GLP-1 analogue fusion protein and application thereof

Abstract

The invention relates to the field of medicines, in particular to a GLP-1 analogue fusion protein and application thereof. The GLP-1 analogue fusion protein is obtained by fusing GLP-1 at N ends of a heavy chain constant region and a light chain constant region of IgG4. A research result shows that the GLP-1 analogue fusion protein has an obvious effect of reducing the body weight of a mouse, the volume and the weight of the liver of the mouse are reduced, and the NAS score and the liver fatty degeneration are obviously improved. Therefore, the traditional Chinese medicine composition can be applied to medicines or products for slimming or treating fatty liver.

Description

Technical Field

[0001] The present application relates to the field of medicine, and specifically to a GLP-1 analog fusion protein and its application. Background Art

[0002] Glucagon-like peptide (GLP) is a polypeptide expressed by the proglucagon gene, which exerts its biological effects by binding to the corresponding G protein-coupled receptors. GLP can significantly increase the first and second phase insulin secretion, reduce glucagon levels, and delay gastric emptying. Because its blood sugar lowering effect is blood sugar-dependent and has the effect of reducing body weight, GLP and its analogs are widely used in the treatment of type 2 diabetes and alleviate insulin resistance caused by obesity.

[0003] There are many weight loss drugs approved overseas, and various categories of weight loss drugs have appeared, such as mitochondrial uncouplers, sympathomimetics, cannabinoid 1 receptor (CB1) antagonists, lipase inhibitors, serotonin (5-HT) receptor agonists and GLP-1 receptor agonists, etc. However, most of the drugs were withdrawn from use after approval due to extremely serious adverse reactions, such as serious cardiovascular side effects (sibutramine, fenfluramine, dexfenfluramine hydrochloride and rainbow pills), suicidal tendencies (rimonabant), and the risk of drug dependence and abuse (methamphetamine).

[0004] Among the weight loss drugs approved at home and abroad, GLP-1 receptor agonists have relatively few side effects. Therefore, it is necessary to develop a GLP-1 receptor agonist drug. Summary of the invention

[0005] Based on the needs of the prior art, the present application provides a GLP-1 analog fusion protein and its application.

[0006] First, the present application discloses a GLP-1 analog fusion protein, which is obtained by fusing glucagon-like peptide GLP-1 to the N-terminus of the heavy chain constant region and the light chain constant region of IgG4, respectively.

[0007] Preferably, the amino acid sequence of the glucagon-like peptide GLP-1 is shown as SEQ ID NO.1; the amino acid sequence of the IgG4 heavy chain constant region is shown as SEQ ID NO.4; and the amino acid sequence of the IgG4 light chain constant region is shown as SEQ ID NO.8.

[0008] Specifically, the N-termini of the IgG4 heavy chain constant region and the light chain constant region are fused to the glucagon-like peptide GLP-1 via a connecting peptide, respectively. Preferably, the amino acid sequence of the connecting peptide is as shown in SEQ ID NO.3.

[0009] The amino acid sequences obtained by fusing the glucagon-like peptide GLP-1 to the N-terminus of the IgG4 heavy chain constant region and the light chain constant region are shown in SEQ ID NO.5 and SEQ ID NO.9, respectively;

[0010] Furthermore, a signal peptide is fused to the N-terminus of GLP-1; preferably, the amino acid sequence of the signal peptide is shown in SEQ ID NO. 2. The present invention has determined through research that the adopted signal peptide enables the expression amount of GLP-1 to reach a higher level.

[0011] The present invention also provides a nucleic acid encoding the GLP-1 analog fusion protein.

[0012] Preferably, the nucleotide sequence encoding the glucagon-like peptide GLP-1 fused to the N-terminus of the IgG4 heavy chain constant region is shown as SEQ ID NO.6, and the nucleotide sequence encoding the glucagon-like peptide GLP-1 fused to the N-terminus of the IgG4 light chain constant region is shown as SEQ ID NO.10.

[0013] The present invention provides a recombinant expression vector encoding the nucleic acid.

