expression level was measured by Fortebio, and 180. mu.L of preheated CD CHO AGT medium was supplied to each well. Screening 48 clones with expression level not less than 9.55ug/mL and relative expression level not less than 20.46, transferring to 24-well plate, and performing propagation.
2. Cloning, expanding culture and batch culture screening
After culturing for 3-4 days in a 24-well plate, transferring 48 clones to a 6-well plate for continuous amplification culture, after culturing for 3-4 days, transferring the clones to a 6-well plate batch culture, after culturing for 7 days, determining the expression level by Fortebio, calculating the SPR value at the same time, selecting 5 better clones with the expression level of more than or equal to 260ug/mL or the SPR value of more than or equal to 35pcd, transferring the 5 better clones to a 125mL shake flask, respectively, culturing at 37 ℃, 140rpm and 10% CO2And (5) performing shaking culture, and performing frozen storage after culturing for 3-4 days.
Example 5 fermentative production of GLP-1 analog Fc fusion proteins
1. Seed liquid preparation
Recovering 5 better monoclonals (C2, C6, C8, C9 and C15) from the selection, and culturing at 37 ℃ for 3-4 days until the cell density is 1-2 multiplied by 106cells/mL are subcultured for 3 days until the cell density is 1-2 × 106cells/mL continues to be passed on, and then cultured for 3 days until the cell density is 1E2×106cells/mL to obtain seed solution.
2. Supplemented fermentation
Mixing the obtained seed liquid at 8 × 105cells/mL are inoculated into 50mL/250mL shake flasks and cultured for 3 days until the cell density is 1.5-2 × 106At the time of cells/mL, sampling is started to determine the growth trend (see figure 5) and the expression quantity (see figure 6) of the cells, meanwhile, biochemical parameters of cell fermentation (see figure 7) are detected, and the cells are fed and cultured, and the Feed proportion is maintained to be 2 percent, the glucose is maintained to be 4g/L, the glutamic acid is maintained to be 4mM, and the cysteine is maintained to be 0.2 percent. Until the culture reaches 10 days, the expression level of 5 monoclonals is in the range of 2.8-3.4 g/L, wherein the expression level of C8 is the highest and reaches 3.4 g/L.
Example 6 purification and biological Activity assay of fusion proteins
1. Separation and purification of fusion protein
After 10 days of fermentation, the culture was terminated, the supernatant was collected by centrifugation, purified and concentrated using FPLC AKTA purification apparatus (GE Healthcare, Pittsburgh, Pa.) loaded with protein A column (Bestchrom Shanghai CN) while being equilibrated with 20mM PBS-0.15M NaCl (pH 7.0). Finally, the fusion protein was eluted with 0.1M HAc-NaAc (pH 3.0) solution.
2. Detection of biological Activity of fusion protein (see FIG. 8)
cAMP measurement: the assay is based on in vitro cell bioassay. The rGLP-1-Fc can activate HEK293 cells (HEK293/GLP-1R, HD BIOSCIENCES (CHINA) CO., LTD) transfected with human GLP-1R and produce cAMP intracellularly in a dose-dependent manner. HEK293/GLP-1R cells were lysed after incubation with rGLP-1-Fc or Troulicity stimulation of different clones (C2, C6, C8, C9, C15), the cAMP produced was measured using the CISBIO cAMP detection kit, the plates were challenged with a multifunctional microplate reader and read, and the results are shown in FIG. 8, which shows that clone C8 exhibited the highest cytological activity with an EC50 value of 0.08675nM, which is about 1.27 fold higher than the control Troulicity activity.
