Preparation method of Jingzhaotoxin polypeptide XI and application of Jingzhaotoxin polypeptide XI in treatment of diabetes

Abstract

The invention belongs to the technical field of polypeptide preparation and biological medicine, and particularly relates to a preparation method of Jingzhaotoxin polypeptide XI (JZTX-XI) and application of Jingzhaotoxin polypeptide XI (JZTX-XI) in treatment of diabetes mellitus. A Jingzhaotoxin polypeptide XI coding gene, a nucleotide sequence of a 6 * His tag, a nucleotide sequence of a thrombin restriction enzyme cutting site and a nucleotide sequence of an MBP hydrotropy tag are connected to a prokaryotic expression vector for prokaryotic expression, and a target polypeptide which is high in purity, complete in disulfide bond folding and uniform in conformation is obtained through purification. The purity and the biological activity are relatively high. Meanwhile, the treatment activity of the JZTX-XI in the aspect of diabetes is verified, and it is found that the JZTX-XI can remarkably reduce fasting blood glucose of mice with type II diabetes, remarkably up-regulate a glucose transport regulation gene Glut2, improve glucose tolerance and insulin sensitivity, relieve polydipsia and polyuria symptoms and promote insulin secretion and islet cell proliferation.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide preparation and biomedical technology, specifically relating to the preparation method of Jingzhao spider toxin polypeptide XI and its application in the treatment of diabetes. Background Technology

[0002] Spider venom-derived peptides are considered a potential source of novel therapeutic molecules due to their structural stability, high target selectivity, and well-defined pharmacological effects. However, natural availability is limited, and chemical synthesis followed by oxidative folding is costly and scale-up production is difficult. Recombinant expression strategies can improve availability; common approaches include using large tags such as MBP to enhance solubility and cleaving with specific proteases such as Factor Xa to obtain mature peptides. However, existing research to the inventors' knowledge lacks low-cost, scalable, and controllable preparation processes, making it difficult to guarantee their biological activity. Therefore, a systematic study and evaluation of the preparation process, folding uniformity, and the relationship between yield and efficacy of venom peptides is needed.

[0003] Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia, which can lead to complications such as neuropathy, nephropathy, and cardiovascular damage in the long term. While existing hypoglycemic drugs can control blood sugar, they struggle to simultaneously protect pancreatic β-cells, delay the progression of complications, and ensure targeted safety. For many years, peptide toxins, as selective ligands for ion channels and other receptors, have attracted considerable interest from researchers. In fact, some venom-derived peptides have already been used to treat hypertension, neuropathic pain, and other diseases, demonstrating potential in clinical trials. Compared to small molecule drugs, peptide drugs have stronger activity and better selectivity, which is beneficial for achieving precise and targeted therapy. Compared to protein drugs, peptide drugs have advantages such as better stability, higher purity, lower production costs, and lower immunogenicity. Drug quality is easier to control, and drug properties can be optimized through chemical modification.

[0004] Jingzhao's hairy spider venom polypeptide XI (JZTX-XI) is derived from Jingzhao's hairy spider ( Chilobrachys jingzhao JZTX-XI is a novel neurotoxin isolated and purified from venom. It possesses a typical ICK motif structure, consisting of 34 amino acid residues, including six cysteine ​​residues forming three disulfide bonds, and exhibiting C-terminal amidation modification. Structural and functional studies of JZTX-XI are of significant research value in the fields of cardiac electrophysiology and neurological diseases. However, the preparation process of JZTX-XI also faces the challenges of preparing spider venom peptides as described above. Therefore, it is necessary to establish a controllable expression system and standardized purification process suitable for peptides with multiple disulfide bonds, such as JZTX-XI, to obtain correctly folded and biologically active target peptides and to develop and validate their pharmacological activity. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing the *Jingzhao* spider venom polypeptide XI and its application in the treatment of diabetes. The method provides a method for preparing *Jingzhao* spider venom polypeptide XI with high purity and ensures its biological activity, while also exploring its medicinal activity in the treatment of diabetes.

[0006] This invention provides a method for preparing the venom polypeptide XI of the spider tufted spider, comprising the following steps: The gene encoding the XI polypeptide from the spider venom of Jingzhao, the nucleotide sequence of the 6×His tag, the nucleotide sequence of the thrombin restriction site, and the nucleotide sequence of the MBP solubilizing tag were ligated into a prokaryotic expression vector for prokaryotic expression, resulting in a highly soluble fusion protein. The highly soluble fusion protein was purified to obtain the Jingzhao taeniatus spider toxin polypeptide XI. The amino acid sequence of the Jingzhao taeniacus spider toxin polypeptide XI is shown in SEQ ID NO:2.

