Long-acting insulin analogue as well as preparation method and application thereof
By performing site mutation of the insulin B chain, eliminating the enzyme degradation site, and combining existing or mutated insulin A chain, the problem of short-acting insulin is solved, the long-acting effect of insulin is achieved and the preparation process is simplified, and the half-life is significantly extended. and usage time.
Patent Information
- Application Number
- CN202411211223.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-30
- Publication Date
- 2024-12-20
AI Technical Summary
The existing insulin products have short-acting defects, which leads to diabetic patients who need to eat half an hour after administration, causing inconvenience. In addition, in order to modify the insulin molecules to achieve long-term effects, it is necessary to transform the A and B chains of insulin at the same time, which is a large workload. .
By performing site mutations on the insulin B chain only, the sensitive sites of enzyme degradation, especially the enzyme cleavage sites of trypsin, obtaining insulin B chain mutants, thereby prolonging the time of action of insulin and with existing or mutated Insulin A chain linkage simplifies the preparation process.
It achieves the long-term effect of insulin molecules, significantly prolongs the half-life, reduces the sensitivity to trypsin, and provides a longer effective use time and convenient treatment plan.
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Figure CN119161447A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to a long-acting insulin analog, a preparation method and application thereof. Background Art
[0002] Insulin is the first protein molecule to be sequenced, and has always been a hot topic for experts and scholars. The study of insulin mutants is an important research direction in modern protein engineering, especially the study of insulin medicinal mutants has become a research hotspot.
[0003] Insulin is a hormone secreted by the human pancreas to regulate blood sugar. Its function is to transfer excess glucose in the blood to cells, provide energy for cells, and maintain blood sugar levels at normal levels. Insulin can control the blood sugar level of diabetic patients at normal levels.
[0004] With the development of recombinant protein manufacturing technology, a variety of insulin products have been put on the market. Existing insulin products have the disadvantage of short-term effect, and people can only eat half an hour after administration, which is seriously inconvenient. Therefore, modifying insulin molecules is of great significance to the research and development of diabetes treatment drugs.
[0005] Insulin consists of insulin A chain and insulin B chain. Currently, if we want to prolong the duration of action, we need to modify both the insulin A chain and the insulin B chain at the same time to obtain long-acting insulin analogs, but this is a lot of work. Summary of the invention
[0006] In order to develop a long-acting insulin analog with a prolonged action time and a simple preparation process, the present invention provides a long-acting insulin analog and its preparation method and application. The present invention obtains a long-acting insulin analog by only subjecting the insulin B chain to site mutation to obtain an insulin B chain mutant, thereby eliminating the sensitive sites of enzyme degradation at the molecular level, especially eliminating the enzyme cleavage sites of trypsin, so that the activity of the insulin molecule can be maintained to the greatest extent, thereby achieving the purpose of long-term effect, and the preparation process is simple.
[0007] The present invention provides a long-acting insulin analog, wherein the long-acting insulin analog comprises insulin A chain and insulin B chain mutants;
[0008] The insulin B chain mutant is obtained by mutation of the insulin B chain shown in SEQ ID NO.8, and the mutation sites are that the 22nd arginine and the 29th lysine on the insulin B chain are replaced by non-basic amino acids;
[0009] The non-basic amino acid is any one of glycine, alanine, valine, leucine, isoleucine, serine, threonine, tyrosine, methionine, phenylalanine, aspartic acid, asparagine, glutamic acid and glutamine.
[0010] The present invention obtains an insulin B chain mutant by subjecting only the insulin B chain to site mutation, thereby obtaining a long-acting insulin analog, eliminating sensitive sites for enzyme degradation at the molecular level, especially eliminating the enzyme cleavage sites of trypsin, so that the activity of the insulin molecule can be maintained to the greatest extent, thereby achieving the purpose of long-term effect, and the preparation process is simple.
[0011] Furthermore, the amino acid sequence of the insulin A chain is shown in SEQ ID NO.7, and can also be replaced by other existing mutants of insulin A chain. The present invention obtains an insulin B chain mutant by mutating only the insulin B chain, and then connects it with a known insulin A chain or a mutant of the insulin A chain to obtain a long-acting insulin analog, which simplifies the preparation process of the long-acting insulin analog while achieving long-acting effect.
[0012] Furthermore, the connection relationship between the insulin A chain and the insulin B chain mutant is as follows: the cysteine at position 6 of the insulin A chain is connected to the cysteine at position 11 of the insulin A chain in the form of a disulfide bond; the cysteine at position 7 of the insulin A chain is connected to the cysteine at position 7 of the insulin B chain mutant in the form of a disulfide bond; the cysteine at position 20 of the insulin A chain is connected to the cysteine at position 19 of the insulin B chain mutant in the form of a disulfide bond.
[0013] The present invention also provides a method for preparing a long-acting insulin analog, comprising the following steps:
[0014] The PCR primers and mutation primers were designed using the human insulin gene sequence shown in SEQ ID NO.6 as a template, wherein the PCR primers included a forward primer F shown in SEQ ID NO.10 and a reverse primer R shown in SEQ ID NO.11;
[0015] The first round of PCR reaction was carried out using forward primer F and mutant primer as primers and the insulin gene shown in SEQ ID NO.6 as a template;
[0016] After the first round of PCR reaction, reverse primer R is added to the reaction system for a second round of PCR reaction;
[0017] The second round of PCR amplification products are connected to the vector to construct a recombinant plasmid, and the products are obtained by transformation and induction of expression, and then enzymatic hydrolysis, separation and purification are performed to obtain long-acting islet analogs;
[0018] The mutation primer contains a mutation site.
[0019] Furthermore, when the mutation site is that the arginine at position 22 on the insulin B chain is replaced by alanine, and the lysine at position 29 on the insulin B chain is replaced by valine, the sequence of the mutation primer is as shown in SEQ ID NO.12;
[0020] When the mutation site is that the arginine at position 22 on the insulin B chain is replaced by leucine, and the lysine at position 29 on the insulin B chain is replaced by serine, the sequence of the mutant primer is as shown in SEQ ID NO.14;
[0021] When the mutation site is that the arginine at position 22 on the insulin B chain is replaced by phenylalanine, and the lysine at position 29 on the insulin B chain is replaced by isoleucine, the sequence of the mutation primer is as shown in SEQ ID NO.16;
[0022] When the mutation site is that the arginine at position 22 on the insulin B chain is replaced by asparagine, and the lysine at position 29 on the insulin B chain is replaced by alanine, the sequence of the mutation primer is as shown in SEQ ID NO.18;
[0023] When the mutation site is that the arginine at position 22 on the insulin B chain is replaced by threonine, and the lysine at position 29 on the insulin B chain is replaced by isoleucine, the mutation primer sequence is shown in SEQ ID NO. 20. The above is a preferred mutation mode of the insulin B chain in the embodiment of the present invention. When the obtained long-acting insulin analog is incubated with trypsin, the half-life of the mutant insulin is extended by at least 7 times compared with the half-life of the insulin molecule under the same measurement environment.
