CN116063562A - Long-acting human recombinant glucagon-like peptide-1 and application thereof - Google Patents

Long-acting human recombinant glucagon-like peptide-1 and application thereof Download PDF

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CN116063562A
CN116063562A CN202211188658.0A CN202211188658A CN116063562A CN 116063562 A CN116063562 A CN 116063562A CN 202211188658 A CN202211188658 A CN 202211188658A CN 116063562 A CN116063562 A CN 116063562A
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巴萨姆法瑞斯
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Shanghai Yanli Pharmaceutical Co ltd
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Abstract

The invention provides a long-acting human recombinant glucagon-like peptide-1 and application thereof, wherein wild GLP-1 is subjected to mutation modification so as not to be sheared by DPP-4 enzyme, and a sequence containing one or more CTP is connected to the N end or/and the C end of the mutant GLP-1; the GLP-1 wild type amino acid sequence is shown as SEQ ID NO. 1; the amino acid sequence of the GLP-1 variant is shown as SEQ ID NO. 2; the amino acid sequence of the CTP is shown as SEQ ID NO. 3. Means were developed for long acting human glucagon-like peptide-1 linked to the coding sequence of mutant glucagon-like peptide-1 through the sequence of hcgβ Carboxy Terminal Peptide (CTP). Analogs were designed by ligating one or more CTP sequences to the C-terminus or/and the N-terminus of GLP-1.

Description

Long-acting human recombinant glucagon-like peptide-1 and application thereof
Technical Field
The invention relates to a long-acting human recombinant glucagon-like peptide-1 and application thereof, belonging to the technical field of gene construction.
Background
Glucagon-like peptide-1 (GLP-1) is a peptide hormone consisting of 30 or 31 amino acids, derived from tissue-specific post-translational processing of the glucagon-like peptide. It is produced and secreted by enteroendocrine L cells and by certain neurons within the solitary nucleus following food consumption. The initial product GLP-1 (1-37) is susceptible to amidation and proteolytic cleavage, resulting in two truncated and equivalent bioactive forms, GLP-1 (7-36) amide and GLP-1 (7-37). Active GLP-1 consists of two alpha-helices, located at positions 13-20 and 24-35 of the amino acid, respectively, separated by a linker region.
GLP-1, once secreted, is extremely sensitive to the catalytic activity of the proteolytic enzyme dipeptidyl peptidase 4 (DPP-4). Specifically, DPP-4 cleaves GLP-1 Ala 8 -Glu 9 Peptide bonds between them, resulting in the formation of a rich GLP-1 (9-36) amide, which can be up to 60-80% of the total GLP-1 in the circulation. DPP-4 is widely expressed in a variety of tissues and cell types and exists in membrane-anchored and soluble circulating forms. More notably, DPP-4 is expressed on the surface of endothelial cells, including cells immediately adjacent to the GLP-1 secretion site.
GLP-1 has various physiological characteristics, so that the GLP-1 (and functional analogues thereof) is the subject of intensive research, can be used as a potential treatment method for diabetes, can improve the condition of diabetes for a long time and has direct effect. It has the ability to lower blood glucose levels in a glucose dependent manner by enhancing insulin secretion. In addition to insulinotropic effects, GLP-1 is involved in many regulatory and protective actions. Unlike GIP (gastric inhibitory peptide), the effects of GLP-1 are preserved in type ii diabetics, and thus a number of drug studies have been directed to developing GLP-1 based therapies.
Endogenous GLP-1 is primarily rapidly degraded by DPP-4 and neutral endopeptidase and renal clearance, with a half-life of about 2 minutes. Thus, only 10-15% of GLP-1 enters the circulation intact, resulting in fasting plasma levels of only 0-15 pmol/L. To overcome this problem, GLP-1 receptor agonists and DPP-4 inhibitors have been developed to increase GLP-1 activity. In contrast to common therapeutic agents such as insulin and sulfonylureas, GLP-1 based therapies are associated with weight loss and reduced risk of hypoglycemia, two important considerations for type ii diabetics.
