Signal peptides for glp-1 and analogs thereof and uses thereof

Abstract

The present application belongs to the technical field of biological medicine. More particularly, it relates to a signal peptide of GLP-1 and its analogs and application thereof. The present application provides a signal peptide of GLP-1 protein or its analogs, the amino acid sequence of which is shown in any one of SEQ ID NO. 1-8. The present application improves the signal peptide of GLP-1 protein, which can promote the production of inclusion bodies, optimize the properties of inclusion bodies, and improve the unit yield of target protein, so that the target protein can be expressed more efficiently. In addition, the present application optimizes the base sequence of the target protein while truncating the signal peptide, changes the properties of the inclusion body, and further optimizes the properties of the inclusion body. The fusion protein of the present application is used to prepare GLP-1 protein analogs, which can greatly improve the proportion of target protein in inclusion bodies while ensuring the original high inclusion body yield, ultimately achieving the purpose of improving productivity from the source, and has good application prospect and value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a signal peptide of GLP-1 and its analogues and their applications. Background Technology

[0002] GLP-1 is a physiological peptide intestinal hormone encoded by the human glucagon gene and secreted by L cells in the intestine. It has a glucose concentration-dependent hypoglycemic effect and can simultaneously suppress appetite and delay gastric emptying. GLP-1 drugs have received much attention in recent years and have made significant progress in the treatment of diabetes and obesity. Due to its huge market demand, there is a supply shortage in many parts of the world, and improving the production rate of GLP-1 and its analogues has become an urgent issue for many pharmaceutical companies.

[0003] The upstream preparation processes for GLP-1 and its analogues are mainly divided into two categories: bio-fermentation and chemical synthesis. Bio-fermentation has the advantages of high safety and low cost, making it suitable for large-scale production. Furthermore, GLP-1 is a polypeptide hormone whose biological activity does not require eukaryotic post-translational modification, making *E. coli* a suitable host bacterium for production.

[0004] Protein fusion technology has been applied to the stable expression of peptides in *E. coli*, which involves fusing the target protein or peptide with the DNA sequence of another protein or short peptide for expression within the bacteria. Fusion proteins not only utilize the host's expression regulation system to increase the expression level of the target protein but also reduce the action of intracellular proteases, thus decreasing the degradation of the target protein. As the source of biofermentation, the bacterial strain, if optimized, can fundamentally and significantly improve production capacity. CN114539357A discloses the application of a signal peptide in the expression of GLP-1 or a GLP-1 fusion protein, which increases the expression level of GLP-1 or the GLP-1 fusion protein by altering a specific amino acid sequence of the signal peptide.

[0005] Currently, the method of constructing fusion proteins by linking GLP-1 or GLP-1 analogs with signal peptides to enhance the expression level of fusion proteins, although it can increase the expression level of fusion proteins, has the problems of low proportion of target protein in inclusion bodies and low overall yield. This problem has become a technical challenge that urgently needs to be solved for the efficient preparation of GLP-1 and its analogs. Summary of the Invention

[0006] The present invention aims to solve the above problems by providing a signal peptide of GLP-1 protein or its analogues, wherein the signal peptide is linked to the target protein GLP-1 to form a fusion protein. This fusion protein can greatly increase the proportion of the target protein in the inclusion bodies while ensuring the original high inclusion body yield.

[0007] The first objective of this invention is to provide a signal peptide of GLP-1 protein or an analogue thereof.

[0008] A second objective of this invention is to provide a polynucleotide.

[0009] A third objective of this invention is to provide a nucleotide combination for expressing the GLP-1 protein.

[0010] The fourth objective of this invention is to provide a GLP-1 protein expression vector.

[0011] The fifth objective of this invention is to provide an expression strain.

[0012] A sixth object of the present invention is to provide the application of the above-mentioned signal peptide, polynucleotide, nucleotide combination, expression vector or expression strain.

[0013] A seventh object of the present invention is to provide a method for preparing GLP-1 protein or analogues thereof.