[0024] Furthermore, the first round of PCR reaction system was: forward primer F 25 pmol 2 μL, mutant primer 1, 50 pmol 1 μL, template DNA 10 ng 0.5 μL, dNTP 200 μmol, 10× PCR buffer 5 μL, ddH 2 O 40.5 μL;
[0025] Furthermore, the reaction conditions of the first round of PCR reaction were: denaturation at 94°C for 60 s, annealing at 56°C for 60 s, extension at 72°C for 60 s, amplification for 30 cycles, and the last cycle was 72°C for 10 min.
[0026] Furthermore, the second round of PCR reaction conditions were denaturation at 94°C for 60 s, annealing for 60 s, extension at 72°C for 60 s, amplification for 30 cycles, and the last cycle was 72°C for 10 min.
[0027] The present invention also provides an application of the long-acting insulin analog in the preparation of insulin medicines.
[0028] Furthermore, the insulin drug has the long-acting insulin analog as the only active ingredient.
[0029] The present invention utilizes the means of protein engineering to mutate the arginine at the 22nd position and the lysine at the 29th position of the basic amino acid B chain of the insulin molecule that are easily degraded, and replaces them with non-basic amino acids at the same time. The basis for the mutation of the present invention is that the arginine at the 22nd position and the lysine at the 29th position of the insulin B chain are located on the surface of the insulin spatial structure, and the substitution mutation is not easy to cause too much change in the insulin spatial structure, and has a certain influence on the three pairs of hydrogen bonds formed between the insulin molecules to form dimers, and can better destroy the hydrogen bonds formed between the insulin molecule dimers, thereby being more conducive to forming monomeric insulin and playing a quick-acting effect, and replacing these two basic amino acids at the same time, eliminating the sensitive sites of enzyme degradation at the molecular level, especially eliminating the enzyme cleavage site of trypsin, so that the activity of the insulin molecule can be maintained to the greatest extent, thereby achieving the purpose of long-acting. Due to the changes in the amino acid polarity of the insulin mutant and the steric hindrance of forming hydrogen bonds, it is also difficult to form hydrogen bonds between the insulin molecules (the insulin molecules exist in the molecular form of monomers) and play a quick-acting effect.
[0030] Compared with the prior art, the present invention has the following beneficial effects:
[0031] The present invention obtains a new insulin B chain mutant by mutating the 22nd arginine and the 29th lysine on the insulin B chain, eliminating the sensitive sites of enzyme degradation at the molecular level, especially eliminating the enzyme cleavage sites of trypsin, so that the activity of the insulin analog molecule prepared by using the insulin B chain mutant can be maintained to the greatest extent, solving the problem of short-acting insulin.
[0032] The invention effectively avoids the degradation of insulin molecules by proteolytic enzymes in vivo, especially the hydrolysis by trypsin, thereby prolonging the survival time of insulin molecules in vivo, achieving the purpose of continuously maintaining activity, and significantly playing a role in the long-term effect of insulin. At the same time, the purpose of changing the structure of the surface of its spatial structure is achieved, without destroying the spiral structure and domain inside the insulin molecule, and changing the spatial structure of the amino acid at the end of the B chain. While ensuring the elimination of the trypsin hydrolysis site to achieve the purpose of long-term effect, the formation of insulin molecule dimers is better eliminated, thereby increasing the number of monomeric insulin molecules, and then playing a corresponding quick-acting role.
[0033] The sensitivity of insulin molecules to enzymes, especially trypsin, is further reduced, making its long-acting effect more obvious. It has a significantly increased half-life in the body, and thus can provide convenience for diabetic patients who directly administer insulin as an injection. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0035] Figure 1 This is a diagram of the molecular structure of natural insulin.
[0036] Figure 2 Identification of the PCR amplification product of long-acting insulin analog No. 1; Lane 1: DNA ladder, Lane 2: PCR amplification product.
[0037] Figure 3 This is the identification of the PCR amplification product of long-acting insulin analog No. 2; lane 1: DNA ladder, lane 2: PCR amplification product.
[0038] Figure 4 This is the identification of the PCR amplification product of the long-acting insulin analog No. 3; Lane 1: DNA ladder, Lane 2: PCR amplification product.
[0039] Figure 5 Identification of the PCR amplification product of long-acting insulin analog No. 4; Lane 1: DNA ladder, Lane 2: PCR amplification product.
[0040] Figure 6 Identification of the PCR amplification product of long-acting insulin analog No. 5; Lane 1: DNA ladder, Lane 2: PCR amplification product.
[0041] Figure 7 A mathematical model of the long-acting insulin analog No. 1 prepared in Example 1 of the present invention and the dimer structure of natural insulin;
[0042] In the figure, A is the mathematical model of the natural insulin dimer structure;
[0043] B is the mathematical model of the dimer structure of the long-acting insulin analog No. 1 prepared in Example 1 of the present invention. DETAILED DESCRIPTION
[0044] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.
[0045] Example 1: Preparation of long-acting insulin analog No. 1.
[0046] 1. Primer Design
[0047] 1. Selection of insulin molecules for modification
[0048] The human insulin gene template was provided by the Protein Engineering Laboratory of Peking University. The GenBank accession number of the insulin gene is V00082, and its gene sequence is shown in SEQ ID NO.6; the insulin sequence is sequentially connected by the insulin B chain (referred to as the B chain), the insulin C chain (referred to as the C chain) and the insulin A chain (referred to as the A chain), and the amino acid sequences of each part are shown in Table 1. The sequencing of the DNA primer fragments and PCR products used in the experiment was completed by Shanghai Shenggong Biotechnology Co., Ltd.
[0049] SEQ ID NO.6:
[0050] 5′-TTTGTCAATCAGCACCTTTGTGGTTCTCACCTGGTGGAGGCTCTGTACCTGGTGTGTGGGGAACGTGGTTTCTTCTACACACCCAAGACCCGTCGTGAAGCTGAAGACCTTCAAGTGGGTCAAGTTGAACTTGGTGGGGGTCCTGGT GAG GGTTCTCTTCAACCTTTGGCTCTCGAGGGATCACTTCAAAAGCGTGGCATTGTGGAGCAGTGCTGCACCAGCATCTGCTCCCTCTACCAACTGGAGAACTACTGCAACTGAG-3'.
