Method for prolonging half-life period of polypeptide by introducing compound N-glycosylation modification
By expressing Trypanosoma brucei oligosaccharyltransferase in insect cells and catalyzing the complex N-glycosylation modification of GLP-1M, the problem of short half-life of GLP-1 was solved, the stability of the polypeptide was significantly enhanced, and the development of glycochemistry and glycobiology was promoted.
Patent Information
- Application Number
- CN202510631330.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-16
- Publication Date
- 2025-09-30
AI Technical Summary
In the existing technology, polypeptide drugs such as GLP-1 have a short half-life in the body, which limits their application in disease treatment. The existing half-life extension strategy needs to be further improved.
Oligosaccharyltransferase from Trypanosoma brucei was expressed in insect cells, and GLP-1 was used to synthesize the mutant GLP-1M containing asparagine. Human complex fatty alcohol oligosaccharides were transferred to GLP-1M by oligosaccharyltransferase, and further modified by sialyltransferase to form complex N-glycosylation modification.
It significantly prolonged the half-life of GLP-1, provided a new strategy for enhancing peptide stability, and promoted the development of glycochemistry and glycobiology.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biochemistry, and specifically relates to a method for introducing complex N-glycosylation modification to prolong the half-life of a polypeptide. Background Art
[0002] Peptides have a wide range of biological activities, and peptide drugs are gaining increasing attention in the pharmaceutical field due to their high activity, target specificity, and safety. Peptides have a molecular weight between small molecules and protein drugs, and their metabolic stability is often poor. They are rapidly metabolized in the body, which greatly limits their application. In recent years, modified peptides have accounted for an increasing proportion of marketed peptide drugs, with glycosylation being a hot topic in peptide drug research: sugar chains can enhance the metabolic stability of peptides and slow their degradation rate. Glucagon-like peptide-1 (GLP-1, HDEYERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG) is a peptide hormone composed of 37 amino acids that can be used to treat type 2 diabetes. It also plays a role in obesity, non-alcoholic fatty liver disease, cardiovascular disease, and neurological diseases.
[0003] Currently, the potential value of GLP-1 and its therapeutic applications have become a research hotspot. Among the main active components in the human body, GLP-1 (7-36, H7AEGTFTSDVSSYLEGQAAKEFIAWLVKGR36), which contains 30 amino acids (aa), is the most prevalent, followed by GLP-1 (7-37), which has the same biological activity. Under physiological conditions, GLP-1 is rapidly degraded in the bloodstream, surviving for only 1-2 minutes before being broken down by dipeptidyl peptidase 4 (DPP4), rendering it inactive. This limits its application in disease treatment.
[0004] To increase circulation time in vivo, existing half-life extension strategies include altering the amino acid sequence, fatty acid modification, and protein fusion. The stability of the GLP-1 protein can be enhanced by introducing sialylated LacNAc structures at positions 26, 34, and 37, for example. Similarly, introducing O-glycosylation at serine position 18 can also extend the half-life of GLP-1, but the current half-life needs to be further improved. Summary of the Invention
[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.
[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.
[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for introducing complex N-glycosylation modification to extend the half-life of polypeptides.
[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:
[0009] Expression of oligosaccharyltransferase from Trypanosoma brucei in insect cells;
[0010] GLP-1 is used to synthesize the asparagine-containing mutant GLP-1M;
[0011] Using oligosaccharyl transferase to catalyze the transfer of oligosaccharides from human-derived complex fatty alcohol oligosaccharides to GLP-1M, a human-derived complex N-glycopeptide with extended half-life was obtained.
[0012] The nucleotide sequence of the oligosaccharyl transferase is shown in SEDID NO.1.
[0013] As a preferred embodiment of the method of introducing complex N-glycosylation modification to prolong the half-life of a polypeptide as described in the present invention, the human complex fatty alcohol oligosaccharide uses PPGn2 as the starting substrate and is catalyzed by recombinant glycosyltransferases ScAlg1, hAlg2, hGnT-I, hGnT-II, and hGalT expressed in Escherichia coli to obtain PPGn2Man3Gn2 and PPGn2Man3Gn2Gal2 human complex fatty alcohol oligosaccharides.
[0014] As a preferred embodiment of the method of introducing complex N-glycosylation modification to prolong polypeptide half-life according to the present invention, the oligosaccharyl transferase is STT3A with 8*His and StrepII tags fused to the N-terminus.