[0007] Preferably, the prokaryotic expression step includes: The expression module was linked to a plasmid vector containing an MBP solubilizing tag to obtain a recombinant vector; The recombinant vector was transformed into Escherichia coli to obtain recombinant engineered bacteria; The recombinant engineered bacteria were induced to express a highly soluble fusion protein. The expression module consists of, from the 5' end to the 3' end, a nucleotide sequence of a 6×His tag, a nucleotide sequence of a thrombin restriction site, and the gene encoding the Jingzhao taeniacus toxin polypeptide XI.

[0008] Preferably, the plasmid vector includes the pMAL-p5x vector.

[0009] Preferably, the expression module containing the gene encoding the XI toxin polypeptide from the spider venom of the genus *Ceratophyllum demersum* is inserted between the BamHI and EcoRI restriction sites of the pMAL-p5x vector.

[0010] Preferably, the purification step includes: The highly soluble fusion protein was initially purified by Ni-NTA affinity chromatography to obtain the preliminarily purified fusion protein. The pre-purified fusion protein was digested with thrombin, and the digested protein was separated by molecular sieve chromatography. The protein separated by the separation sieve chromatography was purified by reversed-phase high-performance liquid chromatography to obtain the Jingzhao taenia toxin polypeptide XI.

[0011] The present invention also provides the application of the above-mentioned technical solution, Jingzhao taeniatus spider toxin polypeptide XI, in the preparation of a drug for treating diabetes; the amino acid sequence of the Jingzhao taeniatus spider toxin polypeptide XI is shown in SEQ ID NO:2.

[0012] Preferably, the *Jingzhao* spider toxin polypeptide XI is the *Jingzhao* spider toxin polypeptide XI prepared by the preparation method described in the above technical solution.

[0013] Preferably, the diabetes includes type 2 diabetes.

[0014] Preferably, the application includes one or more of the following: 1) Promotes insulin secretion and / or promotes insulin cell proliferation; 2) Improve glucose tolerance and / or insulin sensitivity; 3) Lowers blood sugar; 4) Relieve symptoms of excessive thirst and / or excessive urination.

[0015] The present invention also provides a drug for treating diabetes, the active ingredient of which includes the jingzhao taenia velutipes toxin polypeptide XI prepared by the preparation method described in the above technical solution.

[0016] Beneficial effects: This invention involves ligating the encoding gene of the JZTX-XI (a type of spider venom peptide) from the spider (JZTX) into a prokaryotic expression vector, along with the nucleotide sequences of the 6×His tag, the thrombin cleavage site, and the MBP solubilizing tag. The expressed peptide is then purified, successfully yielding a high-purity peptide with intact disulfide bonds and a homogeneous conformation, exhibiting high purity and bioactivity. Furthermore, this invention retains the large MBP tag as a solubilizing tag to improve solubility. The method of purification based on the small affinity tag 6×His and cleavage using the thrombin cleavage site to obtain the mature peptide offers advantages in cost control compared to existing recombinant expression methods for preparing JZTX-XI.

[0017] Based on this, the present invention discovered and verified the therapeutic activity of JZTX-XI in diabetes, especially type II diabetes. Animal experiments verified that JZTX-XI can significantly reduce fasting blood glucose in type II diabetic mice and significantly upregulate glucose transport regulatory genes. Glut2 It improves glucose tolerance and insulin sensitivity, relieves symptoms of polydipsia and polyuria, and promotes insulin secretion and pancreatic islet cell proliferation. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 This is a map of the recombinant expression plasmid in Example 1; Figure 2 The images show SDS-PAGE analysis results of samples from each purification step in Example 1. Figure 3 This is the mass spectrometry analysis chromatogram of the final purified sample JZTX-XI in Example 1; Figure 4 This is the HPLC chromatogram of the final purified sample JZTX-XI from Example 1; Figure 5 The amino acid sequence determination results of JZTX-XI in Example 1; Figure 6 This is a statistical graph showing the body weight and fasting blood glucose levels of mice in each group in Example 2; Figure 7 This is a statistical chart showing the food intake, water consumption, and urine output of mice in each group in Example 2; Figure 8 The graph shows the OGTT and ITT results of each group of mice in Example 2; Figure 9 The graph shows the H&E results of the pancreas in each group of mice in Example 2; Figure 10 The graph shows the results of serum insulin, C-peptide, and glycated hemoglobin in each group of mice in Example 2; Figure 11 This is a graph showing the mRNA expression results of insulin secretion-related genes in each group of mice in Example 2; Figure 12 The images show the immunofluorescence results of insulin and Ki67 in the pancreatic tissues of mice in each group in Example 2. Figure 13 The image shows the immunohistochemical results of cleaved-caspase 3 in the pancreatic tissues of each group of mice in Example 2. Detailed Implementation