The most notable effect of GLP-1 is its ability to promote insulin secretion in a glucose-dependent manner. Since GLP-1 binds to GLP-1 receptor expressed on pancreatic beta cells, the receptor couples to the G protein subunit and activates adenylate cyclase, thereby increasingATP generates cAMP. Subsequently, activation of secondary pathways, including PKA and Epac2, alters ion channel activity, leading to cytosolic Ca 2+ Elevated levels, thereby enhancing exocytosis of the insulin particles. During this process, the inflow of glucose ensures that there is sufficient ATP to sustain the stimulation.
The present invention designs novel long acting GLP-1 variants by mutating wild type GLP-1 and ligating the Carboxy Terminal Peptide (CTP) of the human chorionic gonadotrophin beta subunit. It has been previously approved that linking CTP to the coding sequence of a protein results in an increase in vivo half-life. Furthermore, CTP shows no immunogenicity in humans.
Disclosure of Invention
In order to solve the technical problems, the invention provides a long-acting human recombinant glucagon-like peptide-1, a preparation method and application thereof, which comprises the steps of firstly carrying out mutation modification on wild GLP-1, and carrying out mutation modification on 8 th alanine (Ala) in wild GLP-1 polypeptide 8 ) Transformation to glycine (Gly) 8 ) GLP-1 variants are subsequently designed by attaching one or more CTP sequences to the C-terminus or/and N-terminus of the variant GLP-1 using means for attaching the sequence of the HCG beta Carboxy Terminal Peptide (CTP) to the coding sequence of the variant GLP-1.
The present invention provides a long acting human recombinant glucagon-like peptide-1 comprising one or more CTP sequences may be linked to the N-terminus or/and the C-terminus of GLP-1.
The GLP-1 wild type amino acid sequence is shown as SEQ ID NO. 1;
the amino acid sequence of the GLP-1 variant is shown as SEQ ID NO. 2;
the amino acid sequence of the CTP is shown as SEQ ID NO. 3;
the construction method of the long-acting human recombinant glucagon-like peptide-1 comprises the following steps: construction of a chimeric gene of glucagon-like peptide-1 (GLP-1) containing one or more CTP sequences, wherein CTP sequences I) are C-terminal, II) are N-terminal, III) are two C-terminal, IV) are two N-terminal, V) are two one of which is N-terminal, the other of which is C-terminal, VI) is three of which is C-terminal, VII) is three of which is N-terminal, and the other of which is two of which is C-terminal, IX) is three of which is N-terminal, and the other of which is C-terminal, using gene synthesis techniques, these chimeric genes will be sequenced and cloned into eukaryotic expression vectors, the constructed vectors containing GLP-1 and CTP sequences will be transfected into CHO cells, stable clones will be selected, the medium of stable clones will be collected, and GLP-1 variants will be purified.
The invention further protects the application of the long-acting human recombinant glucagon-like peptide-1 in preparing medicines for treating type II diabetes and obesity.
The invention has the following beneficial effects: means were developed for long acting human glucagon-like peptide-1 linked to the coding sequence of mutant glucagon-like peptide-1 through the sequence of hcgβ Carboxy Terminal Peptide (CTP). Analogs were designed by ligating one or more CTP sequences to the C-terminus or/and the N-terminus of GLP-1.
GLP-1 CTP analogs were transfected into CHO cells and stable GLP-1 CTP analog clones were selected. The biological activity of selected GLP-1 CTP analogs was tested in vivo and in vitro.
Chimeric genes containing GLP-1 and CTP sequences are capable of producing long acting GLP-1 and may be injected into a patient only once a week.