SEQUENCE LISTING
<110> Guangdong Dongyuang pharmaceutical Co., Ltd
<120> optimized gene of recombinant GLP-1 analogue Fc fusion protein and application thereof
<130> 2017
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> Signal peptide A
<400> 1
atggagctgg gcctgagatg ggtgttcctg gtggctatcc tggagggagt gcagtgc 57
<210> 2
<211> 825
<212> DNA
<213> Artificial Sequence
<220>
<223> sequence B
<400> 2
catggcgagg gcacctttac ctccgacgtg tcctcctacc tggaagaaca ggccgccaaa 60
gagtttatcg cctggctcgt gaagggcggt ggtggcggcg gaggatctgg cggaggtgga 120
agcggaggcg gtggatctgc cgagtctaag tacggccctc cctgccctcc ttgtcctgct 180
cctgaagctg ctggcggccc ttccgtgttc ctgttccccc caaagcccaa ggacaccctg 240
atgatctccc ggacccccga agtgacctgc gtggtggtgg atgtgtccca ggaagatccc 300
gaggtgcagt tcaattggta cgtggacggc gtggaagtgc acaacgccaa gaccaagccc 360
agagaggaac agttcaactc cacctaccgg gtggtgtccg tgctgacagt gctgcaccag 420
gactggctga acggcaaaga gtacaagtgc aaggtgtcca acaagggcct gcccagctcc 480
atcgaaaaga ccatctccaa ggccaagggc cagccccggg aaccccaggt gtacacactg 540
cctccaagcc aggaagagat gaccaagaac caggtgtccc tgacctgtct cgtgaaaggc 600
ttctacccct ccgatatcgc cgtggaatgg gagtccaacg gccagcctga gaacaactac 660
aagaccaccc cccctgtgct ggactccgac ggctccttct tcctgtactc ccggctgacc 720
gtggacaagt ccagatggca ggaaggcaac gtgttctcct gctccgtgat gcacgaggcc 780
ctgcacaacc actacaccca gaagtccctg tccctgtctc tggga 825
<210> 3
<211> 294
<212> PRT
<213> Artificial Sequence
<220>
<223> recombinant fusion protein amino acid sequence
<400> 3
Met Glu Leu Gly Leu Arg Trp Val Phe Leu Val Ala Ile Leu Glu Gly
1 5 10 15
Val Gln Cys His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
20 25 30
Leu Glu Glu Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly
35 40 45
Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
50 55 60
Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
65 70 75 80
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
85 90 95
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
100 105 110
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
115 120 125
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
130 135 140
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
145 150 155 160
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
165 170 175
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
180 185 190
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
195 200 205
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
210 215 220
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
225 230 235 240
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
245 250 255
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
260 265 270
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
275 280 285
Leu Ser Leu Ser Leu Gly
290
<210> 4
<211> 903
<212> DNA
<213> Artificial Sequence
<220>
<223> nucleotide sequence C
<400> 4
agcttgccgc caccatggag ctgggcctga gatgggtgtt cctggtggct atcctggagg 60
gagtgcagtg ccatggcgag ggcaccttta cctccgacgt gtcctcctac ctggaagaac 120
aggccgccaa agagtttatc gcctggctcg tgaagggcgg tggtggcggc ggaggatctg 180
gcggaggtgg aagcggaggc ggtggatctg ccgagtctaa gtacggccct ccctgccctc 240
cttgtcctgc tcctgaagct gctggcggcc cttccgtgtt cctgttcccc ccaaagccca 300
aggacaccct gatgatctcc cggacccccg aagtgacctg cgtggtggtg gatgtgtccc 360
aggaagatcc cgaggtgcag ttcaattggt acgtggacgg cgtggaagtg cacaacgcca 420
agaccaagcc cagagaggaa cagttcaact ccacctaccg ggtggtgtcc gtgctgacag 480
tgctgcacca ggactggctg aacggcaaag agtacaagtg caaggtgtcc aacaagggcc 540
tgcccagctc catcgaaaag accatctcca aggccaaggg ccagccccgg gaaccccagg 600
tgtacacact gcctccaagc caggaagaga tgaccaagaa ccaggtgtcc ctgacctgtc 660
tcgtgaaagg cttctacccc tccgatatcg ccgtggaatg ggagtccaac ggccagcctg 720
agaacaacta caagaccacc ccccctgtgc tggactccga cggctccttc ttcctgtact 780
cccggctgac cgtggacaag tccagatggc aggaaggcaa cgtgttctcc tgctccgtga 840
tgcacgaggc cctgcacaac cactacaccc agaagtccct gtccctgtct ctgggatgat 900
gag 903
<210> 5
<211> 6893
<212> DNA
<213> Artificial Sequence
<220>
<223> nucleotide sequence D
<400> 5
aattcattga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac 60
ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg 120
tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 180
gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 240
gtctggcggc cgcgacctgc aggcgcagaa ctggtaggta tggaagatcc ctcgagatcc 300
attgtgctgg cggtaggcga gcagcgcctg cctgaagctg cgggcattcc cgatcagaaa 360
tgagcgccag tcgtcgtcgg ctctcggcac cgaatgcgta tgattctccg ccagcatggc 420
ttcggccagt gcgtcgagca gcgcccgctt gttcctgaag tgccagtaaa gcgccggctg 480
ctgaaccccc aaccgttccg ccagtttgcg tgtcgtcaga ccgtctacgc cgacctcgtt 540
caacaggtcc agggcggcac ggatcactgt attcggctgc aactttgtca tgcttgacac 600
tttatcactg ataaacataa tatgtccacc aacttatcag tgataaagaa tccgcgccag 660
cacaatggat ctcgaggtcg agggatctct agaggatcct ctacgccgga cgcatcgtgg 720
ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg 780
gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg 840
caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg 900
cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata 960
agggagagcg tcgacctcgg gccgcgttgc tggcgttttt ccataggctc cgcccccctg 1020
acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 1080
gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 1140
ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 1200
gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 1260
cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 1320
taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 1380
atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 1440
cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 1500
cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 1560
ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 1620
ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 1680
tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 1740
aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 1800
tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 1860
gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 1920
atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 1980
tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 2040
ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 2100
ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 2160
tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 2220
ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 2280
ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 2340
tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 2400
gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 2460
taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 2520
cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 2580
agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 2640
gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 2700
ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa 2760
ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tgatggctct 2820
ttgcggcacc catcgttcgt aatgttccgt ggcaccgagg acaaccctca agagaaaatg 2880
taatcacact ggctcacctt cgggtgggcc tttctgcgtt tataaggaga cactttatgt 2940
ttaagaaggt tggtaaattc cttgcggctt tggcagccaa gctagatccg gctgtggaat 3000
gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc 3060
atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga 3120
agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc 3180
atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 3240
tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga 3300
ggcttttttg gaggcctagg cttttgcaaa aagctagctt ggggccaccg ctcagagcac 3360
cttccaccat ggccacctca gcaagttccc acttgaacaa aaacatcaag caaatgtact 3420
tgtgcctgcc ccagggtgag aaagtccaag ccatgtatat ctgggttgat ggtactggag 3480
aaggactgcg ctgcaaaacc cgcaccctgg actgtgagcc caagtgtgta gaagagttac 3540
ctgagtggaa ttttgatggc tctagtacct ttcagtctga gggctccaac agtgacatgt 3600
atctcagccc tgttgccatg tttcgggacc ccttccgcag agatcccaac aagctggtgt 3660
tctgtgaagt tttcaagtac aaccggaagc ctgcagagac caatttaagg cactcgtgta 3720
aacggataat ggacatggtg agcaaccagc acccctggtt tggaatggaa caggagtata 3780
ctctgatggg aacagatggg cacccttttg gttggccttc caatggcttt cctgggcccc 3840