[0020] This invention provides a method for preparing the venom polypeptide XI of the spider tufted spider, comprising the following steps: The gene encoding the XI polypeptide from the spider venom of Jingzhao, the nucleotide sequence of the 6×His tag, the nucleotide sequence of the thrombin restriction site, and the nucleotide sequence of the MBP solubilizing tag were ligated into a prokaryotic expression vector for prokaryotic expression, resulting in a highly soluble fusion protein. The highly soluble fusion protein was purified to obtain the Jingzhao taeniatus spider toxin polypeptide XI. The amino acid sequence of the Jingzhao taeniacus spider toxin polypeptide XI is shown in SEQ ID NO:2.

[0021] In one embodiment, the nucleotide sequence of the gene encoding the *Jingzhao* spider venom polypeptide XI is shown in SEQ ID NO:1, specifically: GAATGTCGCAAAATGTTCGGTGGTTGCAGCGTGGATAGCGATTGTTGTGCTCATCTGGGCTGCAAGCCGACTCTGAAGTACTGCGCTTGGGATGGTACCTTCGGCAAG. In another embodiment, the nucleotide sequence of the 6×His tag is shown in SEQ ID NO:3, specifically: catcatcatcatcatca. In another embodiment, the nucleotide sequence of the thrombin restriction site is shown in SEQ ID NO:4, specifically: ctggtgccacgcggttct. In another embodiment, the nucleotide sequence of the MBP solubilizing tag is shown in the italicized underlined portion of SEQ ID NO:5.

[0022] In one embodiment, the prokaryotic expression steps of the present invention include: ligating an expression module to a plasmid vector containing an MBP solubilizing tag to obtain a recombinant vector; transforming the recombinant vector into *E. coli* to obtain recombinant engineered bacteria; and inducing expression in the recombinant engineered bacteria to obtain a highly soluble fusion protein. The expression module, from its 5' end to its 3' end, consists of a nucleotide sequence of a 6×His tag, a nucleotide sequence of a thrombin restriction site, and the gene encoding the *Jingzhao* spider toxin polypeptide XI. In one embodiment, the plasmid vector is a pMAL-p5x vector; when the plasmid vector is a pMAL-p5x vector, the expression module containing the *Jingzhao* spider toxin polypeptide XI gene is inserted between the BamHI and EcoRI restriction sites of the pMAL-p5x vector, and the resulting recombinant vector map is shown below. Figure 1 As shown. In one embodiment, the induced expression step includes: culturing the recombinant engineered bacteria under IPTG induction to obtain a highly soluble fusion protein. In one embodiment, the concentration of IPTG is 0.1~1 mM, or 0.1 mM; the culture temperature is 14~20 ℃, or 16 ℃; the culture time is 16~20 h, or 20 h.

[0023] As one embodiment, the purification step of the present invention includes: performing preliminary purification of the highly soluble fusion protein by Ni-NTA affinity chromatography to obtain the preliminarily purified fusion protein; The pre-purified fusion protein was digested with thrombin, and the digested protein was separated by molecular sieve chromatography. The protein separated by the separation sieve chromatography was purified by reversed-phase high-performance liquid chromatography to obtain the Jingzhao taenia toxin polypeptide XI.

[0024] In one embodiment, the molecular sieve chromatography separation is size exclusion chromatography. In another embodiment, the chromatographic column of the reversed-phase high-performance liquid chromatography is an octadecylsilane-bonded silica gel column (C18 column).