[0015] As a preferred embodiment of the method of introducing complex N-glycosylation modification to prolong the half-life of a polypeptide according to the present invention, the STT3A is derived from Trypanosoma brucei.
[0016] As a preferred embodiment of the method of introducing complex N-glycosylation modification to prolong the half-life of a polypeptide according to the present invention, the oligosaccharyltransferase derived from Trypanosoma brucei is expressed in insect cells, comprising:
[0017] Based on the STT3A nucleic acid sequence from Trypanosoma brucei, a vector was constructed using gene synthesis technology and fusion PCR, and then coated on the corresponding resistance screening plate. The PCR-positive single clones were selected, and the plasmids were extracted and sequenced to obtain the oligosaccharyl transferase.
[0018] The oligosaccharyltransferase gene fragment was constructed into the plasmid pFastBac1 to obtain the recombinant plasmid pFastBac1-8His-stepII-TEV-STT3A;
[0019] The recombinant plasmid FastBac1-8His-stepII-TEV-STT3A was transformed into competent DH10 BAac, and the recombinant STT3A bacmid was extracted. After PCR verification, the recombinant STT3A dry particles were transferred into insect cells using liposome transfection reagent to obtain the first-generation virus. The first-generation virus was then used to infect SF9 cells to obtain the second-generation virus. Finally, the second-generation virus was used to infect SF9 cells. After culture, the cells were collected, lysed, and purified by Step affinity chromatography. The expression and purity of the TbSTT3A protein were confirmed by SDS-PAGE.
[0020] As a preferred embodiment of the method of introducing complex N-glycosylation modification to extend the half-life of a polypeptide according to the present invention, the catalytic reaction system further contains HEPES and MnCl2;
[0021] Relative to the 50 μL system, the final concentration of HEPES is 20 mM, the final concentration of MnCl2 is 10 mM, the final concentration of GLP-1M is 100 μM, the final concentration of human complex fatty alcohol oligosaccharide is 100-200 μM, the final concentration of oligosaccharyltransferase is 40 nM, and the reaction time is 1-4 h.
[0022] As a preferred embodiment of the method of introducing complex N-glycosylation modification to extend the half-life of a polypeptide according to the present invention, wherein:
[0023] The sequence of the GLP-1 is HDEYERHAEGTFTSDVSSYLEGQAAKEFIAWLVK GRG, and the sequence of the GLP-1M is HAEGNYTSDVSSYLEGQAAKEFIAWLVKGR.
[0024] As a preferred embodiment of the method of introducing complex N-glycosylation modification to extend the half-life of a polypeptide according to the present invention, when the human complex N-glycopeptide with extended half-life is galactose-modified GLP-1M, sialic acid-modified GLP-1M is obtained by catalysis using sialyltransferase; the sialyltransferase is derived from Photorhabdus mermaidii, and the sialyltransferase with 15 amino acids truncated from the N-terminus is expressed in Escherichia coli. The nucleotide sequence of the sialyltransferase is shown in SED ID NO.2, and the nucleotide sequence of the sialyltransferase with 15 amino acids truncated from the N-terminus is shown in SEDID NO.3.
[0025] As a preferred embodiment of the method of introducing complex N-glycosylation modification to extend the half-life of a polypeptide according to the present invention, wherein: the catalytic reaction system further contains Tris and MnCl2;
[0026] Relative to the 50 μL system, the final concentration of Tris is 20 mM, the final concentration of MnCl2 is 10 mM, the final concentration of GLP-1M is 500 μM, the final concentration of CMP-NANA is 1000-2000 μM, the final concentration of sialyltransferase is 50 μg / mL, the reaction time is 6 min, and the enzyme and donor are added every 6 minutes for 3-4 times.
[0027] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for introducing complex N-glycosylation modification to extend the half-life of a polypeptide to obtain a humanized complex N-glycopeptide with extended half-life.
[0028] As a preferred embodiment of the humanized complex N-glycopeptide with extended half-life according to the present invention, it has the following characteristics:
[0029] 20 μg of human complex N-glycopeptide was dissolved in 15 μL ddH2O, an equal volume of mouse serum was added and mixed, and the mixture was incubated at 37°C. At different sampling time points, 3 μL of the mixture was taken each time and added to 6 μL of pre-cooled methanol and mixed evenly. The supernatant was collected by centrifugation at 12000×g for HPLC detection and analysis. The half-life of the human complex N-glycopeptide was 365-1564 min.