[0014] The above-mentioned objective of this invention is achieved through the following technical solution:

[0015] This invention addresses the problem of low content and overall low yield of the target protein in inclusion bodies by optimizing the signal peptide and target protein sequences of GLP-1 protein, while minimizing the number of amino acids in the amino acid sequence of the signal peptide. The resulting fusion protein, composed of the optimized signal peptide and target protein, ensures high inclusion body yield while significantly increasing the proportion of the target protein in inclusion bodies, thereby enhancing the production capacity of GLP-1 protein. Therefore, this application claims protection for the following scheme:

[0016] The present invention provides a signal peptide of GLP-1 protein or its analogue, the amino acid sequence of which is shown in any one of SEQ ID NO.1-8.

[0017] Preferably, the amino acid sequence of the signal peptide is as shown in any one of SEQ ID NO. 3-6.

[0018] More preferably, the amino acid sequence of the signal peptide is shown in SEQ ID NO.3.

[0019] The present invention provides a polynucleotide comprising a nucleotide sequence encoding the aforementioned signal peptide.

[0020] As an alternative implementation, the nucleotide sequence encoding the signal peptide is as follows:

[0021] The nucleotide sequence encoding the signal peptide shown in SEQ ID NO.2 is shown in SEQ ID NO.10.

[0022] The nucleotide sequence encoding the signal peptide shown in SEQ ID NO.3 is shown in SEQ ID NO.11.

[0023] The nucleotide sequence encoding the signal peptide shown in SEQ ID NO.4 is shown in SEQ ID NO.12.

[0024] The nucleotide sequence encoding the signal peptide shown in SEQ ID NO.5 is shown in SEQ ID NO.13.

[0025] The nucleotide sequence encoding the signal peptide shown in SEQ ID NO. 6 is shown in SEQ ID NO. 14.

[0026] The nucleotide sequence encoding the signal peptide shown in SEQ ID NO.7 is shown in SEQ ID NO.15.

[0027] The nucleotide sequence encoding the signal peptide shown in SEQ ID NO. 8 is shown in SEQ ID NO. 16.

[0028] The present invention provides a nucleotide combination for expressing GLP-1 protein, comprising a signal peptide and a GLP-1 protein fragment; wherein the signal peptide is a nucleotide sequence expressing any of the amino acid sequences shown in SEQ ID NO. 1-8, and the GLP-1 protein fragment is a nucleotide sequence expressing any of the amino acid sequences shown in SEQ ID NO. 17-18.

[0029] As an alternative implementation, the nucleotide sequence expressing the amino acid sequence shown in SEQ ID NO.17 is shown in SEQ ID NO.20, and the nucleotide sequence expressing the amino acid sequence shown in SEQ ID NO.18 is shown in SEQ ID NO.21.

[0030] As an alternative implementation, the nucleotide sequence of the GLP-1 protein fragment is linked to the 3' end of the signal peptide nucleotide sequence.

[0031] Preferably, the signal peptide is a nucleotide sequence expressing the amino acid sequence shown in SEQ ID NO. 3-6.

[0032] More preferably, the signal peptide is a nucleotide sequence expressing the amino acid sequence shown in SEQ ID NO.3; and the GLP-1 protein fragment is a nucleotide sequence expressing the amino acid sequence shown in SEQ ID NO.17.

[0033] The present invention provides a GLP-1 protein expression vector comprising the above-mentioned nucleotide combination for expressing GLP-1 protein.

[0034] As an alternative implementation, the GLP-1 protein expression vector is a pET vector.

[0035] As an alternative implementation, the pET vector is a pET-24(+) vector, a pET-28(+) vector, or a pET-32(+) vector.

[0036] The present invention provides an expression strain comprising the above-described nucleotide combination for expressing GLP-1 protein or the above-described expression vector.

