A protein composition and its preparation method

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a protein composition and its preparation method. The protein composition comprises the following components in parts by weight: 10-20 parts of autologous blood immunostimulant and 1-5 parts of Motilin analog peptide TCL1; the amino acid sequence of the Motilin analog peptide TCL1 is shown in SEQ ID NO.1. Experimental results show that injection of the protein composition prepared according to this invention can reverse the immune status from a pro-inflammatory Th1 type to an anti-inflammatory Th2 type response, creating a crucial immune microenvironment for β cells, improving insulin secretion capacity, thereby reducing blood glucose levels, and demonstrating significant therapeutic effects on diabetes with broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a protein composition and its preparation method. Background Technology

[0002] Autoimmune diseases are a group of chronic illnesses caused by the body's immune system abnormally attacking its own normal tissues, characterized by local or systemic inflammation and tissue damage. The occurrence of these diseases involves multiple mechanisms, including genetic susceptibility, environmental factors, and imbalances in the immune regulatory network. When immune tolerance is broken, autoreactive T cells and B cells are abnormally activated, producing antibodies and cytotoxic reactions against self-antigens, leading to damage to specific organs or systems.

[0003] Type 1 diabetes is a typical example of an autoimmune disease. Its core pathology lies in the autoimmune system's misidentification and persistent attack on pancreatic beta cells, leading to massive destruction of these cells and an absolute deficiency of insulin. Currently, one treatment strategy for autoimmune diseases involves immunomodulatory methods, such as injecting specific autoantigens, to induce immune tolerance and restore immune balance.

[0004] Autologous blood immunostimulants are personalized immunomodulators prepared by systematically temperature-controlled and standardized processing of the patient's own peripheral blood. Their core mechanism involves delivering "educational signals" from the patient's own mutated cells to the immune system, inducing specific cellular and humoral immune responses, promoting a shift from a Th1 (pro-inflammatory) to a Th2 (anti-inflammatory / modulatory) immune response, thereby rebuilding immune tolerance to self-antigens and blocking the continuous attack of the pancreas by the autoimmune system. However, current single immunomodulatory therapies still have limitations in protecting residual pancreatic function and stabilizing blood glucose. To more effectively address this clinical challenge, this invention aims to provide a novel, synergistic treatment regimen. Summary of the Invention

[0005] To achieve the above objectives, the present invention provides a protein composition that combines a personalized immunomodulator (autologous blood immunostimulant) with a Motilin analog polypeptide (TCL1) with pancreatic islet protection function to achieve a therapeutic effect on diabetes.

[0006] The primary objective of this invention is to provide a protein composition.

[0007] A second objective of this invention is to provide a method for preparing a protein composition.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A protein composition comprising the following components in parts by weight: 10-20 parts of autologous blood immunostimulant and 1-5 parts of Motilin analog peptide TCL1; the amino acid sequence of the Motilin analog peptide TCL1 is shown in SEQ ID NO.1; the method for preparing the autologous blood immunostimulant specifically includes the following steps:

[0010] (1) Isolate PBMCs from peripheral blood and adjust the concentration to 1-3×10⁻⁶ with PBS. 6 / mL;

[0011] (2) The PBMC cells obtained in step (1) were subjected to heat shock in a constant temperature water bath at 40-45℃ for 1-2 hours; then they were subjected to repeated freeze-thaw cycles in liquid nitrogen and a water bath at 35-40℃ for 2-4 times, and the supernatant lysate was collected after centrifugation.

[0012] (3) The supernatant lysate obtained in step (2) is filtered through a 0.22 μm filter membrane for sterilization, and then dispensed and stored at -80℃ for later use.