[0051] Table 1 Sequences of each part of insulin sequence
[0052] Each part Amino acid sequence serial number Insulin A chain GIVEQCCTSICSLYQLENYCN SEQ ID NO.7 Insulin B chain FVNQHLCGSHLVEALYLVCGERGFFYTPKT SEQ ID NO.8 Insulin C chain RREAEDLQVGQVELGGGPGEGSLQPLALEGSLQKR SEQ ID NO.9
[0053] The cysteines of insulin chain A6 (Cys) and A11 (Cys), chain A7 (Cys) and B7 (Cys), chain A20 (Cys) and B19 (Cys) are connected by disulfide bonds (e.g. Figure 1 shown).
[0054] 2. Primer design
[0055] (1) Using the human insulin gene sequence shown in SEQ ID NO.6 as a template, PCR primers were designed, including a forward primer F and a reverse primer R. The specific sequences are as follows:
[0056] F: 5′-GGAATTCCATATGAAGTTTGTC-3′ (SEQ ID NO. 10);
[0057] R: 5'-CAGGTCGACGGATCCTCAGTTG-3' (SEQ ID NO. 11).
[0058] (2) Design of mutation primer 1:
[0059] Different insulin B chain mutants have different introduced sites and sequences. The mutant primer 1 containing the mutation site in which the arginine at the 22nd position is replaced by alanine and the lysine at the 29th position is replaced by valine is shown in SEQ ID NO.12.
[0060] SEQ ID NO.12: 5′-GAA GCT GGTTTCTTCTACACACCC GTT ACC-3'.
[0061] 2. Preparation of Long-Acting Insulin Analog No. 1
[0062] 1. First round of PCR
[0063] The first round of PCR reaction was carried out using the forward primer F shown in SEQ ID NO.10 and the mutant primer 1 shown in SEQ ID NO.12 as primers and the insulin gene shown in SEQ ID NO.6 as a template.
[0064] The PCR reaction system was as follows: forward primer F 25 pmol 2 μL, mutant primer 1, 50 pmol 1 μL, template DNA 10 ng 0.5 μL, dNTP 200 μmol, 10× PCR buffer 5 μL, ddH 2 O 40.5μL; add Taq plus DNA polymerase 3U, paraffin oil, centrifuge and put into PCR instrument for reaction.
[0065] PCR cycle reaction conditions: denaturation at 94°C for 60 s, annealing at 56°C for 60 s, extension at 72°C for 60 s, amplification for 30 cycles, and the last cycle was 72°C for 10 min.
[0066] 2. Second round of PCR
[0067] After the first round of PCR reaction was completed, the reverse primer R shown in SEQ ID NO. 11 was added to the reaction system, 200 μmol of dNTP and 3 U of Taq plus DNA polymerase were supplemented, and then a second round of PCR reaction was carried out.
[0068] The second round of PCR reaction conditions: 94℃ denaturation for 60s, annealing for 60s, 72℃ extension for 60s, amplification for 30 cycles, the last cycle is 72℃ for 10min, and the temperature is set to cool to 4℃ after the cycle reaction is completed. The PCR amplification product is subjected to 1.5% agarose gel electrophoresis to observe the results.
[0069] The PCR amplified product was sequenced and identified. The sequence of the target gene product amplified by PCR is shown in SEQ ID NO.13.
[0070] SEQ ID NO.13:
[0071] 5′-TTTGTCAATCAGCACCTTTGTGGTTCTCACCTGGTGGAGGCTCTGTACCTGGTGTGTGGGGAA GC T GGTTTCTTCTACACACCC GTT ACCCGTCGTGAAGCTGAAGACCTTCAAGTGGGTCAAGTTGAACTTGGTGGGGGTCCTGGT GAG GGTTCTCTTCAACCTTTGGCTCTCGAGGGATCACTTCAAAAGCGTGGCATTGTGGAGCAGTGCTGCACCAGCATCTGCTCCCTCTACCAACTGGAGAACTACTGCAACTGAG-3'.
[0072] 3. Expression and purification of long-acting insulin analog No. 1
[0073] 1. Inducible expression of long-acting insulin analog 1
[0074] The PCR amplification product shown in SEQ ID NO.13 was connected to the EcoRI and HindIII sites of the PBV220 vector to obtain a recombinant plasmid, which was then transformed into Escherichia coli BL21 (DE3) to obtain recombinant bacteria, which were cultured at 37 degrees Celsius for 16 hours and induced to express with IPTG. The product shown in SEQ ID NO.1 (the complete amino acid sequence of insulin mutant 1) was obtained.
[0075] SEQ ID NO.1:
[0076] FVNQHLCGSHLVEALYLVCGEAGFFYTPVTRREAEDLQVGQVELGGGP GEGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN.
[0077] 2. Separation and purification
[0078] The product shown in SEQ ID NO.1 can be separated and purified by conventional protein separation methods to obtain the target product. The expression product of insulin mutant 1 can be operated by the following specific steps to obtain the target product. The details are as follows:
[0079] A1. Obtaining bacteria: After fermentation with E. coli BL21 and induction with IPTG, samples were taken for analysis. When a large number of product bands appeared, the fermentation liquid was centrifuged at more than 5000 rpn, the bacteria were collected and washed.
[0080] B1. Obtaining crude proinsulin: Take the washed bacterial cells, add lysis buffer to lyse them with ultrasonic treatment, centrifuge to remove bacterial fragments, and collect the crude proinsulin.
[0081] C1. Pretreatment of enzymatic hydrolysis: Add guanidine hydrochloride for denaturation treatment, collect the denatured crude proinsulin by centrifugation, remove the denaturant by dialysis, and then concentrate the crude proinsulin to an appropriate concentration by reverse osmosis dehydration.
[0082] D1. Enzymatic cleavage: Add trypsin to the crude proinsulin and incubate for 5 minutes to remove the C peptide. Then add dithiothreitol and incubate for 5 minutes to open the disulfide bonds.
[0083] E1. Separate the two chains A and B. Select molecular sieve dextran gel Sephadex G-25 chromatography to collect and separate the A chain and B chain respectively.
[0084] F1. Selectively cleave the terminal basic amino acid of chain B. Add carboxypeptidase to the collected chain B and control the cleavage conditions to completely cut off the arginine at the C-terminus of chain B.
[0085] G1. Denaturation treatment of insulin: Combine the A chain and B chain of insulin, add appropriate urea and dithiothreitol, and keep warm for 5 minutes to denature the A chain and B chain of insulin. Choose appropriate dialysis conditions to slowly dialyze away urea and dithiothreitol, which is conducive to the formation of correct disulfide bond pairing between the A chain and B chain of insulin.