[0025] This invention also provides the application of the above-mentioned technical solution, Jingzhao trichotillomania toxin polypeptide XI, in the preparation of a drug for treating diabetes; the amino acid sequence of the Jingzhao trichotillomania toxin polypeptide XI is shown in SEQ ID NO:2. As one embodiment, the Jingzhao trichotillomania toxin polypeptide XI of this invention is the Jingzhao trichotillomania toxin polypeptide XI prepared by the preparation method described in the above technical solution.

[0026] In one implementation, the diabetes includes type 2 diabetes. In one implementation, the application includes one or more of the following: 1) promoting insulin secretion and / or promoting insulin cell proliferation; 2) improving glucose tolerance and / or insulin sensitivity; 3) lowering blood glucose; 4) relieving polydipsia and / or polyuria. In one implementation, the blood glucose lowering is lowering fasting blood glucose.

[0027] In this invention, the effect of JZTX-XI in treating type 2 diabetes was evaluated using a BKS-db / db mouse model of type 2 diabetes. During administration, mouse body weight, fasting blood glucose, insulin and glucose tolerance levels, food intake, water intake, and urine output were recorded. After sacrifice, serum and pancreatic tissue were collected. H&E staining was used to observe changes in the number and morphology of islets in the pancreatic tissue. Serum insulin, C-peptide, and glycated hemoglobin (GHb) levels were measured using ELISA. Real-time quantitative PCR was used to detect genes related to insulin secretion. Glut2 , Ins1 , Ins2 , Lapp , Pdx1 , Sox9 , Ngn3 The expression of mRNA in the peptides (e.g., insulin secretion, islet cell apoptosis and proliferation) was detected using immunofluorescence and immunohistochemistry. The effects of the peptides on hypoglycemia, promoting insulin secretion and protecting islet cells were comprehensively evaluated. It was found that JZTX-XI can significantly reduce fasting blood glucose, improve glucose tolerance and insulin sensitivity, alleviate polydipsia and polyuria symptoms, and promote insulin secretion and islet cell proliferation in type II diabetic mice.

[0028] Meanwhile, this invention discovered that JZTX-XI significantly upregulates glucose transport regulatory genes. Glut2The mRNA expression of JZTX-XI was reduced, while the structurally similar spider toxin polypeptide JZTX-IX remained unchanged, thus demonstrating the specificity of JZTX-XI in its hypoglycemic mechanism.

[0029] This invention also provides a drug for treating diabetes, the active ingredient of which includes the *Jingzhao* spider venom polypeptide XI prepared by the preparation method described above. This invention does not specifically limit the content and type of excipients in the drug; conventional limitations are sufficient as needed.

[0030] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0031] Experimental materials used in the following examples: BL21(DE3)pLysS competent cells were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0032] Male BKS-db / db (4-week-old) mice, SPF grade, were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0033] The statistical methods used in the following examples: Gray-scale analysis was performed using ImageJ software, and data processing was conducted using GraphPad Prism 7.0 software. Data results are presented as mean ± standard deviation (mean ± SD). One-way ANOVA was used for statistical analysis and testing. p <0.05 ( ) are considered to have statistical differences. p <0.01 ( These are considered to have statistically significant differences. p <0.001 ( The difference is considered to be statistically significant.

[0034] Example 1 The expression and purification experiments of Jingzhao's spider toxin polypeptide XI were conducted as follows: The toxin polypeptide XI (JZTX-XI) from the spider tufted spider of Jingzhao was expressed in Escherichia coli BL21(DE3) pLysS using recombinant DNA technology, as follows: The encoding gene of JZTX-XI was cloned into the pMAL-p5x vector to construct a recombinant expression plasmid containing an MBP solubilizing tag, a 6×His tag, and a thrombin site. The map of the recombinant expression plasmid is shown below. Figure 1The target gene nucleotide sequence is shown in SEQ ID NO:1, as follows: GAATGTCGCAAAATGTTCGGTGGTTGCAGCGTGGATAGCGATTGTTGTGCTCATCTGGGCTGCAAGCCGACTCTGAAGTACTGCGCTTGGGATGGTACCTTCGGCAAG. The amino acid sequence of JZTX-XI is shown in SEQ ID NO:2, as follows: ECRKMFGGCSVDSDCCAHLGCKPTLKYCAWDGTFGK. The nucleotide sequence of the 6×His tag is shown in SEQ ID NO:3, as follows: catcatcatcatcatcac. The nucleotide sequence of the thrombin restriction site is shown in SEQ ID NO:4, as follows: ctggtgccacgcggttct.