[0013] Furthermore, the preparation method of the Motilin analog peptide TCL1 specifically includes the following steps:

[0014] (1) Add the artificial peptide GTFKFIQWLVTSPP to the end of the amino acid sequence of wild-type Motilin to obtain the amino acid sequence of Motilin analog peptide TCL1, as shown in SEQ ID NO.1;

[0015] (2) Based on the amino acid sequence of the Motilin analog peptide TCL1, the gene sequence was reverse-engineered and the codons were optimized to obtain the nucleotide sequence encoding the Motilin analog peptide TCL1, as shown in SEQ ID NO.2;

[0016] (3) Based on the nucleotide sequence obtained in step (2), design upstream and downstream primers to amplify the corresponding nucleotide sequence of the Motilin analog peptide TCL1, and connect the nucleotide sequence of the Motilin analog peptide TCL1 amplified by the primers to the pET30a vector to construct a recombinant plasmid.

[0017] (4) Transform the recombinant plasmid constructed in step (3) into competent cells to construct a recombinant strain; after IPTG induction, obtain bacterial cell precipitate;

[0018] (5) The bacterial precipitate obtained in step (4) is separated and purified to obtain the Motilin analog peptide TCL1.

[0019] Further, the upstream primer sequence of step (3) is shown in SEQ ID NO.3; the downstream primer sequence is shown in SEQ ID NO.4.

[0020] Furthermore, in step (4), the competent cells are E. coli BL21 (DE3) competent cells.

[0021] Furthermore, in step (4), the concentration of IPTG-induced expression is 0.3-0.6 mmol / L, and the time for IPTG-induced expression is 6-10 h.

[0022] The preparation method of the protein composition described above specifically includes the following steps: weighing autologous blood immunostimulant and Motilin analog peptide TCL1 according to the stated weight proportions, stirring and mixing evenly to obtain the final product.

[0023] Compared with the prior art, the main advantages of the present invention are:

[0024] (1) The Motilin analog peptide TCL1 was modified based on the wild type of Motilin. The artificial peptide GTFKFIQWLVTSPP was added to the end of Motilin to achieve efficient expression of the Motilin analog, prolong its half-life, accelerate gastric emptying, reduce blood glucose fluctuations caused by food retention, and protect the body's β cells, thereby enhancing insulin release.

[0025] (2) The combination of Motilin analog peptide TCL1 and autologous blood immunostimulant has a synergistic effect. After injection, it reverses the immune status from pro-inflammatory Th1 type to anti-inflammatory Th2 type response, thereby correcting the autoimmune imbalance, creating a key immune microenvironment for β cells, improving insulin secretion capacity, thereby reducing blood glucose level, and has a significant therapeutic effect on diabetes, with good market application prospects. Attached Figure Description

[0026] Figure 1 The plasmid map of recombinant plasmid pET30a-TCL1;

[0027] Figure 2 This is an expression diagram of the purified Motilin analog peptide TCL1 prepared in this invention;

[0028] Figure 3 Figure showing the effects of different protein compositions on blood glucose levels in type 1 diabetic mice;

[0029] Figure 4 Figure showing the effect of different protein compositions on serum IFN-γ levels in type 1 diabetic mice;

[0030] Figure 5 The figure shows the effect of different protein compositions on the serum IL-10 level in type 1 diabetic mice. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0032] The method for preparing the autologous blood immunostimulant used in the experiments of this invention specifically includes the following steps:

[0033] (1) Isolate PBMCs from peripheral blood and adjust the concentration to 1×10⁻⁶ with PBS. 6 / mL;

[0034] (2) The PBMC cells obtained in step (1) were subjected to heat shock in a constant temperature water bath at 42℃ for 1 h; then they were repeatedly frozen and thawed in liquid nitrogen and a water bath at 37℃ 3 times, and the supernatant lysate was collected after centrifugation.

[0035] (3) The supernatant lysate obtained in step (2) is filtered through a 0.22 μm filter membrane for sterilization, and then dispensed and stored at -80℃ for later use.