[0086] H1. Separation and purification of mutants: After molecular sieve Sephadex G-25 chromatography, combined with sampling analysis, the peaks of insulin B chain mutants were collected and identified to obtain the target product.
[0087] Thus, long-acting insulin analog No. 1 is obtained.
[0088] The long-acting insulin analog No. 1 includes insulin A chain and insulin B chain mutants, and the amino acid sequences of insulin A chain and insulin B chain mutants (underlined mutation sites) are as follows:
[0089] Insulin A chain:
[0090] H 2 N-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-
[0091] Glu-Asn-Tyr-Cys-Asn-COOH (SEQ ID NO.7);
[0092] Insulin B chain mutants:
[0093] H 2 N-Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-
[0094] Leu-Val-Cys-Gly-Glu- Ala -Gly-Phe-Phe-Tyr-Thr-Pro- Val -Thr-COOH (SEQ ID NO. 22).
[0095] Among them, the cysteine (Cys) at position 6 of the insulin A chain is connected to the cysteine (Cys) at position 11 of the insulin A chain in the form of a disulfide bond;
[0096] The cysteine (Cys) at position 7 of the insulin A chain and the cysteine (Cys) at position 7 of the insulin B chain mutant are linked in the form of a disulfide bond;
[0097] The cysteine (Cys) at position 20 of the insulin A chain and the cysteine (Cys) at position 19 of the insulin B chain mutant are linked in the form of a disulfide bond.
[0098] Example 2: Preparation of long-acting insulin analog No. 2.
[0099] 1. Primer Design
[0100] 1. Design mutation primer 2:
[0101] Mutation primer 2 was designed so that the arginine at position 22 of the insulin B chain was replaced by leucine, and the lysine at position 29 was replaced by serine. The sequence of mutation primer 2 is shown in SEQ ID NO.14.
[0102] SEQ ID NO.14: 5′-GAA TTA GGTTTCTTCTACACACCC TCT ACC-3'.
[0103] 2. PCR reaction
[0104] The PCR reaction process is the same as that in Example 1, except that the mutant primer 1 is replaced by the mutant primer 2.
[0105] The sequence of the target gene product amplified by PCR is shown in SEQ ID NO.15.
[0106] SEQ ID NO.15:
[0107] 5′-TTTGTCAATCAGCACCTTTGTGGTTCTCACCTGGTGGAGGCTCTGTACCTGGTGTGTGGGGAA TT A GGTTTCTTCTACACACCC TCT ACCCGTCGTGAAGC TGAAGACCTTCAAGTGGGTCAAGTTGAACTTGGTGGGGGTCCTGGT GAG GGTTCTCTTCAACCTTTGGCTCTCGAGGGATCACTTCAAAAGCGTGGCATTGTGGAGCAGTGCTGCACCAGCATCTGCTCCCTCTACCAACTGGAGAACTACTGCAACTGAG-3'.
[0108] 3. Expression and purification of long-acting insulin analog No. 2
[0109] 1. Inducible expression of long-acting insulin analog No. 2
[0110] The PCR amplification product shown in SEQ ID NO.15 was connected to the EcoRI and HindIII sites of the PBV220 vector to obtain a recombinant plasmid, which was then transformed into E. coli BL21 (DE3) to obtain a recombinant bacterium, which was induced to express with IPTG after shaking culture at 37 degrees Celsius for 16 hours to obtain the product shown in SEQ ID NO.2.
[0111] SEQ ID NO.2:
[0112] FVNQHLCGSHLVEALYLVCGE L GFFYTPST RR EAEDLQVGQVELGGGPGEGSLQPLALEGSLQ KR GIVEQCCTSICSLYQLENYCN.
[0113] 2. Separation and purification of long-acting insulin analog No. 2
[0114] The product shown in SEQ ID NO.2 can be subjected to conventional protein enzymatic hydrolysis, enzymatic cleavage, separation and purification methods to obtain the target product. The expression product of insulin mutant 1 can be subjected to the following specific steps to obtain the target product. The details are as follows:
[0115] A2. Obtaining bacteria: After fermentation with E. coli BL21 and induction with IPTG, samples were taken for analysis. When a large number of product bands appeared, the fermentation liquid was centrifuged at more than 5000 rpn, the bacteria were collected and washed.
[0116] B2. Obtaining crude proinsulin: Take the washed bacterial cells, add lysis buffer to lyse them with the aid of ultrasonic treatment, centrifuge to remove bacterial fragments, and collect the crude proinsulin.
[0117] C2. Pre-treatment of enzymatic hydrolysis: Add guanidine hydrochloride for denaturation treatment, collect the denatured crude proinsulin by centrifugation, remove the denaturant by dialysis, and then concentrate the crude proinsulin to an appropriate concentration by reverse osmosis dehydration.
[0118] D2. Enzyme cleavage: Add trypsin to the crude proinsulin and incubate for 5 minutes to remove the C peptide. Then add dithiothreitol and incubate for 5 minutes to open the disulfide bonds.
[0119] E2. Separate the two chains A and B: Select molecular sieve Sephadex G-25 chromatography to collect and separate the A chain and B chain respectively.
[0120] F2. Selectively cleave the terminal basic amino acid of chain B. Add carboxypeptidase to the collected chain B and control the cleavage conditions to completely cut off the arginine at the C-terminus of chain B.
[0121] G2. Denaturation treatment of insulin. Combine the A chain and B chain of insulin, add appropriate urea and dithiothreitol, and keep warm for 5 minutes to denature the A chain and B chain of insulin. Select appropriate dialysis conditions to slowly dialyze away urea and dithiothreitol, which is conducive to the formation of correct disulfide bond pairing between the A chain and B chain of insulin.
[0122] H2. Separation and purification of mutants: After molecular sieve Sephadex G-25 chromatography, combined with sampling analysis, the peaks of insulin B chain mutants were collected and identified to obtain the target product.
[0123] Thus, long-acting insulin analog No. 2 was obtained.
[0124] The long-acting insulin analog No. 2 includes insulin A chain and insulin B chain mutants, and the amino acid sequences of insulin A chain and insulin B chain mutants (underlined mutation sites) are as follows:
[0125] Insulin A chain:
[0126] H 2 N-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-
[0127] Glu-Asn-Tyr-Cys-Asn-COOH (SEQ ID NO.7);
[0128] Insulin B chain mutants:
[0129] H 2 N-Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-
[0130] Leu-Val-Cys-Gly-Glu- Leu -Gly-Phe-Phe-Tyr-Thr-Pro- Ser -Thr-COOH (SEQ ID NO. 23).