[0014] The present invention provides a method for preparing a cell line producing a GLP-1 analog fusion protein, which is obtained by transfecting the encoding nucleic acid into mammalian cells, preferably, the mammal is a CHO cell; preferably, by constructing a recombinant expression vector containing the encoding nucleic acid, linearizing it and then transfecting it into mammalian cells.

[0015] The present invention provides a cell line producing a GLP-1 analog fusion protein obtained by the method.

[0016] The present invention particularly provides the use of the GLP-1 analog fusion protein and the cell line in preparing weight loss products or drugs for treating fatty liver.

[0017] The research results of the present invention show that compared with the control, the GLP-1 analog fusion protein of the present application has a significant effect of reducing the weight of mice, the volume and weight of the mouse liver are reduced, and the NAS score and hepatic steatosis are significantly improved. The GLP-1 analog fusion protein of the present application can be used in drugs or products for weight loss and treatment of fatty liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 , the structure of the GLP-1 analog fusion protein of the present application.

[0019] Figure 2 , Reduced SDS-PAGE electrophoresis diagram of the GLP-1 analog fusion protein of the present application.

[0020] Figure 3, body weight changes of mice treated with GLP-1 analog fusion protein.

[0021] Figure 4 , GLP-1 analog fusion protein treated mice liver volume changes. Compared with the control group, "*" P < 0.01, "**" P < 0.05, "***" P < 0.001.

[0022] Figure 5 , Changes in liver weight of mice treated with GLP-1 analog fusion protein.

[0023] Figure 6 , NAS scores of GLP-1 analog fusion protein treatment. DETAILED DESCRIPTION

[0024] The present invention is described below through specific implementation modes in order to better understand the present invention, but it does not constitute a limitation to the present invention.

[0025] Example 1: Construction of an expression vector for a GLP-1 analog fusion protein

[0026] The fusion protein of the GLP-1 analogue of the present application is designed, wherein the amino acid sequence of the glucagon-like peptide GLP-1 polypeptide used is: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG (SEQ ID NO.1). The specific design is as follows:

[0027] (1) The GLP-1 analog fusion protein of the present application:

[0028] The amino acid sequence of the heavy chain IgG4-Hc is as follows:

[0029] PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS

[0030] LSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPAALGGPSVFLFPPK

[0031] PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL

[0032] TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC

[0033] LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV

[0034] MHEALHNHYTQKSLSLSLG (SEQ ID NO. 4).

[0035] The heavy chain fusion protein GLP-Hc is composed of a GLP-1 polypeptide connected to the N-terminus of the heavy chain IgG4-Hc via a connecting peptide (GGGGSGGGGSGGGGS, SEQ ID NO.3), and its amino acid sequence is as follows:

[0036] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSPSVFPLAPCSRS

[0037] TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT

[0038] YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPAALGGPSVFLFPPKPKDTLMISRTPEVTCV

[0039] VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK

[0040] CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE

[0041] SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL

[0042] SLG (SEQ ID NO. 5).

[0043] Through research and comparison, in this example, a signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO. 2) is also connected to the N-terminus of the GLP-1 polypeptide.

[0044] Based on the above amino acid sequence, codon optimization was performed according to the codon preference of CHO cells, which is more conducive to the expression of GLP-Hc fusion protein in CHO cells. The optimized coding nucleotide sequence of GLP-Hc is as follows:

[0045]

[0046] The nucleotide sequence of the signal peptide connected to the N-terminus of GLP-Hc (after screening) is: ATGGGATGGTCATGTAT TATTCTGTTTCTCGTCGCCACAGCCACAGGCGTACACTCA (SEQ ID NO. 7).

[0047] The amino acid sequence of the light chain IgG4-Lc is as follows:

[0048] APSVFIFPPSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSKDSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC (SEQ ID NO. 8).

[0049] The light chain fusion protein GLP-Lc is composed of a GLP-1 polypeptide connected to the N-terminus of the light chain IgG4-Lc through a connecting peptide (GGGGSGGGGSGGGGS), and its amino acid sequence is as follows:

[0050] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO. 9).

[0051] In this example, a signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO. 2) is also connected to the N-terminus of the GLP-1 polypeptide.