[0035] After the encoding gene of JZTX-XI was cloned into the pMAL-p5x vector, the MBP solubilizing tag, BamHI-6×His, thrombin site, and target gene were ligated. The nucleotide sequence is shown in SEQ ID NO:5. The nucleotide sequence of MBP solubilizing tag-BamHI-6×His-thrombin site-target gene is as follows: ATGAAAATAAAAACAGGTGCACGC ATCCTCGCATTATCCGCATTAACGACGATGATGTTTTCCGCCTCGGCTCTCGCCAAAATCGAAGAAGGTAAACTGG TAATCTGGATTAACGGCGATAAAGGCTATAACGGTCTCGCTGAAGTCGGTAAGAAATTCGAGAAAGATACCGGAAT TAAAGTCACCGTTGAGCATCCGGATAAACTGGAAGAGAAATTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGAC ATTATCTTCTGGGCACACGACCGCTTTGGTGGCTACGCTCAATCTGGCCTGTTGGCTGAAATCACCCCGGACAAAG CGTTCCAGGACAAGCTGTATCCGTTTACCTGGGATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGC TGTTGAAGCGTTATCGCTGATTTATAACAAAGATCTGCTGCCGAACCCGCCAAAAACCTGGGAAGAGATCCCGGCG CTGGATAAAGAACTGAAAGCGAAAGGTAAGAGCGCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTGGCCGC TGATTGCTGCTGACGGGGGTTATGCGTTCAAGTATGAAAACGGCAAGTACGACATTAAAGACGTGGGCGTGGATAA CGCTGGCGCGAAAGCGGGTCTGACCTTCCTGGTTGACCTGATTAAAAACAAACACATGAATGCAGACACCGATTAC TCCATCGCAGAAGCTGCCTTTAATAAAGGCGAAACAGCGATGACCATCAACGGCCCGTGGGCATGGTCCAACATCG ACACCAGCAAAGTGAATTATGGTGTAACGGTACTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTCGTTGGCGT GCTGAGCGCAGGTATTAACGCCGCCAGTCCGAACAAAGAGCTGGCAAAAGAGTTCCTCGAAAACTATCTGCTGACT GATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTACGAGGAAGAGTTGG TGAAAGATCCGCGTATTGCCGCCACTATGGAAAACGCCCAGAAAGGTGAAATCATGCCGAACATCCCGCAGATGTC CGCTTTCTGGTATGCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTGAAA GACGCGCAGACT - GGATCC- Catcatcatcatcatcac- Ctggtgccacgcggttct - GAATGTCGCAAAATGTTCGGTGGTTGCAGCGTGGATAGCGATTGTTGTGCTCATCTGGGCTGCAAGCCGACTCTGAAGTACTGCGCTTGGGATGGTACCTTCGGCAAG.

[0036] pass Figure 1 We can conclude that: Figure 1 The locations of the BamHI and EcoRI restriction sites are indicated. Translation frames on the vector show the sequence where the 6×His tag links the thrombin site and the protein. The 6×His tag and the protein are shown in orange and purple, respectively. The thrombin cleavage site is shown in blue.

[0037] The constructed recombinant expression plasmid was transformed into E. coli BL21(DE3)pLysS cells and cultured at 16°C for 20 h under IPTG (0.1 mM) induction to obtain a highly soluble fusion protein. The fusion protein was then initially purified using Ni-NTA affinity chromatography, followed by thrombin digestion to remove MBP and His tags. The pre-purified protein was further separated by size exclusion chromatography to remove impurities and optimize purity. Finally, high-efficiency purification was achieved using HPLC (reversed-phase chromatography, C18 column), utilizing hydrophobic interactions to ensure a target protein purity greater than 95%. The purified peptide was identified by SDS-PAGE and mass spectrometry to confirm its molecular weight and amino acid sequence accuracy.

[0038] SDS-PAGE analysis of samples at each purification step is as follows: Figure 2 As shown, lanes 4 and 5 represent the samples before and after enzyme digestion, respectively, while lane 8 represents the final purified sample. The results indicate high enzyme digestion efficiency, and the final purified sample exhibits high purity with clear bands, meeting expectations.