[0037] The use of the above-mentioned signal peptide, the above-mentioned polynucleotide, the above-mentioned nucleotide combination expressing GLP-1 protein, the above-mentioned GLP-1 protein expression vector, or the above-mentioned expression strain in the preparation of GLP-1 protein or its analogues should also be within the scope of protection of this invention.

[0038] This invention provides a method for preparing GLP-1 protein or its analogues, comprising transforming the above-mentioned nucleotide combination for expressing GLP-1 protein into an expression strain, and culturing the transformed expression strain to prepare GLP-1 protein or its analogues.

[0039] Specifically, the GLP-1 protein analog is a truncated form of GLP-1 protein, which is one of the active forms of GLP-1 and has complete biological activity. As an alternative implementation, the amino acid sequence of the GLP-11 protein analog is shown in any of SEQ ID NO.17-19.

[0040] As an alternative implementation, the above expression vector is transformed into an expression strain, and the transformed expression strain is cultured to prepare GLP-1 protein or its analogues.

[0041] As an alternative implementation, the above-mentioned expression strains are cultured to prepare GLP-1 protein or its analogues.

[0042] As an alternative implementation, the expressed strain is Escherichia coli.

[0043] The present invention has the following beneficial effects:

[0044] This invention improves the signal peptide of the GLP-1 protein, enabling it to both promote inclusion body formation and optimize inclusion body properties, while also increasing the unit yield of the target protein, ultimately leading to more efficient expression of the target protein. This optimization is achieved by truncating the amino acids involved in stable and efficient protein expression from the initial signal peptide. Furthermore, this invention optimizes the target protein while truncating the signal peptide, altering the inclusion body characteristics (such as color and water content) to further enhance the inclusion body's properties.

[0045] This invention designed various fusion proteins and conducted fusion expression tests on different signal peptides with GLP-1 analogs of various lengths (e.g., 7-37, 9-37, and 11-37). Suitable GLP-1 analogs were identified, and it was confirmed that efficient protein expression could be achieved on commonly used expression vectors such as pET vectors and commonly used expression bacteria such as *E. coli*. Through dual optimization of the signal peptide and the target protein, this invention finally obtained a fusion protein that significantly improves the expression of GLP-1 and its analogs. Using the fusion protein of this invention to prepare GLP-1 protein analogs can greatly increase the proportion of the target protein in the inclusion bodies while ensuring the original high inclusion body yield, ultimately achieving the goal of improving production capacity from the source, and has excellent application prospects and value. Attached Figure Description

[0046] Figure 1 The protein expression results are shown for connecting the initial signal peptide to different GLP-1 analogs.

[0047] Figure 2 The protein expression results of GLP-1(9-37) linked to signal peptide 1.

[0048] Figure 3 The protein expression results for GLP-1(9-37) linked to signal peptide 2.

[0049] Figure 4 The protein expression results for GLP-1(9-37) linked to signal peptide 3.

[0050] Figure 5 The protein expression results for GLP-1(9-37) linked to signal peptide 4.

[0051] Figure 6 The protein expression results for signal peptide 5 linked to GLP-1(9-37).

[0052] Figure 7 The protein expression results of GLP-1(7-37), GLP-1(9-37), and GLP-1(11-37) linked to signal peptide 6.

[0053] Figure 8 The protein expression results of GLP-1(7-37), GLP-1(9-37), and GLP-1(11-37) linked to signal peptide 7.

[0054] Figure 9 The results of protein expression of GLP-1 analogs using different vectors.

[0055] Figure 10 The results of linking signal peptide 2 to different target proteins for expression (Figure A shows the electrophoresis results of GLP-1(9-37); Figure B shows the electrophoresis results of GLP-1(7-37)). Detailed Implementation

[0056] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0057] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0058] The initial signal peptide used a portion of the oxidoreductase protein as the signal peptide.

[0059] The pET-28a(+) vector, commercially available, is 5369 bp in size and contains a replication origin site, a kanamycin resistance gene, a ROP protein-coding gene, and a T7 promoter element. Its robust T7 promoter, controlled by T7 RNA polymerase, allows almost all of the host cell's resources to be used for expressing the target protein, resulting in high expression efficiency.