[0052] Based on the above amino acid sequence, codon optimization was performed according to the codon preference of CHO cells, which is more conducive to the expression of GLP-Lc fusion protein in CHO cells. The optimized coding nucleotide sequence of GLP-Lc is as follows:

[0053] CACGGTGAGGGAACCTTCACTAGTGACGTGAGTAGTTATCTGGAGGAACAGGCCGCCAAAGAGTTCATCGCCTGGCTCGTCAAAGGAGGCGGCGGAGGTGGAGGGTCAGGAGGAGGGGGATCAGGAGGGGGCGGCTCTGCACCCTCTGTCTTTATCTTTCCCCCTAGCGATGAGCAGCTTAAATCCGGGACCGCAAGCGTCGTCTGTCTGCTGAACAATTTTTAT CCCAGAGAGGCCAAGGTGCAGTGGAAAGTGGACAACGCTCTCCAGAGTGGCAACAGTCAGGAGAGCGTAACAGAGCAGGATAGCAAGGACTCTACTTACTCCCTTAGCTCCACTCTGACCCTGTCAAAAGCTGACTATGAGAAGCACAAAGTGTACGCATGCGAGGTGACACATCAGGGTTTGAGCTCCCCAGTCACAAAGTCTTTTAACCGAGGAGAGTGT(SEQ ID NO.10).

[0054] The nucleotide sequence of the signal peptide (after screening) connected to the N-terminus of GLP-Lc is the same as above (SEQ ID NO.7).

[0055] The genes encoding the above fusion proteins are all artificially optimized with CHO cell preferred codons, and the full-length sequences are obtained by chemical synthesis. The synthesized GLP-Hc nucleotide sequence containing a signal peptide is cloned into the HindIII / EcoRI site of pKS001 to obtain the vector pKS001-GLP-Hc; the synthesized GLP-Lc nucleotide sequence containing a signal peptide is cloned into the XbaI / NotI site of pKS001-GLP-Hc to obtain the fusion protein expression vector pKS-GLP-IgG4. The related experimental methods use conventional molecular biological experimental methods.

[0056] (2) Dulaglutide (trade name Eli Lilly) was purchased directly. The amino acid sequence of Dulaglutide is:

[0057] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(dlu-Fc)(SEQ IDNO.11).

[0058] Example 2: Construction of a cell line stably expressing a GLP-1 analog fusion protein

[0059] The recombinant expression plasmid pKS-GLP-IgG4 was linearized by PvuI and then transfected into CHO-K1 cells. For the specific transfection process, please refer to the instruction manual of Lipofectamine2000 (Invitrogen). 48 hours after transfection, the cells began to be cultured in the screening medium. The screening medium used was a medium containing 25 μM L-methionine sulfoximide (MSX), and the screening medium was replaced every 3 days. After transfection, the cell viability first decreased and then increased under the action of the drug. When the cell viability rose to more than 90%, the transfected cells were plated on 96-well plates for monoclonal screening using the limiting dilution method. The cell density of the plate was 0.5 per well. After the cells were statically cultured in the incubator for 14 days, the expression of the fusion protein in the culture supernatant of the monoclonal cells was detected by ELISA. According to the test results, the 10 monoclonal cell lines with the highest expression level were screened out for each transfected cell for gradual expansion culture. After 3 days of culture in a 6-well plate, the cells were counted and the protein expression level was detected. The 3 cell lines with the highest protein expression level were selected for expansion culture in a T125 shake flask. After 3 days of culture, the cells were counted and the protein expression level was detected. The cells with the highest expression level were selected as stable cell lines.

[0060] Example 3. Expression and purification of GLP-1 analog fusion protein

[0061] The high-expressing cells were cultured in serum-free CDOptiCHO and the culture supernatant was collected after a certain period of time. The fusion protein was purified by the following Protein A affinity chromatography method: chromatography column: ATProteinADiamond affinity chromatography medium, column volume CV: 5 ml, flow rate: 5 ml / min, pressure limit: ≤0.3 MPa.

[0062] Pretreatment: Rinse with purified water for at least 5 CV.

[0063] Equilibration: First elute with elution buffer for 1 CV, then fully equilibrate with binding buffer for at least 10 CV before loading.