[0039] Mass spectrometry analysis results as follows Figure 3 As shown, the molecular weight of the final purified sample was consistent with that of the target protein, and the mass spectrometry peaks were clear and free of impurity peaks, verifying the high purity and accuracy of the purified protein. Simultaneously, the free thiol content of the sample was determined using the DTNB method, and the results showed it to be close to zero, indicating that the polypeptide disulfide bonds were fully folded and the conformation was stable.

[0040] HPLC analysis chromatogram of purified sample as shown Figure 4 As shown, the final purified sample showed good separation on the C18 reversed-phase column, with only one single peak, indicating that the target protein had high purity and extremely low impurity levels.

[0041] The amino acid sequence determination results are as follows Figure 5 As shown, sequence alignment and identification were performed using Byonic software. The results showed that the target polypeptide sequence was consistent with the expected sequence, and the sequence matching degree of JZTX-XI was 99.97%.

[0042] In summary, the method described in this invention provides an efficient and economical approach that successfully overcomes the difficulties in extracting natural toxins, and is suitable for subsequent structural and activity studies. Finally, the purified JZTX-XI peptide was stored at -80°C to maintain its stability and biological activity for use in subsequent pharmacodynamic experiments.

[0043] Example 2 Application of Jingzhao's spider venom polypeptide XI in the treatment of type II diabetes This embodiment was validated using a BKS-db / db mouse type 2 diabetes experiment, and the steps are as follows: Eighteen male BKS-db / db mice were randomly divided into two groups after one week of acclimatization: (1) control group and (2) JZTX-XI group (6 mice per group). The control group received intraperitoneal injection of JZTX-XI (6.08 μg / kg), while the control group received an equal volume of physiological saline. The drugs were administered once daily. From day 1 to day 17, all groups were fed a 60% high-fat diet. After day 17, all groups were fed a normal diet. During the drug administration period, body weight, fasting blood glucose, food intake, water intake, and urine output were recorded every 4 days. On day 16 and day 32, glucose tolerance tests (OGTT) and glucose tolerance tests (ITT) were performed, respectively. After 37 days of continuous drug administration, serum, pancreas, heart, liver, spleen, and kidney tissues were collected for further histological and biochemical analysis.

[0044] Changes in body weight and fasting blood glucose in each group of mice are as follows: Figure 6 As shown, the body weight of mice in both the control group and the treatment group showed a trend of first increasing and then decreasing. However, the body weight change trend in the treatment group was slower than that in the control group. Furthermore, the fasting blood glucose in the control group reached its peak on day 13 and remained at a high level after the diet was changed to normal. In contrast, the fasting blood glucose in the treatment group reached its peak on day 17 and then gradually decreased after the diet was changed to normal.

[0045] The results of food intake, water intake, and urine output of mice in each group are as follows: Figure 7 As shown, compared with the control group, the mice in the drug treatment group did not show significant changes in food intake, but their water intake and urine output were significantly reduced.

[0046] The results of OGTT and ITT tests in each group of mice are as follows: Figure 8 As shown, compared with the control group, the glucose tolerance and insulin sensitivity of the mice in the drug-treated group were significantly improved.

[0047] Pancreatic tissue from each group of mice was stained with hematoxylin and eosin (HE) and scanned using a pathological section scanner. The results are shown in the figure below. Figure 9 The H&E results showed that, compared with the control group, the mice in the treatment group had abundant pancreatic islets, regular shapes, varying areas, and a significant increase in the number of islet cells.

[0048] The levels of insulin, C-peptide, and GHb in serum were determined by ELISA, and the HOMA-IR was calculated. Results are shown in [Figure number missing]. Figure 10 As can be seen, compared with the control group, the serum insulin level of mice in the drug treatment group was significantly increased and the insulin resistance index (HOMA-IR) was significantly decreased, but the C-peptide and glycated hemoglobin levels remained unchanged.

[0049] Real-time quantitative PCR was used to detect the mRNA expression of genes related to insulin secretion in pancreatic tissue. The primer sequences used are shown in Table 1.

[0050] Table 1 Primers for real-time quantitative PCR assay of mRNA expression of genes related to insulin secretion

[0051] See results Figure 11 As can be seen, compared with the control group, JZTX-XI significantly upregulated the mRNA expression of the glucose transport regulatory gene Glut2. All treatment groups showed significant upregulation of the mRNA expression of genes regulating insulin secretion, such as Ins1, Ins2, Lapp, and Pdx1, while significantly downregulating the mRNA expression of genes regulating pancreatic β-cell dedifferentiation, such as Ngn3 and Oct4.