[0060] Seed culture medium: Add 5g glucose, 20g peptone, 10g yeast powder, 3.5g KH2PO4, 5g K2HPO4, 3.5g (NH4)HPO4, 5g NaCl, and 1g MgSO4·7H2O to 1L of distilled water.

[0061] Example 1: Design of different signal peptides

[0062] In order to improve the yield of GLP-1 target protein, after a lot of exploration and analysis, a signal peptide (shown in SEQ ID NO.1) that can guide the expression of GLP-1 protein was obtained. However, the yield of GLP-1 target protein is not high (generally 4g / L), and its practical application value is not high.

[0063] We conducted further research and analysis based on the signal peptide of this GLP-1 protein, and found that by appropriate truncation optimization, a signal peptide with significantly improved protein yield can be obtained. Details are shown below.

[0064] Table 1. Signal peptide sequences obtained using different truncation strategies.

[0065]

[0066]

[0067] Based on the amino acid sequences of the signal peptide in Table 1, the nucleotide sequences encoding its amino acids are obtained as follows:

[0068] The nucleotide sequence of the initial signal peptide is shown in SEQ ID NO.9, SEQ ID NO.9:

[0069] GCCACCAAAGCAGTGAGTGTGCTGAAAGGTGATGGCCCGGTTCAGGGTATTATTAATTTTGAACAGAAGGAAAGCAACGGCCCGGTTAAAGTTTGGGGCAGTATTAAAGGTCTGACCGAAGGTCTGCATGGTTTTCATGTGCATGAATTTGGCGATAATACCGCCGGTAGCACCAGCGCCGGTACC.

[0070] The nucleotide sequence of signal peptide 1 is shown in SEQ ID NO. 10.

[0071] GCGACCAAGGCTGTGAGCGTTCTGAAAGGTGACGGCCCGGTGCAGGGTATCATTAACTTCGAGCAAAAGGAAAGCAACGGCCCGGTGAAGGTTTGGGGTAGCATCAAAGGCCTGACCGAGGGTCTGCACGGCTTCCACGTGCACGAATTCGGCGATAAT.

[0072] The nucleotide sequence of signal peptide 2 is shown in SEQ ID NO. 11.

[0073] GCGACCAAGGCTGTGAGCGTTCTGAAAGGTGACGGCCCGGTGCAGGGTATCATTAACTTCGAGCAAAAGGAAAGCAACGGCCCGGTGAAGGTTTGGGGTAGCATCAAAGGCCTGACCGAGGGTCTGCACGGCTTCCACGTGCACGAA.

[0074] The nucleotide sequence of signal peptide 3 is shown in SEQ ID NO. 12.

[0075] GCGACCAAGGCTGTGAGCGTTCTGAAAGGTGACGGCCCGGTGCAGGGTATCATTAACTTCGAGCAAAAGGAAAGCAACGGCCCGGTGAAGGTTTGGGGTAGCATCAAAGGCCTGACCGAGGGTCTGCACGGCTTCCACGTGCACACC.

[0076] The nucleotide sequence of signal peptide 4 is shown in SEQ ID NO. 13.

[0077] GCGACCAAGGCTGTGAGCGTTCTGAAAGGTGACGGCCCGGTGCAGGGTATCATTAACTTCGAGCAAAAGGAAAGCAACGGCCCGGTGAAGGTTTGGGGTAGCATCAAAGGCCTGACCGAGGGTCTGCACGGCTTCCACGTG.

[0078] The nucleotide sequence of signal peptide 5 is shown in SEQ ID NO. 14.

[0079] GCGACCAAGGCTGTGAGCGTTCTGAAAGGTGACGGCCCGGTGCAGGGTATCATTAACTTCGAGCAAAAGGAAAGCAACGGCCCGGTGAAGGTTTGGG GTAGCATCAAAGGCCTGACCGAGGGTCTG.