[0030] Beneficial effects of the present invention:
[0031] The present invention breaks the conventional complex biosynthesis pathway of human N-glycans in vivo, and synthesizes fatty alcohol oligosaccharides containing human complex N-glycans in vitro. Subsequently, the complex N-glycans are transferred from the fatty alcohol oligosaccharide to the GLP-1M polypeptide using the oligosaccharyltransferase TbSTT3A with high substrate adaptability to obtain glycosylated GLP-1M. The glycosylated GLP-1M can significantly enhance the stability of GLP-1M, providing a new strategy to prolong the half-life of GLP-1 and promoting the development of glycochemistry and glycobiology. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:
[0033] Figure 1This is a flow chart of the synthesis of the mutant GLP-1M polypeptide purchased from Anhui Guoping Pharmaceutical Co., Ltd. in the present invention.
[0034] Figure 2 This is an SDS-PAGE Coomassie blueprint of the purified TbSTT3A protein in Example 1 of the present invention.
[0035] Figure 3 Flowchart (A), liquid chromatography (B) and mass spectrum (C) of TbSTT3A-catalyzed conversion of PPGn2Man3Gn2 to GLP-1M to generate Gn2Man3Gn2-GLP-1M in Example 2 of the present invention.
[0036] Figure 4 Flowchart (A), liquid chromatography (B) and mass spectrum (C) of the process of TbSTT3A catalyzing the conversion of PPGn2Man3Gn2Gal2 to GLP-1M to generate Gal2Gn2Man3Gn2-GLP-1M in Example 3 of the present invention.
[0037] Figure 5 The SDS-PAGE image (A), flow chart (B), liquid chromatography (C) and mass spectrum (D) of Sia2Gal2Gn2Man3Gn2-GLP-1M formed by SiaT-catalyzed Gal2Gn2Man3Gn2-GLP-1M in Example 4 of the present invention.
[0038] Figure 6 This is a graph showing the half-life determination of GLP-1M in Example 5 of the present invention. DETAILED DESCRIPTION
[0039] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.
[0040] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.
[0041] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.
[0042] The GLP-1M polypeptide used in the present invention was purchased from Anhui Guoping Pharmaceutical Co., Ltd. and was synthesized as follows: Figure 1shown.
[0043] The restriction endonucleases, Prime Star DNA polymerase, and ligase used in the present invention were purchased from Takara Biotechnology Co., Ltd. (Dalian, Japan).
[0044] The plasmid extraction kit and IPTG used in the present invention were purchased from Shanghai Bioengineering; sequencing was performed by Suzhou Andasheng.
[0045] The human-derived complex fatty alcohol oligosaccharides PPGn2Man3Gn2 and PPGn2Man3Gn2Gal2 in the present invention are various fatty alcohol oligosaccharides obtained by using PPGn2 as a starting substrate and catalyzing recombinant glycosyltransferases ScAlg1, hAlg2, hGnT-I, hGnT-II, and hGalT expressed in Escherichia coli. The gene sequence is obtained from NCBI and the protein sequence is obtained from Uniprot.
[0046] Example 1 Expression and purification of oligosaccharyltransferase TbSTT3A
[0047] Vector construction: Based on the nucleic acid sequence of Trypanosoma brucei STT3A (NCBI accession number: ADG03035.1) at NCBI, the vector was constructed using gene synthesis technology and fusion PCR. The vector was spread on the corresponding resistance screening plate, and single colonies positive for colony PCR were selected. The plasmid was extracted and sent for sequencing;
[0048] Oligosaccharyltransferase TbSTT3A is fused with His and StrepII tags, and its nucleotide sequence is shown in SEQ ID NO.1;
[0049] The TbSTT3A gene fragment was constructed into the plasmid pFastBac1 to obtain the recombinant plasmid pFastBac1-8His-strepII-TEV-STT3A.
[0050] The recombinant STT3A bacmid was extracted from the recombinant competent cell DH10BAac. After PCR verification, the recombinant STT3A bacmid was transferred into insect SF9 cells using a liposome transfection reagent. After 96 hours of culture, the cell culture supernatant was collected to obtain the first-generation virus. The SF9 cells were then infected with the first-generation virus. After 60 hours of culture, the cell culture supernatant was collected to obtain the second-generation virus. Finally, the SF9 cells were infected with the second-generation virus and the cells were collected after 72 hours of culture.