aaggtccgta ttactgtggt gtgggcgcag acaaagccta tggcagggat atcgtggagg 3900
ctcactaccg cgcctgcttg tatgctgggg tcaagattac aggaacaaat gctgaggtca 3960
tgcctgccca gtgggagttc caaataggac cctgtgaagg aatccgcatg ggagatcatc 4020
tctgggtggc ccgtttcatc ttgcatcgag tatgtgaaga ctttggggta atagcaacct 4080
ttgaccccaa gcccattcct gggaactgga atggtgcagg ctgccatacc aactttagca 4140
ccaaggccat gcgggaggag aatggtctga agcacatcga ggaggccatc gagaaactaa 4200
gcaagcggca ccggtaccac attcgagcct acgatcccaa ggggggcctg gacaatgccc 4260
gtcgtctgac tgggttccac gaaacgtcca acatcaacga cttttctgct ggtgtcgcca 4320
atcgcagtgc cagcatccgc attccccgga ctgtcggcca ggagaagaaa ggttactttg 4380
aagaccgccg cccctctgcc aattgtgacc cctttgcagt gacagaagcc atcgtccgca 4440
catgccttct caatgagact ggcgacgagc ccttccaata caaaaactaa ttagactttg 4500
agtgatcttg agcctttcct agttcatccc accccgcccc agagagatct ttgtgaagga 4560
accttacttc tgtggtgtga cataattgga caaactacct acagagattt aaagctctaa 4620
ggtaaatata aaatttttaa gtgtataatg tgttaaacta ctgattctaa ttgtttgtgt 4680
attttagatt ccaacctatg gaactgatga atgggagcag tggtggaatg cctttaatga 4740
ggaaaacctg ttttgctcag aagaaatgcc atctagtgat gatgaggcta ctgctgactc 4800
tcaacattct actcctccaa aaaagaagag aaaggtagaa gaccccaagg actttccttc 4860
agaattgcta agttttttga gtcatgctgt gtttagtaat agaactcttg cttgctttgc 4920
tatttacacc acaaaggaaa aagctgcact gctatacaag aaaattatgg aaaaatattc 4980
tgtaaccttt ataagtaggc ataacagtta taatcataac atactgtttt ttcttactcc 5040
acacaggcat agagtgtctg ctattaataa ctatgctcaa aaattgtgta cctttagctt 5100
tttaatttgt aaaggggtta ataaggaata tttgatgtat agtgccttga ctagagatca 5160
taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc 5220
ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt 5280
ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 5340
tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatctcta 5400
gcttcgtgtc aaggacggtg aggcgcgcct actgagtcat tagggacttt ccaatgggtt 5460
ttgcccagta cataaggtca ataggggtga atcaacagga aagtcccatt ggagccaagt 5520
acactgagtc aatagggact ttccattggg ttttgcccag tacaaaaggt caataggggg 5580
tgagtcaatg ggtttttccc attattggca cgtacataag gtcaataggg gtgagtcatt 5640
gggtttttcc agccaattta attaaaacgc catgtacttt cccaccattg acgtcaatgg 5700
gctattgaaa ctaatgcaac gtgaccttta aacggtactt tcccatagct gattaatggg 5760
aaagtaccgt tctcgagcca atacacgtca atgggaagtg aaagggcagc caaaacgtaa 5820
caccgccccg gttttcccct ggaaattcca tattggcacg cattctattg gctgagctgc 5880
gttctacgtg ggtataagag gcgcgaccag cgtcggtacc gtcgcagtct tcggtctgac 5940
caccgtagaa cgcagcctca ggacctccat agaagacacc gggaccgatc cagcctccgc 6000
ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca agagtgacgt aagtaccgcc 6060
tatagagtct ataggcccac ccccttggct tcttatgcat gctatactgt ttttggcttg 6120
gggtctatac acccccgctt cctcatgtta taggtgatgg tatagcttag cctataggtg 6180
tgggttattg accattattg accactcccc tattggtgac gatactttcc attactaatc 6240
cataacatgg ctctttgcca caactctctt tattggctat atgccaatac actgtccttc 6300
agagactgac acggactctg tatttttaca ggatggggtc tcatttatta tttacaaatt 6360
cacatataca acaccaccgt ccccagtgcc cgcagttttt attaaacata acgtgggatc 6420
tccacgcgaa tctcgggtac gtgttccgga catgggctct tctccggtag cggcggagct 6480
tctacatccg agccctgctc ccatgcctcc agcgactcat ggtcgctcgg cagctccttg 6540
ctcctaacag tggaggccag acttaggcac agcacgatgc ccaccaccac cagtgtgccg 6600
cacaaggccg tggcggtagg gtatgtgtct gaaaatgagc tcggggagcg ggcttgcacc 6660
gctgacgcat ttggaagact taaggcagcg gcagaagaag atgcaggcag ctgagttgtt 6720
gtgttctgat aagagtcaga ggtaactccc gttgcggtgc tgttaacggt ggagggcagt 6780
gtagtctgag cagtactcgt tgctgccgcg cgcgccacca gacataatag ctgacagact 6840
aacagactgt tcctttccat gggtcttttc tgcagtcacc gtccttgaca cga 6893
Claims (9)
1. A recombinant fusion protein gene is characterized in that the recombinant fusion protein gene has a gene sequence with an A-B structure, wherein A is a signal peptide gene, and B is a gene for encoding GLP-1 analogue Fc fusion protein; in the fusion protein gene, the nucleotide sequence of the signal peptide A is shown in SEQ ID No.1, and the nucleotide sequence of the gene B for encoding the GLP-1 analogue Fc fusion protein is shown in SEQ ID No. 2.