[0036] Example 1

[0037] A protein composition comprising the following components in parts by weight: 15 parts of autologous blood immunostimulant and 3 parts of Motilin analog peptide TCL1; the amino acid sequence of the Motilin analog peptide TCL1 is shown in SEQ ID NO. 1.

[0038] The preparation method of the Motilin analog peptide specifically includes the following steps:

[0039] (1) Add the artificial peptide GTFKFIQWLVTSPP to the end of the amino acid sequence of wild-type Motilin to obtain the amino acid sequence of Motilin analog peptide TCL1, as shown in SEQ ID NO.1;

[0040] (2) Based on the amino acid sequence of the Motilin analog peptide TCL1 (SEQ ID NO.1), a reverse gene sequence was designed. Codon optimization was performed on the corresponding base sequences according to the codon usage frequency in *E. coli* to obtain the nucleotide sequence encoding the Motilin analog peptide TCL1, as shown in SEQ ID NO.2. Upstream and downstream primers for amplifying the Motilin analog peptide TCL1 were designed based on the optimized sequence. The upstream primer sequence is shown in SEQ ID NO.3, containing the Nde I restriction site CATATG; the downstream primer sequence is shown in SEQ ID NO.4, containing the Hind III restriction site AAGCTT. The Motilin analog peptide TCL1 gene was amplified by PCR, and the gene fragment was purified using a gel extraction kit.

[0041] Table 1 Sequence List

[0042]

[0043] (3) The expression vector pET30a was double-digested with Nde I and Hind III using restriction nucleic acids. The double digestion reaction system is shown in Table 2. The amplified and recovered Motilin analog peptide TCL1 gene fragment was ligated with the digested expression vector pET30a fragment using T4 DNA ligase. The T4 DNA ligase reaction system is shown in Table 3. The recombinant plasmid pET30a-TCL1 was obtained after identification and sequencing. The map of the recombinant plasmid pET30a-TCL1 is shown in Table 3. Figure 1 As shown.

[0044] Table 2. Double enzyme digestion reaction system

[0045]

[0046] Table 3. T4 DNA ligase reaction system

[0047]

[0048] (4) Transform the recombinant plasmid pET30a-TCL1 constructed in step (3) into E. coli BL21 (DE3) competent cells, spread it on LB plates containing 100 μg / mL kanamycin, and incubate it in an inverted incubator at 37°C overnight to obtain the recombinant strain, named pET30a-TCL1 / BL21.

[0049] (5) Select positive monoclonal colonies and inoculate them into LB broth containing 100 μg / mL kanamycin. Incubate overnight at 37°C with shaking to obtain seed culture. Inoculate the seed culture into LB broth containing 100 μg / mL kanamycin at a 5 v / v inoculation rate and incubate at 37°C with shaking to obtain OD. 600 The concentration was set to 0.8, the temperature was lowered to 25°C, and 0.5 mM IPTG was added to induce the expression of the Motilin analog peptide for 8 h.

[0050] (6) After induction, the bacterial cells were collected by centrifugation, sonicated, and centrifuged again to collect the supernatant. The Motilin analog peptide TCL1 was purified by affinity chromatography. The purified Motilin analog peptide TCL1 was detected by SDS-PAGE small molecule protein gel electrophoresis, and the results are as follows: Figure 2 As shown.

[0051] This embodiment also provides a method for preparing a protein composition, specifically including the following steps:

[0052] Weigh out 15 parts of autologous blood immunostimulant and 3 parts of Motilin analog peptide TCL1 according to the stated weight proportions, stir and mix evenly to obtain the final product.

[0053] Example 2

[0054] A protein composition comprising the following components in parts by weight: 10 parts of autologous blood immunostimulant and 1 part of Motilin analog peptide TCL1; the amino acid sequence of the Motilin analog peptide TCL1 is shown in SEQ ID NO. 1.

[0055] The preparation method of the Motilin analog peptide TCL1 is the same as in Example 1.