[0051] The cells were collected and lysed with lysis buffer (100 mM HEPES (pH 7.5), 150 mM NaCl, 1% DDM, 0.2% CHS, protease inhibitor). The proteins were bound to StrepII beads and washed with wash buffer (100 mM HEPES (pH 7.5), 150 mM NaCl, 0.05% DDM, 0.005% CHS). Finally, the proteins were eluted with elution buffer (100 mM HEPES (pH 7.5), 150 mM NaCl, 0.05% DDM, 0.005% CHS, 50 mM biotin). Figure 2 The results showed that the TbSTT3A protein was successfully expressed and purified.
[0052] Example 2: Under the catalysis of oligosaccharyltransferase TbSTT3A, the sugar chain in PPGn2Man3Gn2 is transferred to GLP-1M to form GLP-1M with acetylglucosamine modification at the end:
[0053] The standard reaction conditions are as follows: a 50 μL reaction system contains 20 mM HEPES (pH 7.5), 10 mM MnCl2, 200 μM PPGn2Man3Gn2, 100 μM GLP-1M, and 40 nM TbSTT3A.
[0054] like Figure 3 As shown in (A), under the above standard reaction conditions, the reaction was carried out at 30°C for 4 hours, inactivated at 100°C for 5 minutes, and the supernatant was collected by centrifugation to obtain a reaction solution containing GLP-1M-Gn2Man3Gn2, the product glycopeptide. The reaction solution was purified using a high performance liquid chromatograph U3000 (Thermo Fisher Scientific), an ultraviolet detector UltiMate 3000VWD, a detection wavelength of 220 nm, a liquid chromatography column C18 (Shimadzu, 5 μm 4.6×100 mm), and elution conditions were acetonitrile (CH 3 CN)-0.1% TFA linear gradient elution (solution A: CH 3 CN; solution B: 0.1% TFA; elution conditions: 0-3 min, 35% A; 3-30 min, 10%-50% A; 30-33 min, 50%-100% A; 33-35 min, 100%-100% A; 35-40 min, 100%-10% A; flow rate: 1 mL / min) to separate the substrate and product, as shown in FIG. Figure 3 (B) The effluent was detected by UV detector, and the target peak was simultaneously detected by MALDI-TOF MS for molecular weight identification.
[0055] Figure 3(C) The results show that the product Gn2Man3Gn2-GLP-1M plus H + The mass-to-charge ratio is consistent with the predicted molecular weight.
[0056] Example 3: Under the catalysis of oligosaccharyltransferase TbSTT3A, the sugar chain of PPGn2Man3Gn2Gal2 was transferred to GLP-1M to form GLP-1M with a galactose terminal modification:
[0057] The standard reaction conditions are as follows: a 50 μL reaction system contains 20 mM HEPES (pH 7.5), 10 mM MnCl2, 200 μM PPGn2Man3Gn2Gal2, 100 μM GLP-1M, and 40 nM TbSTT3A.
[0058] like Figure 4 As shown in (A), under the above standard reaction conditions, the reaction was carried out at 30°C for 4 hours. The reaction was inactivated at 100°C for 5 minutes, and the supernatant was collected by centrifugation to obtain a reaction solution containing GLP-1M-Gn2Man3Gn2Gal2 and the product glycopeptide. The reaction solution was separated by high performance liquid chromatography, as shown in FIG. Figure 4 (B) The effluent was detected by UV detector, and the target peak was simultaneously detected by MALDI-TOF MS for molecular weight identification.
[0059] Figure 4 (C) The results show that the product Gal2Gn2Man3Gn2-GLP-1M glycopeptide is added with H + The mass-to-charge ratio is consistent with the predicted molecular weight.
[0060] Example 4 Expression and purification of sialyltransferase SiaT and activity testing
[0061] Vector construction: Based on the nucleic acid sequence of the mermaid SiaT (NCBI accession number: BAA25316.1) on NCBI, the vector was constructed using gene synthesis technology and fusion PCR. The vector was spread on the corresponding resistance screening plate, and the single clones positive for colony PCR were selected. The plasmid was extracted and sent for sequencing.
[0062] The nucleotide sequence of sialyltransferase SiaT is shown in SEQ ID NO.2; the nucleotide sequence of SiaTΔTM with 15 amino acids truncated at the N-terminus is shown in SEQ ID NO.3.