[0131] Among them, the cysteine at position 6 of the insulin A chain is connected to the cysteine at position 11 of the insulin A chain in the form of a disulfide bond;
[0132] The cysteine at position 7 of the insulin A chain is linked to the cysteine at position 7 of the insulin B chain mutant in the form of a disulfide bond;
[0133] The cysteine at position 20 of the insulin A chain is linked to the cysteine at position 19 of the insulin B chain mutant in the form of a disulfide bond.
[0134] Example 3: Preparation of long-acting insulin analog No. 3.
[0135] 1. Primer Design
[0136] 1. Design mutation primer 3:
[0137] Mutation primer 3 was designed so that the arginine at position 22 of the insulin B chain was replaced by phenylalanine, and the lysine at position 29 was replaced by isoleucine. The sequence of mutation primer 3 is shown in SEQ ID NO.16.
[0138] SEQ ID NO.16: 5′-GAA TTT GGTTTCTTCTACACACCC ATT ACC-3'.
[0139] 2. PCR reaction
[0140] The PCR reaction process is the same as that in Example 1, except that the mutant primer 1 is replaced by the mutant primer 3.
[0141] The sequence of the target gene product amplified by PCR is shown in SEQ ID NO.17.
[0142] SEQ ID NO.17:
[0143] 5′-TTTGTCAATCAGCACCTTTGTGGTTCTCACCTGGTGGAGGCTCTGTACCTGGTGTGTGGGGAA TT T GGTTTCTTCTACACACCC ATT ACCCGTCGTGAAGCTGAAGACCTTCAAGTGGGTCAAGTTGAACTTGGTGGGGGTCCTGGTGAGGGTTCTCTTCAACCTTTGGCTCTCGAGGGATCACTTCAAAAGCGTGGCATTGTGGAGCAGTGCTGCACCAGCATCTGCTCCCTCTACCAACTGGAGAACTACTGCAACTGAG-3'.
[0144] 3. Expression and purification of long-acting insulin analog No. 3
[0145] 1. Inducible expression of long-acting insulin analog No. 3
[0146] The PCR amplification product shown in SEQ ID NO.17 was connected to the EcoRI and HindIII sites of the PBV220 vector to obtain a recombinant plasmid, which was then transformed into E. coli BL21 (DE3) to obtain a recombinant bacterium, which was induced to express with IPTG after shaking culture at 37 degrees Celsius for 16 hours to obtain the product shown in SEQ ID NO.3.
[0147] SEQ ID NO.3:
[0148] FVNQHLCGSHLVEALYLVCGEFGFFYTPITRREAEDLQVGQVELGGGPGEGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN.
[0149] 2. Separation and purification
[0150] The product shown in SEQ ID NO.3 can be separated and purified by conventional protein separation methods to obtain the target product. The expression product of insulin mutant 3 can be processed through the following specific steps to obtain the target product.
[0151] A3. Obtaining bacterial cells: After fermentation with E. coli BL21 and induction with IPTG, samples were taken for analysis. When a large number of product bands appeared, the fermentation liquid was centrifuged at more than 5000 rpn, the bacterial cells were collected, and washed.
[0152] B3. Obtaining crude proinsulin. Take the washed bacterial cells, add lysis solution and ultrasonic treatment, centrifuge to remove bacterial fragments, and collect crude proinsulin.
[0153] C3. Pretreatment of enzymatic hydrolysis. Add guanidine hydrochloride for denaturation treatment, collect the denatured crude proinsulin by centrifugation. Remove the denaturant by dialysis, and then use reverse osmosis dehydration to concentrate the crude proinsulin to an appropriate concentration.
[0154] D3. Enzyme cleavage. Trypsin was added to the crude proinsulin and incubated for 5 minutes to remove the C peptide. Dithiothreitol was then added and incubated for 5 minutes to open the disulfide bonds.
[0155] E3. Separate the two chains A and B. Select molecular sieve dextran gel Sephadex G-25 chromatography to collect and separate the A chain and B chain respectively.
[0156] F3. Selectively cleave the terminal basic amino acid of chain B. Add carboxypeptidase to the collected chain B and control the cleavage conditions to completely cut off the arginine at the C-terminus of chain B.
[0157] G3. Denaturation of insulin. Combine the A and B chains of insulin, add appropriate urea and dithiothreitol, and keep warm for 5 minutes to denature the A and B chains of insulin. Select appropriate dialysis conditions to slowly dialyze away urea and dithiothreitol, which is conducive to the formation of correct disulfide bond pairing between the A and B chains of insulin.
[0158] H3. Separation and purification of mutants. Then, the peaks of insulin B chain mutants were collected through molecular sieve Sephadex G-25 chromatography combined with sampling analysis, and the target product was obtained after identification.
[0159] At this point, long-acting insulin analog No. 3 was obtained.
[0160] Then the long-acting insulin analog No. 3 is obtained by separation and purification.
[0161] The long-acting insulin analog No. 3 includes insulin A chain and insulin B chain mutants, and the amino acid sequences of the insulin A chain and insulin B chain mutants are as follows:
[0162] Insulin A chain:
[0163] H 2 N-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-
[0164] Glu-Asn-Tyr-Cys-Asn-COOH (SEQ ID NO.7);
[0165] Insulin B chain mutants:
[0166] H 2 N-Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-
[0167] Leu-Val-Cys-Gly-Glu- Phe -Gly-Phe-Phe-Tyr-Thr-Pro- Ile -Thr-COOH (SEQ ID NO. 24).
[0168] Among them, the cysteine at position 6 of the insulin A chain is connected to the cysteine at position 11 of the insulin A chain in the form of a disulfide bond;
[0169] The cysteine at position 7 of the insulin A chain is linked to the cysteine at position 7 of the insulin B chain mutant in the form of a disulfide bond;
[0170] The cysteine at position 20 of the insulin A chain is linked to the cysteine at position 19 of the insulin B chain mutant in the form of a disulfide bond.
[0171] Example 4: Preparation of long-acting insulin analog No. 4.
[0172] 1. Primer Design
[0173] 1. Design mutation primer 4:
[0174] Mutation primer 4 was designed so that the arginine at position 22 of the insulin B chain was replaced by asparagine, and the lysine at position 29 was replaced by alanine. The sequence of mutation primer 4 is shown in SEQ ID NO.18.
[0175] SEQ ID NO.18: 5′-GAA AAT GGTTTCTTCTACACACCC GCT ACC-3'.
[0176] 2. PCR reaction
[0177] The PCR reaction process is the same as that in Example 1, except that the mutant primer 1 is replaced by the mutant primer 4.
[0178] The sequence of the target gene product amplified by PCR is shown in SEQ ID NO.19.