[0080] The nucleotide sequence of signal peptide 6 is shown in SEQ ID NO. 15.

[0081] GCGACCAAGGCTGTGAGCGTTCTGAAAGGTGACGGCCCGGTGCAGGGTATCATTAACTTCGAGCAAAAGGAA.

[0082] The nucleotide sequence of signal peptide 7 is shown in SEQ ID NO. 16.

[0083] GCGACCAAGGCTGTGAGC.

[0084] Example 2

[0085] Using genetic engineering techniques, DNA sequences encoding the desired protein were synthesized based on the sequences of signal peptides 1-7 from Example 1. The DNA sequences of signal peptides 1-7 were then ligated to the target protein sequences and inserted into pET series vectors to construct the GLP-1 target protein expression vector. The GLP-1 target protein expression vector was transformed into *E. coli*, and the target protein was expressed using the genetically engineered *E. coli* bacteria.

[0086] The specific method is as follows:

[0087] (1) Obtaining the expression strain

[0088] The codon sequence of the fusion protein (GLP-1 protein or its analogue with a tandem signal peptide) was designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The obtained DNA fragment was then transformed into pET series vectors (pET-28a, pET-24a, and pET-32a) and subsequently into competent *E. coli* BL21(DE3). This resulted in the acquisition of an expression strain capable of expressing the GLP-1 protein.

[0089] (2) Fermentation

[0090] The expression strain was inoculated at a volume ratio of 0.1-0.3% into a shake flask containing 50 mL of seed culture medium. The flask was incubated at 37°C and 220 rpm for 4-8 hours with shaking. Aseptic samples were then taken to measure the OD (exposure potential). 600 Value, determine the OD of seed liquid 600 The value is between 1 and 3.

[0091] Inoculate the seed culture at a ratio of 0.1-1.0% into a fermenter containing seed culture medium under aseptic conditions (generally 30-40 mL). Initially maintain an incubation temperature of 37.0℃, an aeration rate of 1.0 vvm, a rotation speed of 150 rpm, a pH of 6.7, and an optimal DO value of 30-80%. When the pH rises to 6.90±0.10 and the OD of the bacterial culture increases... 600 Feeding should begin when the pH reaches 10-15. The bacteria will grow to the OD value. 600 When the pH value is between 90 and 120, add IPTG at a final concentration of approximately 0.5 mM to induce protein expression. Terminate fermentation 10-12 hours after induction.

[0092] (3) Homogenization and collection of bacterial cells

[0093] The bacterial culture from the fermenter was collected by centrifugation using a high-speed refrigerated centrifuge. The centrifugation temperature was set at 15℃, the centrifugation speed at 8000 rpm, and the centrifugation time at 15 minutes. The OD of the centrifuged supernatant was controlled. 600 <2. Record the weight of the collected bacteria.

[0094] Add homogenization buffer (Tris 50mM, EDTA 5mM, pH=7.0) to the bacterial cell storage tank, bring the volume to the required level, stir well, and resuspend the bacterial cells. Perform homogenization four times, with the first homogenization at 500 bar, followed by adjustments to 1000 bar. Maintain the homogenization buffer temperature between 2-10℃.

[0095] (4) Inclusion body collection, washing and preservation

[0096] Inclusion bodies were collected by centrifuging the homogenized bacterial culture using a high-speed refrigerated centrifuge. The centrifugation temperature was set at 15℃, the centrifugation speed at 8000 rpm, and the centrifugation time at 1 hour. The OD of the centrifuged supernatant was controlled. 600<2, collect the solid inclusion bodies. Record the OD of the centrifuged supernatant. 600 Inclusion body volume. Add inclusion body wash buffer, resuspend the collected inclusion bodies in the wash buffer, and bring the volume to the required level. Collect the inclusion bodies using a high-speed refrigerated centrifuge. Set the centrifugation temperature to 15℃, the centrifugation speed to 8000 rpm, and the centrifugation time to 40 minutes. Control the OD of the supernatant. 600 <1. Collect solid inclusion bodies. Add inclusion body wash buffer, resuspend the collected inclusion bodies in the wash buffer, and bring the volume to the required level. Collect the inclusion bodies using a high-speed refrigerated centrifuge. Set the centrifugation temperature to 15℃, the centrifugation speed to 8000 rpm, and the centrifugation time to 40 minutes. Control the OD of the supernatant. 600 <1. Collect solid inclusion bodies. Store the collected inclusion bodies in a freezer at -20°C.