Drawings
FIG. 1 is a sequence structural diagram of GLP-1-CTP in example 1 of the present invention;
FIG. 2 is a sequence structural diagram of CTP-CLP-1 in example 2 of the present invention;
FIG. 3 is a sequence structural diagram of GLP-1- (CTP) 2 in example 3 of the present invention;
FIG. 4 is a diagram showing the structure of the (CTP) 2-GLP-1 sequence in example 4 of the present invention;
FIG. 5 is a diagram showing the sequence structure of CTP-GLP-1-CTP in example 5 of the present invention;
FIG. 6 is a diagram showing the structure of GLP-1- (CTP) 3 sequence in example 6 of the present invention;
FIG. 7 is a block diagram showing the sequence structure of (CTP) 3-GLP-1 in example 7 of the present invention;
FIG. 8 is a diagram showing the structure of CTP-GLP-1- (CTP) 2 sequence in example 8 of the present invention;
FIG. 9 is a block diagram showing the sequence of (CTP) 2-GLP-1-CTP in example 9 of the present invention.
Detailed Description
In the following, the technical solutions of the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the embodiments are only representative embodiments of some but not all embodiments of the present invention, and all other embodiments obtained by those skilled in the art without making any creative effort are within the protection scope of the present invention.
A long-acting human recombinant glucagon-like peptide-1 of this example would employ overlapping Polymerase Chain Reaction (PCR) to construct a GLP-1 chimeric gene and the carboxy-terminal peptide (CTP) sequence of the HCG beta subunit, ligating the coding sequence of CTP to the N-terminus or/and C-terminus of the coding sequence of GLP-1, sequencing and cloning the chimeric gene into eukaryotic expression vectors containing LTR/CMV promoter, transfecting the constructed vector into CHO cells, and selecting stable clones. Culture medium from the stable clones was collected and GLP-1 CTP analogs purified and the concentration of GLP-1 was detected by commercial kits. The biological activity of GLP-1 variants was tested in vitro and in vivo.
Example 1: referring to FIG. 1, the GLP-1-CTP is a CTP sequence and a cassette gene with the GLP-1 sequence as a template is used as template synthesis, wherein the cassette gene comprises 1 CTP sequence at the C end of the GLP-1 sequence.
Example 2: referring to FIG. 2, the CTP-CLP-1 is a box gene with a CTP sequence and a GLP-1 sequence as templates, wherein the box gene contains 1 CTP sequence at the N-terminal of the GLP-1 sequence, which is used as a template for synthesis.
Example 3: referring to FIG. 3, the GLP-1- (CTP) 2 is a cassette gene with two CTP sequences and GLP-1 sequences as templates, wherein the cassette gene comprises 2 CTP sequences at the C-terminal end of the GLP-1 sequence, and is used as a template for synthesis.
Example 4: the (CTP) 2-GLP-1 is a box gene taking two CTP sequences and a GLP-1 sequence as templates, and is used for template synthesis, wherein 2 CTP sequences are contained at the N end of the GLP-1 sequence.
Example 5: the CTP-GLP-1-CTP is synthesized by using two CTP sequences and a box gene taking the GLP-1 sequence as templates, wherein 2 CTP sequences are respectively added at the N end of the GLP-1 sequence, and the other CTP sequence is added at the C end of the GLP-1 sequence.
Example 6: the GLP-1- (CTP) 3 is a box gene taking 3 CTP sequences and the GLP-1 sequences as templates, and is used for template synthesis, wherein the box gene comprises 3 CTP sequences added at the C end of the GLP-1 sequences.
Example 7: the (CTP) 3-GLP-1 is a box gene taking 3 CTP sequences and GLP-1 sequences as templates, and is used for template synthesis, wherein the box gene comprises 3 CTP sequences added at the N end of the GLP-1 sequences.
Example 8: the CTP-GLP-1- (CTP) 2 is a box gene taking 3 CTP sequences and a GLP-1 sequence as templates, and is used for template synthesis, wherein the box gene comprises 3 CTP sequences, 2 CTPs are respectively added at the C end of the GLP-1 sequence, and the other CTP sequence is added at the N end of the GLP-1 sequence.