[0179] SEQ ID NO.19:
[0180] 5′-TTTGTCAATCAGCACCTTTGTGGTTCTCACCTGGTGGAGGCTCTGTACCTGGTGTGTGGGGAA AA T GGTTTCTTCTACACACCC GCT ACCCGTCGTGAAGCTGAAGACCTTCAAGTGGGTCAAGTTGAACTTGGTGGGGGTCCTGGT GAG GGTTCTCTTCAACCTTTGGCTCTCGAGGGATCACTTCAAAAGCGTGGCATTGTGGAGCAGTGCTGCACCAGCATCTGCTCCCTCTACCAACTGGAGAACTACTGCAACTGAG-3'.
[0181] 3. Expression and purification of long-acting insulin analog No. 4
[0182] 1. Inducible expression of long-acting insulin analog No. 4
[0183] The PCR amplification product shown in SEQ ID NO.19 was connected to the EcoRI and HindIII sites of the PBV220 vector to obtain a recombinant plasmid, which was then transformed into E. coli BL21 (DE3) to obtain a recombinant bacterium, which was induced for expression with IPTG after shaking culture at 37 degrees Celsius for 16 hours to obtain the product shown in SEQ ID NO.4.
[0184] SEQ ID NO.4:
[0185] FVNQHLCGSHLVEALYLVCGENGFFYTPATRREAEDLQVGQVELGGGPGEGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN.
[0186] 2. Separation and purification
[0187] The product shown in SEQ ID NO. 4 can be separated and purified by conventional protein separation methods to obtain the target product. The expression product of insulin mutant 4 can be processed through the following specific steps to obtain the target product.
[0188] A4. Obtaining bacterial cells: After fermentation with E. coli BL21 and induction with IPTG, samples were taken for analysis. When a large number of product bands appeared, the fermentation liquid was centrifuged at more than 5000 rpn, the bacterial cells were collected, and washed.
[0189] B4. Obtaining crude proinsulin. Take the washed bacterial cells, add lysis solution and ultrasonic treatment, centrifuge to remove bacterial fragments, and collect crude proinsulin.
[0190] C4. Pretreatment of enzymatic hydrolysis. Add guanidine hydrochloride for denaturation treatment, collect the denatured crude proinsulin by centrifugation. Remove the denaturant by dialysis, and then use reverse osmosis dehydration to concentrate the crude proinsulin to an appropriate concentration.
[0191] D4. Enzyme cleavage. Trypsin was added to the crude proinsulin and incubated for 5 minutes to remove the C peptide. Dithiothreitol was then added and incubated for 5 minutes to open the disulfide bonds.
[0192] E4. Separate the two chains A and B. Select molecular sieve Sephadex G-25 chromatography to collect and separate the A chain and B chain respectively.
[0193] F4. Selectively cleave the terminal basic amino acid of chain B. Add carboxypeptidase to the collected chain B and control the cleavage conditions to completely cut off the arginine at the C-terminus of chain B.
[0194] G4. Denaturation treatment of insulin. Combine the A chain and B chain of insulin, add appropriate urea and dithiothreitol, and keep warm for 5 minutes to denature the A chain and B chain of insulin. Select appropriate dialysis conditions to facilitate the formation of correct disulfide bond pairing between the A chain and B chain of insulin while slowly dialysing to remove urea and dithiothreitol.
[0195] H4. Separation and purification of mutants. Then, the peaks of insulin B chain mutants were collected through molecular sieve Sephadex G-25 chromatography and combined with sampling analysis. After identification, the target product was obtained.
[0196] At this point, long-acting insulin analog No. 4 was obtained.
[0197] The long-acting insulin analog No. 4 includes insulin A chain and insulin B chain mutants, and the amino acid sequences of the insulin A chain and insulin B chain mutants are as follows:
[0198] Insulin A chain:
[0199] H 2 N-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-
[0200] Glu-Asn-Tyr-Cys-Asn-COOH (SEQ ID NO.7);
[0201] Insulin B chain mutants:
[0202] H 2 N-Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu- Asp -Gly-Phe-Phe-Tyr-Thr-Pro- Ala -Thr-COOH (SEQ ID NO. 25).
[0203] Among them, the cysteine at position 6 of the insulin A chain is connected to the cysteine at position 11 of the insulin A chain in the form of a disulfide bond;
[0204] The cysteine at position 7 of the insulin A chain is linked to the cysteine at position 7 of the insulin B chain mutant in the form of a disulfide bond;
[0205] The cysteine at position 20 of the insulin A chain is linked to the cysteine at position 19 of the insulin B chain mutant in the form of a disulfide bond.
[0206] Example 5: Preparation of long-acting insulin analog No. 5.
[0207] 1. Primer Design
[0208] 1. Design mutation primer 5:
[0209] Mutation primer 5 was designed so that the arginine at position 22 of the insulin B chain was replaced by threonine, and the lysine at position 29 was replaced by isoleucine. The sequence of mutation primer 5 is shown in SEQ ID NO.20.
[0210] SEQ ID NO.20: 5′-GAA ACT GGTTTCTTCTACACACCC ATT ACC-3'.
[0211] 2. PCR reaction
[0212] The PCR reaction process is the same as that in Example 1, except that mutant primer 1 is replaced by mutant primer 5.
[0213] The sequence of the target gene product amplified by PCR is shown in SEQ ID NO.21.
[0214] SEQ ID NO.21:
[0215] 5′-TTTGTCAATCAGCACCTTTGTGGTTCTCACCTGGTGGAGGCTCTGTACCTGGTGTGTGGGGAA AC T GGTTTCTTCTACACACCC ATT ACCCGTCGTGAAGCTGAAGACCTTCAAGTGGGTCAAGTTGAACTTGGTGGGGGTCCTGGTGAGGGTTCTCTTCAACCTTTGGCTCTCGAGGGATCACTTCAAAAGCGTGGCATTGTGGAGCAGTGCTGCACCAGCATCTGCTCCCTCTACCAACTGGAGAACTACTGCAACTGAG-3'.
[0216] 3. Expression and purification of long-acting insulin analog No. 5
[0217] 1. Inducible expression of long-acting insulin analog No. 5
[0218] The PCR amplification product shown in SEQ ID NO.21 was connected to the EcoRI and HindIII sites of the PBV220 vector to obtain a recombinant plasmid, which was then transformed into E. coli BL21 (DE3) to obtain a recombinant bacterium, which was induced to express with IPTG after shaking culture at 37 degrees Celsius for 16 hours to obtain the product shown in SEQ ID NO.5.
[0219] SEQ ID NO.5:
[0220] FVNQHLCGSHLVEALYLVCGETGFFYTPITRREAEDLQVGQVELGGGPGEGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN.