[0052] The expression of insulin and Ki67 in islet cells of pancreatic tissue from mice in each group was detected by immunofluorescence. The results are shown in [Figure number missing]. Figure 12 As can be seen, compared with the control group, the expression of Insulin and Ki67 in pancreatic islet cells of the pancreatic tissue of mice in the drug-treated group was significantly increased.

[0053] Immunohistochemistry was used to detect the expression of Cleaved-caspase 3 in islet cells of pancreatic tissue from mice in each group. The results are shown in the table below. Figure 13 As can be seen, compared with the control group, the expression of Cleaved-caspase 3 in pancreatic islet cells of the pancreatic tissue of mice in the drug-treated group was significantly reduced.

[0054] Based on the above experimental results, it can be seen that the present invention successfully obtained the target polypeptide with high purity, intact disulfide bond folding, and uniform conformation by combining the expression-purification strategy of MBP-assisted solubilization and 6×His affinity purification. Each liter of E. coli culture can produce more than 15 mg of polypeptide JZTX-XI, which is suitable for subsequent in vitro and in vivo pharmacodynamic studies.

[0055] JZTX-XI can significantly reduce fasting blood glucose in type II diabetic mice, improve glucose tolerance and insulin sensitivity, alleviate polydipsia and polyuria symptoms, and promote insulin secretion and pancreatic islet cell proliferation.

[0056] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method of making a Gifftanoxin polypeptide XI, comprising, The method comprises the following steps: The gene of the Jingeantuo spider toxin polypeptide XI, the nucleotide sequence of the 6×His tag, the nucleotide sequence of the thrombin cleavage site and the nucleotide sequence of the MBP solubilization tag are connected to a prokaryotic expression vector for prokaryotic expression, so as to obtain a high-solubility fusion protein; The high-solubility fusion protein is purified to obtain the Jingeantuo spider toxin polypeptide XI; The amino acid sequence of the Jingeantuo spider toxin polypeptide XI is shown as SEQ ID NO:

2.

2. The production method according to claim 1, wherein The prokaryotic expression step comprises: The expression module is connected to a plasmid vector containing the MBP solubilization tag to obtain a recombinant vector; The recombinant vector is transformed into E. coli to obtain a recombinant engineering bacterium; The recombinant engineering bacterium is induced for expression to obtain a high-solubility fusion protein; the expression module comprises, from 5' end to 3' end, the nucleotide sequence of the 6×His tag, the nucleotide sequence of the thrombin cleavage site and the gene of the Jingeantuo spider toxin polypeptide XI.

3. The production method according to claim 2, wherein The plasmid vector comprises a pMAL-p5x vector.

4. The production method according to claim 3, wherein The expression module containing the gene of the Jingeantuo spider toxin polypeptide XI is inserted between the BamHI and EcoRI enzyme cleavage sites of the pMAL-p5x vector.

5. The production method according to claim 1, wherein The purification step comprises: The high-solubility fusion protein is preliminarily purified by Ni-NTA affinity chromatography to obtain a fusion protein after preliminary purification; The fusion protein after preliminary purification is cleaved by thrombin, and the cleaved protein is separated by molecular sieve chromatography; The protein separated by the molecular sieve chromatography is purified by reverse-phase high-performance liquid chromatography to obtain the Jingeantuo spider toxin polypeptide XI.

6. The Jingeantuo spider toxin polypeptide XI in the preparation of a drug for treating diabetes; the amino acid sequence of the Jingeantuo spider toxin polypeptide XI is shown as SEQ ID NO:

2.

7. Use according to claim 6, wherein The Jingeantuo spider toxin polypeptide XI is prepared by the preparation method in any one of claims 1-5.

8. Use according to claim 6 or 7, wherein the compound is ###0002### The diabetes comprises type II diabetes.

9. Use according to claim 8, wherein the compound is ###0002### The application comprises one or more of the following items: 1) promoting insulin secretion and / or promoting insulin cell proliferation; 2) improving glucose tolerance and / or insulin sensitivity; 3) reducing blood sugar; 4) relieving polydipsia and / or polyuria symptoms.

10. A medicament for treating diabetes, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The effective component comprises the Jingeantuo spider toxin polypeptide XI prepared by the preparation method in any one of claims 1-5.

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