Example 9: the (CTP) 2-GLP-1-CTP is a box gene taking 3 CTP sequences and GLP-1 sequences as templates, and is used for template synthesis, wherein 3 CTP sequences are contained, 1 CTP is added at the C end of the GLP-1 sequence respectively, and 2 CTP sequences are added at the N end of the GLP-1 sequence.
Various modifications may be made to the above disclosure by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is therefore not limited to the above description, but is instead determined by the scope of the claims.

Claims (9)

1. A long-acting human recombinant glucagon-like peptide-1, characterized in that: modifying wild type GLP-1 in a variant way so that the wild type GLP-1 is not sheared by DPP-4 enzyme, and connecting a sequence containing one or more CTP to the N end or/and the C end of the variant GLP-1;
the GLP-1 wild type amino acid sequence is shown as SEQ ID NO. 1;
the amino acid sequence of the GLP-1 variant is shown as SEQ ID NO. 2;
the amino acid sequence of the CTP is shown as SEQ ID NO. 3.
2. A method for preparing a long-acting human recombinant glucagon-like peptide-1 according to claim 1, characterized in that: the specific method comprises the following steps: construction of chimeric genes for mutated human glucagon-like peptide-1 (GLP-1) using gene synthesis techniques contains the sequence of the hcgβ Carboxy Terminal Peptide (CTP), which can be i) one at the C-terminus; II) one at the N-terminal; III) two at the C-terminus; IV) two are at N end; v) two of which are at N-terminal and one at C-terminal; VI) three at the C-terminus; VII) three are at the N end; VIII) three of which are at the N-terminus and two of which are at the C-terminus; IX) three of which are N-terminal and one at the C-terminal, these chimeric genes will be sequenced and cloned into eukaryotic expression vectors, the constructed vectors containing GLP-1 and CTP sequences will be transfected into CHO cells, then stable clones will be selected, the medium of the stable clones will be collected and GLP-1 variants will be purified.
3. The method for preparing the long-acting human recombinant glucagon-like peptide-1 according to claim 2, wherein the construction of the variant chimeric gene comprises the following steps: (S1) constructing GLP-1-CTP; (S2) constructing GLP-1-CTP; (S3) constructing GLP-1-CTP; (S4) constructing CTP-GLP-1; (S5) constructing CTP-GLP-1; (S6) constructing CTP-GLP-1; (S7) constructing CTP-GLP-1-CTP; (S8) constructing CTP-GLP-1-CTP; (S9) constructing CTP-CTP-GLP-1-CTP.
4. A method of preparing a long-acting human recombinant glucagon-like peptide-1 according to claim 3, wherein: the step (S1) comprises the following steps: the GLP-1-CTP is formed by taking a gene of an HCG beta sequence and a gene of a GLP-1 sequence as a template, and the construction method comprises three steps: (1) Primer 1 and primer 2 take HCG beta gene as a template, primer 1 comprises the last 4 base codes of GLP-1 sequence and the first 4 base codes of CTP 5 'end sequence, and primer 2 comprises the last 4 base codes of CTP 3' end sequence and antisense base codes of a restriction site;
(2) Primer 3 and primer 4 take GLP-1 gene as a template, primer 3 comprises the first 4 base codes of GLP-1 sequence and a restriction site, and primer 4 comprises the last 4 base codes of GLP-1 sequence and the antisense base codes of the first 4 base codes of CTP 5' end sequence;
(3) The gene products reacted in (1) and (2) are used as templates of primer 2 and primer 3 for amplifying GLP-1-CTP;
the step (S2) comprises the following steps: the GLP-1-CTP-CTP is a gene of an HCG beta sequence and a GLP-1-CTP sequence, which is used as a template, and the construction method is carried out in three steps: (1) Primer 1 and primer 2 take HCG beta gene as template, primer 1 contains 4 base codes behind CTP 3' end sequence and 4 base codes in front of CTP 5' end sequence, primer 2 contains 4 base codes behind CTP 3' end sequence and antisense base code of a restriction site; (2) Primer 3 and primer 4 take GLP-1-CTP gene as template, primer 3 contains first 4 base codes of GLP-1 sequence and a restriction site, primer 4 contains antisense base codes of first 4 base codes of CTP 3 'end sequence and CTP 5' end sequence; (3) The gene products reacted in (1) and (2) are used as templates of primer 2 and primer 3 for amplifying GLP-1-CTP-CTP;