[0056] This embodiment also provides a method for preparing a protein composition, specifically including the following steps:

[0057] Weigh out 10 parts of autologous blood immunostimulant and 1 part of Motilin analog peptide TCL1 according to the stated weight proportions, stir and mix evenly to obtain the final product.

[0058] Example 3

[0059] A protein composition comprising the following components in parts by weight: 20 parts of autologous blood immunostimulant and 5 parts of Motilin analog peptide TCL1; the amino acid sequence of the Motilin analog peptide TCL1 is shown in SEQ ID NO. 1.

[0060] The preparation method of the Motilin analog peptide TCL1 is the same as in Example 1.

[0061] This embodiment also provides a method for preparing a protein composition, specifically including the following steps:

[0062] Weigh out 20 parts of autologous blood immunostimulant and 5 parts of Motilin analog peptide TCL1 according to the stated weight proportions, stir and mix evenly to obtain the final product.

[0063] Comparative Example 1

[0064] The difference between Comparative Example 1 and Example 1 is that the Motilin analog peptide TCL1 in the protein composition was omitted, while all other components were the same as in Example 1.

[0065] Comparative Example 2

[0066] The difference between Comparative Example 1 and Example 1 is that the Motilin analog peptide TCL1 in the protein composition was replaced with wild-type Motilin, while all other aspects were the same as in Example 1.

[0067] The amino acid sequence of wild-type Motilin is FVPIFTYGELQRMQEKERNKGQ.

[0068] Experimental Example 1

[0069] Effects of Motilin analogue peptide TCL1 on secretion by pancreatic β-cell line (INS-1)

[0070] To evaluate the effect of the synthesized Motilin analog peptide TCL1 on insulin secretion in INS-1 cells, the specific experimental steps were as follows: INS-1 cells were incubated at 2.5 × 10⁶ cells per cell line. 5 INS-1 cells were seeded at a density of 1 / 2 wells in 24-well plates. Before the experiment, INS-1 cells were incubated twice with serum-free and glucose-free KRB buffer for 30 minutes each time. Subsequently, the buffer was replaced with KRB buffer containing 0, 5, and 10 mM glucose and different concentrations (0, 1, and 10 nM) of the motilin analog peptide TCL1 (Example 1), and incubated for another hour. The culture supernatant was collected, and insulin concentration was detected using an ELISA kit. Each experiment had three replicates, and the results are shown in Table 4.

[0071] Table 4. Effects of Motilin analogue peptide TCL1 on insulin secretion by pancreatic β-cell line (INS-1)

[0072]

[0073] The results are shown in Table 4, which presents the effect of the motilin analog peptide TCL1 on insulin secretion in the pancreatic β-cell line (INS-1). Table 4 shows that in the absence of glucose, the motilin analog peptide TCL1 could not stimulate insulin secretion from INS-1 cells. However, in the presence of 5 mM or 10 mM glucose, the motilin analog peptide TCL1 significantly promoted insulin secretion. This result indicates that the motilin analog peptide TCL1 possesses the biological activity to promote glucose-dependent insulin secretion from pancreatic β-cell lines.

[0074] Experimental Example 2

[0075] The following are efficacy tests of the protein compositions prepared in Examples 1-3 and Comparative Examples 1-2:

[0076] (1) Seventy NOD mice weighing 18-22 g were selected for the experiment. All NOD mice were housed in separate cages in a standard SPF-grade animal laboratory, with the ambient temperature controlled at 22±2℃ and the relative humidity at 50-60%. The mice were allowed free access to food and water, with a 12-hour light-dark cycle.