2. The recombinant fusion protein gene as claimed in claim 1, wherein the amino acid sequence of the recombinant fusion protein gene is shown in SEQ ID No. 3.
3. A recombinant expression vector comprising the recombinant fusion protein gene of claim 1.
4. The recombinant expression vector of claim 3, wherein the recombinant expression vector is pXC17.4.
5. A host cell comprising the recombinant expression vector of claim 3 or 4.
6. The host cell of claim 5, wherein the host cell is a CHOK1SV GS-KO cell.
7. Use of the recombinant fusion protein gene of claim 1 for the production of a GLP-1 analogue Fc fusion protein.
8. The use according to claim 7, comprising: recombining the gene with the signal peptide gene of claim 1 and the Fc fusion protein encoding GLP-1 analogue to obtain an optimized recombinant fusion protein gene; inserting the recombinant fusion protein gene into an expression vector to obtain a recombinant expression vector; carrying out PvuI linearization on the recombinant expression vector, transforming host cells, and screening to obtain a positive host strain; culturing the positive host strain, inducing the expression of the GLP-1 analogue Fc fusion protein, and recovering and purifying the expressed GLP-1 analogue Fc fusion protein.
9. Use according to claim 8, characterized in that: the recombinant expression vector is pXC17.4; the host cell is a CHOK1SV GS-KO cell.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201711390118.XA CN108103088B (en) | 2017-12-21 | 2017-12-21 | Optimized gene of recombinant GLP-1 analogue Fc fusion protein and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201711390118.XA CN108103088B (en) | 2017-12-21 | 2017-12-21 | Optimized gene of recombinant GLP-1 analogue Fc fusion protein and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN108103088A CN108103088A (en) | 2018-06-01 |
CN108103088B true CN108103088B (en) | 2021-03-26 |
Family
ID=62210743
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201711390118.XA Active CN108103088B (en) | 2017-12-21 | 2017-12-21 | Optimized gene of recombinant GLP-1 analogue Fc fusion protein and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN108103088B (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110878127B (en) * | 2018-09-06 | 2022-06-28 | 浙江柏拉阿图医药科技有限公司 | Long-acting recombinant GLP1-Fc-CD47 protein and preparation and application thereof |
CN113005140B (en) * | 2019-12-19 | 2024-03-15 | 鲁南制药集团股份有限公司 | GS expression vector with double expression cassettes and application thereof |
WO2022110499A1 (en) * | 2020-11-27 | 2022-06-02 | 广州汉腾生物科技有限公司 | Application of signal peptide in expression of glp-1 fusion protein |
CN117143242B (en) * | 2023-10-30 | 2024-03-29 | 南京佰抗生物科技有限公司 | Monoclonal antibody composition for resisting Galectin-3 protein and application thereof |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1302333A (en) * | 1998-01-23 | 2001-07-04 | 诺沃挪第克公司 | Process for making desired polypeptides in yeast |
CN103804488A (en) * | 2012-11-15 | 2014-05-21 | 刘占良 | Glucagon-like peptides (GLP-1 and GLP-2) produced and delivered by using food grade lactic acid bacteria and preparation method thereof |
CN104327187A (en) * | 2014-10-11 | 2015-02-04 | 上海兴迪金生物技术有限公司 | Recombinant human GLP-1-Fc fusion protein |
CN104854133A (en) * | 2012-10-12 | 2015-08-19 | 新加坡科技研究局 | Optimised heavy chain and light chain signal peptides for the production of recombinant antibody therapeutics |
CN106008717A (en) * | 2016-01-22 | 2016-10-12 | 北京正旦国际科技有限责任公司 | Long-acting recombinant GLP-1 fusion protein, preparation method and use thereof |
-
2017
- 2017-12-21 CN CN201711390118.XA patent/CN108103088B/en active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1302333A (en) * | 1998-01-23 | 2001-07-04 | 诺沃挪第克公司 | Process for making desired polypeptides in yeast |
CN104854133A (en) * | 2012-10-12 | 2015-08-19 | 新加坡科技研究局 | Optimised heavy chain and light chain signal peptides for the production of recombinant antibody therapeutics |