[0097] Example 3

[0098] I. Testing the broad-spectrum applicability of the initial signal peptide

[0099] After being secreted in the human body, GLP-1 protein can be rapidly inactivated by DPP-4. Therefore, there are various GLP-1 analogs in the human body, such as GLP-1(7-36) and GLP-1(7-37) which are produced by cleaving six additional amino acids from the N-terminus of GLP-1(1-37), further GLP-1(7-36) and GLP-1(7-37) which are further amination by the C-terminus, and the truncated form GLP-1(9-36) which is rapidly inactivated by DPP-4.

[0100] Different truncated forms of GLP-1 protein can affect the characteristics of its inclusion bodies (such as color and water content). Therefore, selecting a suitable truncated form of GLP-1 protein can further optimize the characteristics of the inclusion bodies. This invention designs various GLP-1 analogs of different lengths, including GLP-1(7-37), GLP-1(9-37), and GLP-1(11-37).

[0101] GLP-1 (7-37) analogs are truncated forms of GLP-1 starting from the 7th amino acid and are one of the active forms of GLP-1, possessing complete biological activity. The amino acid sequence is shown in SEQ ID NO.17, SEQ ID NO.17: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG.

[0102] GLP-1 (9-37) analogs are truncated forms of GLP-1 starting from the 9th amino acid. They are one of the active forms of GLP-1 and have complete biological activity. The amino acid sequence is shown in SEQ ID NO.18: EGTFTSDVSSYLEGQAAKEFIAWLVKGRG.

[0103] GLP-1 (11-37) analogs are truncated forms of GLP-1 starting from the 11th amino acid. They are one of the active forms of GLP-1 and have complete biological activity. The amino acid sequence is shown in SEQ ID NO.19, SEQ ID NO.19: TFTSDVSSYLEGQAAKEFIAWLVKGRG.

[0104] The nucleotide sequence encoding the amino acid sequence of the GLP-1(7-37) analog is shown in SEQ ID NO.20, SEQ ID NO.20:

[0105] CACGCTGAGGGTACCTTTACCAGCGATGTTAGCAGCTACCTGGAGGGTCAGGCGGCGAAAGAATTTATTGCGTGGCTGGTTCGTGGTCGTGGC.

[0106] The nucleotide sequence encoding the amino acid sequence of the GLP-1(9-37) analog is shown in SEQ ID NO.21, SEQ ID NO.21:

[0107] GAGGGTACCTTTACCAGCGATGTTAGCAGCTACCTGGAGGGTCAGGCGGCGAAAGAATTTATTGCGTGGCTGGTTCGTGGTCGTGGC.

[0108] The nucleotide sequence encoding the amino acid sequence of the GLP-1(11-37) analog is shown in SEQ ID NO.22, SEQ ID NO.22:

[0109] ACCTTTACCAGCGATGTTAGCAGCTACCTGGAGGGTCAGGCGGCGAAAGAATTTATTGCGTGGCTGGTTCGTGGTCGTGGC.