[0063] The SiaTΔTM gene fragment was constructed onto the plasmid pET28a to obtain the recombinant plasmid pET28a-SiaTΔTM. pET28a-SiaTΔTM was transferred into E. coli Rosetta and then transformed into E. coli Rosetta (DE3) competent cells. The cells were plated on LB solid medium containing kanamycin and chloramphenicol resistance. After overnight culture, a single colony was picked for inoculation. The culture was expanded again after shaking overnight. After IPTG induction, the cells were collected and ultrasonically disrupted. The supernatant was collected by centrifugation and incubated with Ni-NTA at 4°C for 1-1.5 hours. The protein was purified according to conventional protein purification procedures. SDS-PAGE showed that the sialyltransferase SiaT was successfully purified. Figure 5 (A) shown.
[0064] Under the catalysis of sialyltransferase SiaT, two sialic acids are added to the Gal2Gn2Man3Gn2-GLP-1M glycopeptide to form GLP-1M with sialic acid modification at the end:
[0065] The standard reaction conditions are as follows: a 50 μL reaction system contains 20 mM Tris (pH 8.5), 10 mM MnCl2, 500 μM Gal2Gn2Man3Gn2-GLP-1M, 1000 μM CMP-NANA, and 50 mg / mL SiaT.
[0066] like Figure 5 As shown in (B), under the above standard reaction conditions, the reaction was carried out at 30°C for 6 minutes, and the corresponding sialyltransferase and donor CMP-NANA were added every 6 minutes. The reaction was inactivated at 100°C for 5 minutes, and the supernatant was collected by centrifugation to obtain a reaction solution containing the product glycopeptide Sia2Gal2Gn2Man3Gn2-GLP-1M. The reaction solution was separated into substrate and product using high performance liquid chromatography, as shown in FIG. Figure 5 The effluent was detected by UV detector, and the target peak was simultaneously detected by MALDI-TOF MS for molecular weight identification.
[0067] Figure 5 (D) The results showed that the product Sia2Gal2Gn2Man3Gn2-GLP-1M glycopeptide reduced H + The mass-to-charge ratio is consistent with the predicted molecular weight.
[0068] Example 5
[0069] 20 μg of each of the purified glycopeptides Gn2Man3Gn2-GLP-1M and Sia2Gal2Gn2Man3Gn2-GLP-1M were dissolved in 15 μL ddH2O, and an equal volume of mouse serum was added and mixed. The mixture was then incubated at 37°C. At different sampling time points, 3 μL of the mixture was added to 6 μL of pre-cooled methanol and mixed evenly. The supernatant was collected by centrifugation at 12000 × g for HPLC analysis. The experiment was repeated three times for each sample. The results are shown in Figure 2. Figure 6 It can be clearly seen from the figure that the half-life of the glycosylated GLP-1M of the present invention is significantly prolonged.
[0070] In summary, the present invention introduces N-glycosylation sites through GLP-1 amino acid mutations, and uses oligosaccharyltransferase TbSTT3A to catalyze the transfer of oligosaccharides on the biantennary complex LLO to the N-glycosylation site of the mutant GLP-1 to generate glycosylated GLP-1, i.e., glycopeptides containing human complex N-glycan chains. This provides a new strategy for the efficient assembly of uniform glycopeptides / glycoproteins containing complex N-glycan chains, and a means of glycosylation modification to enhance the stability of small drug molecules. It is of great significance for comprehensively and accurately exploring the biological activity of human glycoproteins and promoting the development of glycochemistry and glycobiology.
[0071] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.
Claims
1. A method for extending the half-life of a polypeptide by introducing complex N-glycosylation modification, characterized in that: include, Expression of oligosaccharyltransferase from Trypanosoma brucei in insect cells; GLP-1 is used to synthesize the asparagine-containing mutant GLP-1M; Using oligosaccharyl transferase to catalyze the transfer of oligosaccharides from human-derived complex fatty alcohol oligosaccharides to GLP-1M, a human-derived complex N-glycopeptide with extended half-life was obtained. The nucleotide sequence of the oligosaccharyl transferase is shown in SEDID NO.
1.
2. The method for introducing complex N-glycosylation modification to extend polypeptide half-life according to claim 1, characterized in that: The human-derived complex fatty alcohol oligosaccharide is PPGn2Man3Gn2 or PPGn2Man3Gn2Gal2 human-derived complex fatty alcohol oligosaccharide obtained by using PPGn2 as a starting substrate and catalyzed by recombinant glycosyltransferases ScAlg1, hAlg2, hGnT-I, hGnT-II, and hGalT expressed in Escherichia coli.