the step (S3) comprises the following steps: the GLP-1-CTP-CTP-CTP is a gene of an HCG beta sequence and a GLP-1-CTP-CTP sequence, which is used as a template, and the construction method comprises three steps: (1) Primer 1 and primer 2 take HCG beta gene as template, primer 1 contains 4 base codes behind CTP 3' end sequence and 4 base codes in front of CTP 5' end sequence, primer 2 contains 4 base codes behind CTP 3' end sequence and antisense base code of a restriction site; (2) Primer 3 and primer 4 take GLP-1-CTP-CTP gene as template, primer 3 contains first 4 base codes of GLP-1 sequence and a restriction site, primer 4 contains last 4 base codes of CTP 3 'end sequence and antisense base codes of first 4 base codes of CTP 5' end sequence; (3) The gene products reacted in (1) and (2) are used as templates for primer 2 and primer 3 for amplifying GLP-1-CTP-CTP-CTP.
5. A method of preparing a long-acting human recombinant glucagon-like peptide-1 according to claim 3, wherein: the step (S4) comprises the following steps: the CTP-GLP-1 is a gene of an HCG beta sequence and a GLP-1 sequence, which is used as a template, and the construction method is carried out in three steps: (1) Primer 1 and primer 2 take HCG beta gene as a template, primer 1 comprises the first 4 base codes of CTP 5 'end sequence and a restriction site, and primer 2 comprises the last 4 base codes of CTP 3' end sequence and antisense base codes of the first 4 base codes of GLP-1 sequence; (2) Primer 3 and primer 4 take GLP-1 gene as templates, primer 3 contains the last 4 base codes of CTP 3' end sequence and the first 4 base codes of GLP-1 sequence, primer 4 contains the last 4 base codes of GLP-1 sequence and antisense base codes of a restriction site; (3) The gene products reacted in (1) and (2) are used as templates for primer 1 and primer 4 for amplifying CTP-GLP-1;
the step (S5) comprises the following steps: the CTP-CTP-GLP-1 is a gene of an HCG beta sequence and a CTP-GLP-1 sequence, which is used as a template, and the construction method comprises three steps: (1) Primer 1 and primer 2 take HCG beta gene as template, primer 1 contains the first 4 base codes of CTP 5' end sequence and a restriction site, primer 2 contains the last 4 base codes of CTP 3' end sequence and antisense base codes of the first 4 base codes of CTP 5' end sequence; (2) Primer 3 and primer 4 take CTP-GLP-1 gene as template, primer 3 contains the last 4 base codes of CTP 3 'end sequence and the first 4 base codes of CTP 5' end sequence, primer 4 contains the last 4 base codes of GLP-1 sequence and antisense base code of a restriction site; (3) The gene products reacted in (1) and (2) are used as templates for primer 1 and primer 4 for amplifying CTP-CTP-GLP-1;
the step (S6) comprises the following steps: the CTP-CTP-CTP-GLP-1 is a gene of an HCG beta sequence and a CTP-CTP-GLP-1 sequence which are used as templates, and the construction method comprises three steps: (1) Primer 1 and primer 2 take HCG beta gene as template, primer 1 contains the first 4 base codes of CTP 5' end sequence and a restriction site, primer 2 contains the last 4 base codes of CTP 3' end sequence and antisense base codes of the first 4 base codes of CTP 5' end sequence; (2) Primer 3 and primer 4 take CTP-CTP-GLP-1 gene as template, primer 3 contains the last 4 base codes of CTP 3 'end sequence and the first 4 base codes of CTP 5' end sequence, primer 4 contains the last 4 base codes of GLP-1 sequence and antisense base code of a restriction site; (3) The gene products reacted in (1) and (2) are used as templates for primer 1 and primer 4 for amplifying CTP-CTP-CTP-GLP-1.