CN103804488A (en) * | 2012-11-15 | 2014-05-21 | 刘占良 | Glucagon-like peptides (GLP-1 and GLP-2) produced and delivered by using food grade lactic acid bacteria and preparation method thereof |
CN104327187A (en) * | 2014-10-11 | 2015-02-04 | 上海兴迪金生物技术有限公司 | Recombinant human GLP-1-Fc fusion protein |
CN106008717A (en) * | 2016-01-22 | 2016-10-12 | 北京正旦国际科技有限责任公司 | Long-acting recombinant GLP-1 fusion protein, preparation method and use thereof |
Non-Patent Citations (2)
Title |
---|
Dong-Myung Kim等.Fc fusion to Glucagon-like peptide-1 inhibits degradation by human DPP-IV, increasing its half-life in serum and inducing a potent activity for human GLP-1 receptor activation.《BMB reports》.2009,第42卷(第4期),第212-216页. * |
Fc fusion to Glucagon-like peptide-1 inhibits degradation by human DPP-IV, increasing its half-life in serum and inducing a potent activity for human GLP-1 receptor activation;Dong-Myung Kim等;《BMB reports》;20090430;第42卷(第4期);第212-216页 * |
Also Published As
Publication number | Publication date |
---|---|
CN108103088A (en) | 2018-06-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2019283892B2 (en) | Anti-human papillomavirus 16 E7 T cell receptors | |
CN108103088B (en) | Optimized gene of recombinant GLP-1 analogue Fc fusion protein and application thereof | |
US6573058B1 (en) | Antibody to herpes virus entry receptor protein | |
CN110945127B (en) | Viral vectors encoding recombinant FIX with increased expression for gene therapy of hemophilia B | |
KR101078202B1 (en) | Recombinant lubricin molecules and uses thereof | |
US20020147326A1 (en) | Hexameric fusion proteins and uses therefor | |
CN108913718A (en) | A kind of preparation method and application of the CAR-T cell of targeting EGFR v III | |
CN106591371A (en) | CD16A/GPC3 double-antibody lentivirus expression vector, and construction method and application thereof | |
CN113943737A (en) | Application of chicken CTGF gene in inhibiting differentiation of chicken preadipocytes | |
CN107936122A (en) | A kind of slow virus and its preparation method and application | |
CN114540424A (en) | IGFBP7 muscle tissue specific knockout mouse animal model and construction method thereof | |
CN109022363A (en) | A kind of CD-133-CAR-T system constituting method based on PiggyBac carrier | |
CN115916985A (en) | Fibroblast growth factor 21 (FGF 21) gene therapy for central nervous system disorders | |
CN108034678A (en) | A kind of expression vector, system and method for expressing recombinant human nerve growth factor | |
CN110819589B (en) | Method for enhancing immune effector cell function | |
CN112342245B (en) | CRISPR-Cas13d system for promoting suspension of CHO cells and recombinant CHO cells | |
RU2773956C2 (en) | Viral vectors encoding recombinant fix variants with increased expression for gene therapy of hemophilia b | |
CN115074385A (en) | Epigenetic vector for reducing expression level of new coronavirus receptor ACE2 | |
CN115634281A (en) | Anti-tumor effect of CTRP3 protein, recombinant oncolytic virus encoding same, preparation method and application | |
CN117377683A (en) | New antigenic peptide and application thereof in treating BRAF gene mutation related diseases | |
CN113862298A (en) | Recombinant expression vector | |
CN114891114A (en) | Intracellular signal domain for construction of chimeric antigen receptor | |
CN114891115A (en) | Nucleic acid construct for CAR cell therapy, lentiviral vector, cell preparation and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CP03 | Change of name, title or address | ||
CP03 | Change of name, title or address |
Address after: 523808 No.1, Gongye North Road, Songshanhu Park, Dongguan City, Guangdong Province Patentee after: Guangdong Dongyangguang Pharmaceutical Co.,Ltd. Address before: 523808 No. 1 Industrial North Road, Songshan Industrial Park, Songshan, Guangdong, Dongguan, Hubei Patentee before: SUNSHINE LAKE PHARMA Co.,Ltd. |