3. The method for introducing complex N-glycosylation modification to extend polypeptide half-life according to claim 1, characterized in that: The oligosaccharyl transferase is STT3A with 8*His and StrepII tags fused to the N-terminus.
4. The method of introducing complex N-glycosylation modification to extend polypeptide half-life according to claim 3, characterized in that: The STT3A is derived from Trypanosoma brucei.
5. The method of introducing complex N-glycosylation modification to extend polypeptide half-life according to claim 1, characterized in that: The method for expressing the oligosaccharyl transferase derived from Trypanosoma brucei in insect cells comprises: Based on the STT3A nucleic acid sequence from Trypanosoma brucei, a vector was constructed using gene synthesis technology and fusion PCR, and then coated on the corresponding resistance screening plate. The PCR-positive single clones were selected, and the plasmids were extracted and sequenced to obtain the oligosaccharyl transferase. The oligosaccharyltransferase gene fragment was constructed into the plasmid pFastBac1 to obtain the recombinant plasmid pFastBac1-8His-stepII-TEV-STT3A; The recombinant plasmid FastBac1-8His-stepII-TEV-STT3A was transformed into competent DH10 BAac, and the recombinant STT3A bacmid was extracted. After PCR verification, the recombinant STT3A dry particles were transferred into insect cells using liposome transfection reagent to obtain the first-generation virus. The first-generation virus was then used to infect SF9 cells to obtain the second-generation virus. Finally, the second-generation virus was used to infect SF9 cells. After culture, the cells were collected, lysed, and purified by Step affinity chromatography. The expression and purity of the TbSTT3A protein were confirmed by SDS-PAGE.
6. The method of introducing complex N-glycosylation modification to extend polypeptide half-life according to claim 1, characterized in that: The catalytic reaction system also contains HEPES and MnCl2; Relative to the 50 μL system, the final concentration of HEPES is 20 mM, the final concentration of MnCl2 is 10 mM, the final concentration of GLP-1M is 100 μM, the final concentration of human complex fatty alcohol oligosaccharide is 100-200 μM, the final concentration of oligosaccharyltransferase is 40 nM, and the reaction time is 1-4 h.
7. The method of introducing complex N-glycosylation modification to extend polypeptide half-life according to claim 1, characterized in that: The sequence of the GLP-1 is HDEYERHAEGTFTSDVSSYLEGQAAK EFIAWLVKGRG, and the sequence of the GLP-1M is HAEGNYTSDVSSYLEGQAAKEFIAW LVKGR.
8. The method of introducing complex N-glycosylation modification to extend polypeptide half-life according to claim 1, characterized in that: When the humanized complex N-glycopeptide with extended half-life is a galactose-modified GLP-1M, a GLP-1M with a sialic acid-modified terminal is obtained by catalysis using sialyltransferase; the sialyltransferase is derived from Photorhabdus mermaidae, and the sialyltransferase with 15 amino acids truncated at the N-terminus is expressed in Escherichia coli. The nucleotide sequence of the sialyltransferase is shown in SED ID NO.2, and the nucleotide sequence of the sialyltransferase with 15 amino acids truncated at the N-terminus is shown in SED ID NO.
3.
9. The method for introducing complex N-glycosylation modification to extend polypeptide half-life according to claim 8, characterized in that: The catalytic reaction system also contains Tris and MnCl2; Relative to the 50 μL system, the final concentration of Tris is 20 mM, the final concentration of MnCl2 is 10 mM, the final concentration of GLP-1M is 500 μM, the final concentration of CMP-NANA is 1000-2000 μM, the final concentration of sialyltransferase is 50 μg / mL, the reaction time is 6 min, and the enzyme and donor are added every 6 minutes for 3-4 times.
10. The humanized complex N-glycopeptide with extended half-life obtained by the method according to any one of claims 1 to 9, characterized in that: It has the following characteristics, 20 μg of human complex N-glycopeptide was dissolved in 15 μL ddH2O, an equal volume of mouse serum was added and mixed, and the mixture was incubated at 37°C. At different sampling time points, 3 μL of the mixture was taken each time and added to 6 μL of pre-cooled methanol and mixed evenly. The supernatant was collected by centrifugation at 12000×g for HPLC detection and analysis. The half-life of the human complex N-glycopeptide was 365-1564 min.