6. A method of preparing a long-acting human recombinant glucagon-like peptide-1 according to claim 3, wherein: the step (S7) comprises the following steps: the CTP-GLP-1-CTP is a gene of an HCG beta sequence and a GLP-1-CTP sequence, and is used as a template, and the construction method is carried out in three steps: (1) Primer 1 and primer 2 take HCG beta gene as a template, primer 1 comprises the first 4 base codes of CTP 5 'end sequence and a restriction site, and primer 2 comprises the last 4 base codes of CTP 3' end sequence and antisense base codes of the first 4 base codes of GLP-1 sequence; (2) Primer 3 and primer 4 take GLP-1-CTP gene as template, primer 3 contains the last 4 base codes of CTP 3 'end sequence and the first 4 base codes of GLP-1 sequence, primer 4 contains the last 4 base codes of CTP 3' end sequence and antisense base code of a restriction site; (3) The gene products reacted in (1) and (2) are used as templates for primer 1 and primer 4 for amplifying CTP-GLP-1-CTP.
7. A method of preparing a long-acting human recombinant glucagon-like peptide-1 according to claim 3, wherein: the step (S8) comprises the following steps: the CTP-GLP-1-CTP-CTP is a gene of an HCG beta sequence and a CTP-GLP-1-CTP sequence, which is used as a template, and the construction method comprises three steps: (1) Primer 1 and primer 2 take CTP-GLP-1-CTP sequence gene as a template, wherein primer 1 comprises the first 4 base codes of CTP 5' end sequence and a restriction site, and primer 2 comprises the last 4 base codes of CTP 3' end sequence and the antisense base codes of the first 4 base codes of CTP 5' end sequence; (2) Primer 3 and primer 4 take HCG beta gene as template, primer 3 contains the last 4 base codes of CTP 3' end sequence and the first 4 base codes of CTP 5' end sequence, primer 4 contains the last 4 base codes of CTP 3' end sequence and antisense base code of a restriction site; (3) The gene products reacted in (1) and (2) are used as templates for primer 1 and primer 4 for amplifying CTP-GLP-1-CTP-CTP.
8. A method of preparing a long-acting human recombinant glucagon-like peptide-1 according to claim 3, wherein: the step (S9) comprises the following steps: the CTP-CTP-GLP-1-CTP is a gene of an HCG beta sequence and a CTP-GLP-1-CTP sequence, which is used as a template, and the construction method comprises three steps: (1) Primer 1 and primer 2 take HCG gene as template, primer 1 contains the first 4 base codes of CTP 5' end sequence and a restriction site, primer 2 contains the last 4 base codes of CTP 3' end sequence and antisense base codes of the first 4 base codes of CTP 5' end sequence; (2) Primer 3 and primer 4 take the gene of the CTP-GLP-1-CTP sequence as a template, primer 3 comprises the last 4 base codes of the CTP 3' end sequence and the first 4 base codes of the CTP 5' end sequence, and primer 4 comprises the last 4 base codes of the CTP 3' end sequence and an antisense base code of a restriction site; (3) The gene products reacted in (1) and (2) are used as templates for primer 1 and primer 4 for amplifying CTP-CTP-GLP-1-CTP.
9. Use of a long-acting human recombinant glucagon-like peptide-1 of claim 1 for the preparation of a medicament for the treatment of type ii diabetes and obesity.
CN202211188658.0A 2022-09-28 2022-09-28 Long-acting human recombinant glucagon-like peptide-1 and application thereof Pending CN116063562A (en)

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