CN108103088B

CN108103088B - Optimized gene of recombinant GLP-1 analogue Fc fusion protein and application thereof

Info

Publication number: CN108103088B
Application number: CN201711390118.XA
Authority: CN
Inventors: 肖海鹏; 陈英; 张奕敏; 张玮; 龚庆伟; 张晓焰; 马利; 陈小锋; 李文佳
Original assignee: Sunshine Lake Pharma Co Ltd
Current assignee: Guangdong HEC Pharmaceutical
Priority date: 2017-12-21
Filing date: 2017-12-21
Publication date: 2021-03-26
Anticipated expiration: 2037-12-21
Also published as: CN108103088A

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

Optimized gene of recombinant GLP-1 analogue Fc fusion protein and application thereof

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

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.