[0221] 2. Separation and purification of long-acting insulin analog No. 5
[0222] The product shown in SEQ ID NO.5 can be separated and purified by conventional protein separation methods to obtain the target product. The expression product of insulin mutant 5 can be processed by the following specific steps to obtain the target product. The details are as follows:
[0223] A5. Obtaining bacterial cells: After fermentation with E. coli BL21 and induction with IPTG, samples were taken for analysis. When a large number of product bands appeared, the fermentation liquid was centrifuged at more than 5000 rpn, the bacterial cells were collected, and washed.
[0224] B5. Obtaining crude proinsulin. Take the washed bacterial cells, add lysis solution and ultrasonic treatment, centrifuge to remove bacterial fragments, and collect crude proinsulin.
[0225] C5. Pretreatment of enzymatic hydrolysis. Add guanidine hydrochloride for denaturation treatment, collect the denatured crude proinsulin by centrifugation. Remove the denaturant by dialysis, and then use reverse osmosis dehydration to concentrate the crude proinsulin to an appropriate concentration.
[0226] D5. Enzyme cleavage. Trypsin was added to the crude proinsulin and incubated for 5 minutes to remove the C peptide. Dithiothreitol was then added and incubated for 5 minutes to open the disulfide bonds.
[0227] E5. Separate the two chains A and B. Select molecular sieve Sephadex G-25 chromatography to collect and separate the A chain and B chain respectively.
[0228] F5. Selectively cleave the terminal basic amino acid of chain B. Add carboxypeptidase to the collected chain B and control the cleavage conditions to completely cut off the arginine at the C-terminus of chain B.
[0229] G5. Denaturation treatment of insulin. Combine the A chain and B chain of insulin, add appropriate urea and dithiothreitol, and keep warm for 5 minutes to achieve denaturation of both the A chain and the B chain of insulin. Select appropriate dialysis conditions to facilitate the formation of correct disulfide bond pairing between the A chain and the B chain of insulin while slowly dialysing to remove urea and dithiothreitol.
[0230] H5. Separation and purification of mutants. Then, the peaks of insulin B chain mutants were collected through molecular sieve Sephadex G-25 chromatography combined with sampling analysis, and the target product was obtained after identification.
[0231] At this point, long-acting insulin analog No. 5 was obtained.
[0232] The long-acting insulin analog No. 5 includes insulin A chain and insulin B chain mutants, and the amino acid sequences of the insulin A chain and insulin B chain mutants are as follows:
[0233] Insulin A chain:
[0234] H 2 N-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-
[0235] Glu-Asn-Tyr-Cys-Asn-COOH (SEQ ID NO.7);
[0236] Insulin B chain mutants:
[0237] H 2 N-Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-
[0238] Leu-Val-Cys-Gly-Glu- Thr -Gly-Phe-Phe-Tyr-Thr-Pro- Ile -Thr-COOH (SEQ ID NO. 26).
[0239] Among them, the cysteine at position 6 of the insulin A chain is connected to the cysteine at position 11 of the insulin A chain in the form of a disulfide bond;
[0240] The cysteine at position 7 of the insulin A chain is linked to the cysteine at position 7 of the insulin B chain mutant in the form of a disulfide bond;
[0241] The cysteine at position 20 of the insulin A chain is linked to the cysteine at position 19 of the insulin B chain mutant in the form of a disulfide bond.
[0242] 1. PCR product identification
[0243] The above Examples 1 to 5 were subjected to two rounds of PCR biosynthesis to obtain the DNA fragment of insulin, i.e., the PCR amplification product (target gene product) obtained in the second round of PCR. After electrophoresis analysis, the results were as follows: Figure 2 to Figure 6 As shown, combined with the sequencing results, it can be seen that the PCR amplification product was successfully identified.
[0244] 2. In vitro long-term efficacy verification experiment
[0245] Take the natural insulin molecule and the long-acting insulin analog prepared in Examples 1 to 5 and keep them warm with trypsin, take samples every 3 minutes, and measure the residual amount of the long-acting insulin analog molecule to determine the long-term activity of the long-acting insulin analog molecule. The specific steps are as follows:
[0246] (1) Experimental setup
[0247] Take the natural insulin molecule and use ddH 2 O prepared 2 mL of a solution with a concentration of 1.0 mg / mL and divided it into two groups, a control group and a treatment group. The natural insulin molecule control group was recorded as N1, and the natural insulin molecule treatment group was recorded as N2.
[0248] The long-acting insulin analogs prepared in Examples 1 to 5 were each treated with ddH 2 O prepared 2 mL of a solution with a concentration of 1.0 mg / mL and divided it into a control group and a treatment group.
[0249] The control group of the long-acting insulin analog No. 1 prepared in Example 1 is recorded as CK1, and the long-acting insulin analog No. 1 treatment group is recorded as T1; the control group of the long-acting insulin analog No. 2 prepared in Example 2 is recorded as CK2, and the long-acting insulin analog No. 2 treatment group is recorded as T2; the control group of the long-acting insulin analog No. 3 prepared in Example 3 is recorded as CK3, and the long-acting insulin analog No. 3 treatment group is recorded as T3; the control group of the long-acting insulin analog No. 4 prepared in Example 4 is recorded as CK4, and the long-acting insulin analog No. 4 treatment group is recorded as T4; the control group of the long-acting insulin analog No. 5 prepared in Example 5 is recorded as CK5, and the long-acting insulin analog No. 5 treatment group is recorded as T5;
[0250] The control group was added with 0.5 mL ddH 2 O and 0.5mL 50mmol / L Tris-HCl pH8.0 buffer solution; 0.5mL 1mg / mL trypsin solution and 0.5mL 50mmol / L Tris-HCl pH8.0 buffer solution were added to the treatment group for enzyme degradation treatment.
[0251] The control group and the treatment group were shaken separately, and then kept in a 37°C water bath for reaction. 100 μL of the sample was taken every 2 minutes, 900 μL of 8 mol / L urea solution was added, and the mixture was rapidly shaken (denaturation treatment) and then quickly stored at -20°C.
[0252] The contents of insulin molecules in the samples to be tested were detected respectively. The results are shown in Table 2.
[0253] Table 2 Insulin or insulin analog content at different holding times (%)
[0254]
[0255]
[0256] The analysis of the insulin content in Table 2 shows that compared with natural insulin, the long-acting insulin analogs prepared in Examples 1 to 5 have better resistance to degradation under the action of trypsin, and the integrity of the molecular structure of the long-acting insulin analogs prepared in Examples 1 to 5 is better maintained, thereby achieving the purpose of long-term effect.