[0110] To investigate the broad applicability of the signal peptide of this invention, fusion expression tests were conducted on the signal peptide with various GLP-1 analogs. Specifically, the sequence of the initial signal peptide was ligated into a PET vector along with the sequences of GLP-1 analogs GLP-1(7-37), GLP-1(9-37), and GLP-1(11-37), respectively, to obtain a recombinant vector. This recombinant vector was then transformed into *E. coli* to obtain recombinant bacteria. The recombinant bacteria were added to LB liquid medium at a volume ratio of 1:300 and cultured at 37°C and 220 rpm / h until approximately OD200. 600The value was between 0.5 and 1. IPTG was added for induction, with a final IPTG concentration of 1 mmol / L. A treatment group without IPTG was set up as a negative control. Four hours after induction (at least 4 hours), a 1 mL sample was centrifuged and the OD of the bacterial culture was measured. 600 After centrifuging the bacterial culture sample, discard the supernatant and add LB medium to the OD value. 600 The value is 8. Wash and blow the pipette tip to form a suspension. Take 80 μL of the suspension and add 20 μL of Loading Buffer (5×). Gently tap to mix. Incubate in a 100°C water bath for 10 min. Remove and centrifuge slightly for 5 s. Cool and then perform electrophoresis.

[0111] The PET carrier can be pET-24(+) carrier, pET-28(+) carrier, or pET-32(+) carrier. In this embodiment, pET-28a(+) carrier is selected.

[0112] II. Test Results

[0113] The protein expression results of initial signal peptides linked to different GLP-1 analogs are as follows: Figure 1 As shown, the results indicate that GLP-1(7-37) and GLP-1(9-37) are both suitable for protein expression in tandem with the initial signal peptide, while (11-37) has poor expression performance.

[0114] Example 4: Expression results of the target protein in tandem with different signal peptides

[0115] I. Experimental Methods

[0116] Protein expression was performed using the signal peptides listed in Table 1 tandem with GLP-1(9-37). Specifically, the sequences of signal peptides 1-5 from Table 1 were ligated into the pET vector along with the sequences of GLP-1(9-37). Additionally, the sequences of signal peptides 6 and 7 from Table 1 were ligated into the pET vector along with the sequences of GLP-1(7-37), GLP-1(9-37), and GLP-1(11-37), resulting in a recombinant vector. This recombinant vector was then transformed into *E. coli* to obtain the corresponding expression strain. The culture method for the expression strain and the detection method for the target protein expression are described in Example 3.

[0117] The pET vector can be pET-24(+) vector, pET-28(+) vector, or pET-32(+) vector. In this embodiment, pET-28a(+) vector is selected.

[0118] II. Experimental Results

[0119] The protein expression results of signal peptides 1-5 linked to GLP-1(9-37) are as follows: Figure 2 , Figure 3 , Figure 4 , Figure 5 and Figure 6 As shown, the results indicate that proteins linked to signal peptides 1-5 all exhibited good expression performance and successfully expressed the target protein. Among these, the target protein band linked to signal peptide 2 was the deepest and widest, indicating that signal peptide 2 was more effective in promoting the expression of the target protein.

[0120] Signal peptides 6 and 7 are linked to three protein entries (7-37, 9-37, and 11-37), and the protein expression results are as follows: Figure 7 and Figure 8 As shown, the results indicate that both signal peptide 6 and signal peptide 7 can express the three target proteins in tandem, and the expression of the target proteins is not affected.

[0121] Using the software ImageJ Figures 2 to 8 The bands were analyzed and compared, and the total protein and target protein band areas of a single pore were calculated. The results are shown in Table 2. The results show that the target protein expression ratios of tandem signal peptides 1-7 are all good, with signal peptide 2 having the largest target protein ratio and the best protein expression.

[0122] Table 2. Expression of target proteins with different tandem signal peptides.

[0123]

[0124]

[0125] Example 5: Expression of the target protein using different vectors

[0126] I. Experimental Methods

[0127] Protein expression was performed using signal peptide 2 tandemly with GLP-1(9-37) from Table 1. Specifically, the sequences of signal peptide 2 and GLP-1(9-37) from Table 1 were ligated into pET-24(+) expression vector, pET-28(+) expression vector, and pET-32(+) expression vector, respectively, to obtain recombinant vectors. These three different recombinant vectors were then transformed into *E. coli* to obtain the corresponding expression strains. The culture methods for the expression strains and the methods for detecting the expression of the target protein are described in Example 3.