[0064] Loading: Load the filtered culture supernatant onto the equilibrated chromatography column, collect the load flow-through, and sample 100 μl for electrophoresis detection.

[0065] Elution: After loading, increase the flow rate to 5 ml / min and elute with binding buffer for at least 6 CV until the UV baseline is flat.

[0066] Elution: Use 100% elution buffer for elution until the UV baseline is flat, collect the eluted components in separate tubes, and add an appropriate amount of neutralization buffer to each collection tube according to the collection volume and mix well.

[0067] The GLP-1 analog fusion protein was purified by reducing SDS-PAGE electrophoresis. Figure 2 shown.

[0068] Example 4: Effects of different signal peptides on the expression of GLP-1 analog fusion protein

[0069] An expression vector of a GLP-1 analog fusion protein containing a signal peptide was constructed according to the method of Example 1 above, wherein in the expression vector of the GLP-1 analog fusion protein, the heavy chain sequence GLP-Hc (SEQ ID NO.6) and the light chain sequence GLP-Lc (SEQ ID NO.10) were the same as those in Example 1, the only difference being the sequence of the N-terminal fusion signal peptide.

[0070] A cell line stably expressing a GLP-1 analog fusion protein containing a signal peptide was constructed according to the method of Example 2. The GLP-1 analog fusion protein containing a signal peptide was expressed according to the method of Example 3, and the high-expressing cells were cultured in serum-free CDOptiCHO, and the culture supernatant was collected after 14 days.

[0071] The expression level of GLP-1 analog fusion protein in the culture supernatant was detected by ELISA. The specific steps of ELISA detection include: diluting anti-human IgG (Fc specific) goat antibody (I2136, Sigma) with PBS at 1:2000, and adding 100 μl / well to the ELISA plate. Incubate overnight at 4°C for coating. After washing the plate three times with PBST, add 50g / L BSA blocking solution, 200 μl / well, and incubate at 37°C for 2h. After washing the plate three times with PBST, add cell expression supernatant and standard curve protein diluted at appropriate concentrations, 100 μl / well, and incubate at 37°C for 1h. After washing the plate three times with PBST, add secondary antibody, anti-human IgG (Fc specific)-peroxidase antibody goat antibody (A0170, Sigma) with PBS at 1:8000, 100 μl / well, and incubate at 37°C for 1h. After washing the plate 3 times with PBST, add 100 μl / well TMB substrate colorimetric solution and incubate at room temperature in the dark for 10-15 minutes. Add 50 μl / well stop solution to terminate. Place in a microplate reader and detect the OD value at a wavelength of 450 nm. The expression level of the GLP-1 analog fusion protein in the culture supernatant can be calculated using the standard curve.

[0072] Table 1 Expression levels of GLP-1 analog fusion proteins in culture supernatants obtained with different signal peptides

[0073] Signal peptide Expression level of GLP-1 analog fusion protein (g / L) MGWSCIILFLVATATGVHS(SEQ ID NO.2) 2.5 MPLLLLLPLLWAGALA 1.2 MAHIRGLWLPGCLALAALCSLVHS 1.5

[0074] From the results in Table 1, it can be seen that the expression level of the GLP-1 analog fusion protein obtained by the signal peptide (SEQ ID NO. 2) finally screened and used in the present application is much greater than the expression levels of the GLP-1 analog fusion proteins obtained by the other two signal peptides.

[0075] Example 5: Therapeutic efficacy test of GLP-1 analog fusion protein

[0076] In this experiment, C57BL / 6JNifdc-DIO male mice induced by high-fat diet for 10 weeks were selected, and the efficacy of the drug was investigated in this model mouse by subcutaneous injection for 5 weeks with a dosing frequency of 2 times / week.