[0257] The analysis results show that when the long-acting insulin analog prepared by the present invention is incubated with trypsin, the half-life of the mutant insulin is extended by at least 88 / 12=7 times, calculated as more than 5 times, compared with the half-life of the insulin molecule under the same measurement environment. The half-life extension time of different mutants is different. The experimental data show that the life of the insulin mutant is extended by at least 5 times that of the insulin molecule, which reduces the degradation rate of insulin in the body and prolongs the effective action time of the insulin molecule in the body, thereby achieving the purpose of long-term effect.
[0258] Studies have shown that insulin analog derivatives obtained by replacing one or more amino acids in the A chain and B chain of natural insulin and combining changes in the structural domain have a significantly increased half-life in vivo, thereby achieving a long-acting effect.
[0259] 3. Structural analysis of the insulin analog dimer obtained after mutation of the present invention
[0260] Analyze the mathematical model of the long-acting insulin analog No. 1 prepared in Example 1 and the natural insulin dimer structure;
[0261] The results are as follows Figure 7 As shown, the design of long-acting insulin analog No. 1 achieves the purpose of changing the structure of the surface of its spatial structure without destroying the helical structure and domain inside the insulin molecule, and changes the spatial structure of the amino acid at the terminal of the B chain. While ensuring the elimination of trypsin hydrolysis sites to achieve the purpose of long-term effect, it also better eliminates the formation of insulin molecule dimers, thereby increasing the number of monomeric insulin molecules, and then playing a corresponding rapid-acting role.
[0262] The present invention mutates two basic amino acids at specific sites in the insulin molecule at the same time, further reducing the sensitivity of the insulin molecule to enzymes, especially trypsin, and making its long-acting effect more obvious.
[0263] The two amino acid mutation sites selected by the present invention are both located in the surface part of the molecule in the spatial structure of insulin, and have little effect on the spatial structure of the insulin molecule, and do not have much effect on the original activity of the insulin molecule. From the data analysis of the spatial structure, the mutant structure of the present invention has a good effect on the polymerization of the insulin molecule itself, thereby playing a certain quick-acting effect at the same time.
[0264] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.
[0265] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.
Claims
1. A long-acting insulin analog, It is characterized in that The long-acting insulin analogs include insulin A chain and insulin B chain mutants; The insulin B chain mutant is obtained by mutation of the insulin B chain shown in SEQ ID NO.8, and the mutation sites are that the 22nd arginine and the 29th lysine on the insulin B chain are replaced by non-basic amino acids; The non-basic amino acid is any one of glycine, alanine, valine, leucine, isoleucine, serine, threonine, tyrosine, methionine, phenylalanine, aspartic acid, asparagine, glutamic acid and glutamine.
2. The long-acting insulin analog according to claim 1, It is characterized in that The amino acid sequence of the insulin A chain is shown in SEQ ID NO.
7.
3. The long-acting insulin analog according to claim 2, It is characterized in that The connection relationship between the insulin A chain and the insulin B chain mutant is as follows: the cysteine at the 6th position of the insulin A chain is connected to the cysteine at the 11th position of the insulin A chain in the form of a disulfide bond; the cysteine at the 7th position of the insulin A chain is connected to the cysteine at the 7th position of the insulin B chain mutant in the form of a disulfide bond; the cysteine at the 20th position of the insulin A chain is connected to the cysteine at the 19th position of the insulin B chain mutant in the form of a disulfide bond.
4. A method for preparing the long-acting insulin analogue according to any one of claims 1 to 3, It is characterized in that The steps include: The PCR primers and mutation primers were designed using the human insulin gene sequence shown in SEQ ID NO.6 as a template, wherein the PCR primers included a forward primer F shown in SEQ ID NO.10 and a reverse primer R shown in SEQ ID NO.11; The first round of PCR reaction was carried out using forward primer F and mutant primer as primers and the insulin gene shown in SEQ ID NO.6 as a template; After the first round of PCR reaction, reverse primer R is added to the reaction system for a second round of PCR reaction; The second round of PCR amplification products are connected to the vector to construct a recombinant plasmid, and the products are obtained by transformation and induction of expression, and then enzymatic hydrolysis, separation and purification are performed to obtain long-acting islet analogs; The mutation primer contains a mutation site.
5. The method for preparing the long-acting insulin analog according to claim 4, It is characterized in that When the mutation site is that the arginine at position 22 on the insulin B chain is replaced by alanine, and the lysine at position 29 on the insulin B chain is replaced by valine, the sequence of the mutation primer is as shown in SEQ ID NO.12; When the mutation site is that the arginine at position 22 on the insulin B chain is replaced by leucine, and the lysine at position 29 on the insulin B chain is replaced by serine, the sequence of the mutant primer is as shown in SEQ ID NO.14; When the mutation site is that the arginine at position 22 on the insulin B chain is replaced by phenylalanine, and the lysine at position 29 on the insulin B chain is replaced by isoleucine, the sequence of the mutation primer is as shown in SEQ ID NO.16; When the mutation site is that the arginine at position 22 on the insulin B chain is replaced by asparagine, and the lysine at position 29 on the insulin B chain is replaced by alanine, the sequence of the mutant primer is as shown in SEQ ID NO.18; When the mutation site is that the arginine at position 22 on the insulin B chain is replaced by threonine, and the lysine at position 29 on the insulin B chain is replaced by isoleucine, the sequence of the mutant primer is as shown in SEQ ID NO.
20.
6. The method for preparing the long-acting insulin analog according to claim 4, It is characterized in that The first round of PCR reaction system was: forward primer F 25pmol 2μL, mutant primer 1, 50pmol 1μL, template DNA 10ng 0.5μL, dNTP 200μmol, 10×PCR buffer 5μL, ddH 2 O 40.5 μL.
7. The method for preparing the long-acting insulin analog according to claim 4, It is characterized in that The reaction conditions of the first round of PCR reaction were: denaturation at 94°C for 60 s, annealing at 56°C for 60 s, extension at 72°C for 60 s, amplification for 30 cycles, and the last cycle was 72°C for 10 min.
8. The method for preparing the long-acting insulin analog according to claim 4, It is characterized in that The reaction conditions for the second round of PCR were denaturation at 94°C for 60 s, annealing for 60 s, extension at 72°C for 60 s, amplification for 30 cycles, and the last cycle was at 72°C for 10 min.
9. Use of the insulin B chain mutant according to claim 1 or the long-acting insulin analogue according to any one of claims 1 to 3 in the preparation of insulin drugs.
10. The use according to claim 9, It is characterized in that The insulin drug has the long-acting insulin analog as the only effective ingredient.