[0128] II. Experimental Results

[0129] Results of expressing target proteins using different vectors, such as Figure 9 As shown, the results indicate that the signal peptide 2 tandem with the GLP-1 (9-37) sequence can efficiently express the target protein in pET-24(+) expression vector, pET-28(+) expression vector, and pET-32(+) expression vector, and the expression of the target protein in different vectors is not significantly different.

[0130] Example 6: Yield of signal peptide 2 linking to different target proteins

[0131] I. Experimental Methods

[0132] Protein expression was performed using signal peptide 2 from Table 1, tandemly with GLP-1 (9-37) and GLP-1 (7-37) sequences, respectively. Specifically, signal peptide 2 from Table 1 was ligated into the pET-28(+) expression vector along with the GLP-1 (9-37) and GLP-1 (7-37) sequences to obtain recombinant vectors. These recombinant vectors were then transformed into *E. coli* to obtain the corresponding expression strains. The expression strains underwent fermentation, homogenization, and collection of cells, followed by inclusion body collection, washing, and weighing, as detailed in Example 2.

[0133] II. Experimental Results

[0134] The experimental results are shown in Table 3. The results indicate that the fermentation efficiency of the strain expressing signal peptide 2 in tandem with GLP-1(7-37) is significantly better than that of the strain expressing signal peptide 2 in tandem with GLP-1(9-37). The inclusion body yield of the strain expressing signal peptide 2 in tandem with GLP-1(7-37) reaches 62.38 g / L.

[0135] Table 3. Yields of signal peptide 2 linked to different target proteins

[0136]

[0137] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A signal peptide of GLP-1 protein or its analogue, characterized in that, Its amino acid sequence is shown in any one of SEQ ID NO. 3-8; the amino acid sequence of the GLP-1 protein or its analogue is shown in SEQ ID NO. 17 or SEQ ID NO.

18.

2. A polynucleotide, characterized in that, The polynucleotide is a nucleotide sequence encoding the signal peptide of claim 1.

3. A nucleotide sequence for expressing GLP-1 protein, characterized in that, It includes a signal peptide and a GLP-1 protein fragment; the signal peptide is a nucleotide sequence expressing any of the amino acid sequences shown in SEQ ID NO. 3-8, and the amino acid sequence of the GLP-1 protein fragment is shown in SEQ ID NO. 17 or SEQ ID NO. 18; the nucleotide sequence of the GLP-1 protein fragment is linked to the 3' end of the signal peptide nucleotide sequence.

4. A GLP-1 protein expression vector, characterized in that, It comprises the nucleotide combination of claim 3.

5. An expression strain, characterized in that, Includes the nucleotide combination for expressing GLP-1 protein as described in claim 3 or the expression vector as described in claim 4.

6. The use of the signal peptide of claim 1, the polynucleotide of claim 2, the nucleotide combination for expressing GLP-1 protein of claim 3, the GLP-1 protein expression vector of claim 4, or the expression strain of claim 5 in the preparation of GLP-1 protein or its analogues; wherein the amino acid sequence of the GLP-1 protein or its analogues is shown in SEQ ID NO. 17 or SEQ ID NO.

18.

7. A method for preparing GLP-1 protein or its analogues, characterized in that, The nucleotide combination of claim 3 is transformed into the expression strain, and the transformed expression strain is cultured to prepare GLP-1 protein or its analogues; the amino acid sequence of the GLP-1 protein or its analogues is shown in SEQ ID NO.17 or SEQ ID NO.

18.

8. The method according to claim 7, characterized in that, The expression vector of claim 4 is transformed into the expression strain, and the transformed expression strain is cultured to prepare GLP-1 protein or its analogues.

9. The method according to claim 7, characterized in that, The expression strain of claim 5 is cultured to prepare GLP-1 protein or its analogues.

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