[0077] The fusion proteins used in this experiment include the GLP-1 analog fusion protein (GLP-lgG4) of this application and Eli Lilly's dulaglutide (dlu-Fc, purchased directly). The fusion proteins used were diluted to the required concentration using a diluent (30mMPB, 0.04% Tween80, 3% mannitol, pH7.0) to obtain each fusion protein solution. 25 SPF-grade C57BL / 6JNifdc-DIO male mice with a body weight range of 30-40g and induced for 10 weeks using ResearchDiet-D12492 feed were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. and raised by Beijing Huilin Zegu Biotechnology Co., Ltd. 25 mice were randomly divided into 4 experimental groups and 1 blank control group. The mice in the 4 experimental groups were injected with 40μg dlu-Fc, 75μg dlu-Fc, 40μg GLP-IgG4 and 75μg GLP-IgG4, respectively. The injection volume of the fusion protein solution in the 4 experimental groups was 100μl. The blank control group was injected with 100μl saline. Injection procedure: 2 injections per week for 5 consecutive weeks. The mice were weighed before each injection. The results are shown in Figure 3 After 5 weeks, the mice were killed and their livers were removed. The volume and weight of the livers were measured. Figures 4-5 The excised liver tissue was stained with HE and Sirius red. After taking pictures of each mouse liver slice, NAS score was performed (NAS score = fatty degeneration score + inflammation score). The results are shown in Figure 6 .

[0078] Figure 3 The results showed that after 5 weeks, the GLP-IgG4 75 μg group had a 14.7% weight loss compared to the blank control group. Figures 4-5 As shown in the results, compared with the blank control group, the liver volume of the dlu-Fc75μg, GLP-IgG4 40μg and GLP-IgG4 75μg groups decreased, and the liver volume and weight of the GLP-IgG4 75μg group decreased most significantly.

[0079] Figure 6 The results showed that the NAS scores of the GLP-IgG4 40μg and GLP-IgG4 75μg groups were significantly better than those of the blank control group, and the NAS score and hepatic steatosis of the GLP-IgG4 75μg group were significantly improved.

[0080] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned various embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A GLP-1 analog fusion protein, characterized in that: The glucagon-like peptide GLP-1 is fused to the N-terminus of the IgG4 heavy chain constant region and the light chain constant region, respectively.

2. The GLP-1 analog fusion protein according to claim 1, characterized in that: The amino acid sequence of the glucagon-like peptide GLP-1 is shown in SEQ ID NO.1; The amino acid sequence of the IgG4 heavy chain constant region is shown in SEQ ID NO.4; the amino acid sequence of the IgG4 light chain constant region is shown in SEQ ID NO.

8.

3. The GLP-1 analog fusion protein according to claim 1, characterized in that: The N-termini of the IgG4 heavy chain constant region and the light chain constant region are fused to the glucagon-like peptide GLP-1 via a connecting peptide, respectively. Preferably, the amino acid sequence of the connecting peptide is as shown in SEQ ID NO.3; The amino acid sequences obtained by fusing glucagon-like peptide GLP-1 to the N-terminus of the IgG4 heavy chain constant region and the light chain constant region are shown in SEQ ID NO.5 and SEQ ID NO.9, respectively.

4. The GLP-1 analog fusion protein according to claim 1, characterized in that: The N-terminus of the glucagon-like peptide GLP-1 is fused with a signal peptide; preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO.

2.

5. A nucleic acid encoding the GLP-1 analog fusion protein according to any one of claims 1 to 4.

6. The encoding nucleic acid according to claim 5, characterized in that The nucleotide sequence encoding the glucagon-like peptide GLP-1 fused to the N-terminus of the IgG4 heavy chain constant region is shown in SEQ ID NO.6, and the nucleotide sequence encoding the glucagon-like peptide GLP-1 fused to the N-terminus of the IgG4 light chain constant region is shown in SEQ ID NO.

10.

7. A recombinant expression vector encoding the nucleic acid according to claim 5 or 6.

8. A method for preparing a cell line producing a GLP-1 analog fusion protein, characterized in that: The method is obtained by transfecting a mammalian cell with the encoding nucleic acid as claimed in claim 5 or 6, preferably, the mammal is a CHO cell; preferably, the method is obtained by constructing a recombinant expression vector containing the encoding nucleic acid as claimed in claim 5 or 6, and then transfecting the vector into a mammalian cell after linearization.

9. The cell line producing GLP-1 analog fusion protein obtained by the method according to claim 8.

10. Use of the GLP-1 analog fusion protein according to any one of claims 1 to 4, or the cell line according to claim 9 in the preparation of a weight loss product, or a drug for treating fatty liver.

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