[0077] (2) After acclimatizing the NOD mice obtained in step (1) for 3 days, they were injected intraperitoneally with streptozotocin for 5 consecutive days at a dose of 40 mg / kg / day. When the blood glucose concentration of the mice was greater than 12 mmol, it was determined to be hyperglycemia, and the type 1 diabetes model was considered to have been successfully established. Sixty type 1 diabetes mice that were successfully modeled were randomly divided into model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, and Comparative Example 2 group, with 10 mice in each group. Except for the model group, the mice in the other groups were injected subcutaneously into the abdomen with the protein composition of the corresponding group at a dose of 2 mg / kg on the 1st, 4th and 7th days after the successful modeling.

[0078] (3) Fasting blood glucose levels were measured in all mice on days 17 and 24 following subcutaneous injection into the abdomen. The method was as follows: After fasting for 6 hours, blood was collected from the orbital venous plexus, and blood glucose levels were immediately measured using a portable blood glucose meter. The readings were recorded. The results are as follows: Figure 3 As shown.

[0079] (4) On day 7 after the first administration, 6 mice were randomly selected from each group. After anesthesia, blood was collected through the orbital venous plexus, serum was separated, aliquoted, and stored at -80°C. The levels of pro-inflammatory factor IFN-γ and anti-inflammatory factor IL-10 in the serum were detected using an ELISA kit. The results are as follows: Figure 4 , Figure 5 As shown.

[0080] The results are as follows Figure 3 The image shows the effect of different protein compositions on blood glucose levels in type 1 diabetic mice. Figure 3 It can be seen that, compared with the model group, comparative example 1 group, and comparative example 2 group, the protein compositions prepared in Examples 1-3 of the present invention can significantly reduce the blood glucose level of NOD diabetic mice and have significant therapeutic effects.

[0081] The results are as follows Figure 4 The image shows the effect of different protein compositions on the serum levels of the pro-inflammatory cytokine IFN-γ in type 1 diabetic mice. Figure 4 It can be seen that, compared with the model group, comparative example 1 group and comparative example 2 group, the protein compositions prepared by examples 1-3 of the present invention can significantly reduce the content of IFN-γ pro-inflammatory factors.

[0082] The results are as follows Figure 5 The image shows the effect of different protein compositions on the serum levels of the anti-inflammatory factor IL-10 in type 1 diabetic mice. Figure 5 It can be seen that, compared with the model group, comparative example 1 group and comparative example 2 group, the protein compositions prepared in Examples 1-3 of the present invention can significantly increase the content of IL-10 anti-inflammatory factor.

[0083] In summary, the protein composition of this invention exhibits comprehensive therapeutic effects in a type 1 diabetic mouse model. It significantly downregulates serum levels of the pro-inflammatory cytokine IFN-γ and upregulates levels of the anti-inflammatory cytokine IL-10, reversing the immune status from a pro-inflammatory Th1 response to an anti-inflammatory Th2 response, thereby correcting autoimmune imbalance and creating a crucial immune microenvironment for β-cells. This improves insulin secretion capacity, reduces blood glucose levels in type 1 diabetic mice, and demonstrates significant therapeutic effects on diabetes, showing promising market application prospects.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A protein composition, characterized in that, The protein composition comprises the following components in parts by weight: 10-20 parts of autologous blood immunostimulant and 1-5 parts of Motilin analog peptide TCL1; the amino acid sequence of the Motilin analog peptide TCL1 is shown in SEQ ID NO.1; the preparation method of the autologous blood immunostimulant specifically includes the following steps: (1) PBMC cells are isolated from peripheral blood, and the concentration is adjusted to 1-3 x 10 6 / mL with PBS; (2) The PBMC cells obtained in step (1) were subjected to heat shock in a constant temperature water bath at 40-45℃ for 1-2 hours; then they were subjected to repeated freeze-thaw cycles in liquid nitrogen and a water bath at 35-40℃ for 2-4 times, and the supernatant lysate was collected after centrifugation. (3) The supernatant lysate obtained in step (2) is filtered through a 0.22 μm filter membrane for sterilization, and then dispensed and stored at -80℃ for later use.

2. The protein composition according to claim 1, characterized in that, The preparation method of the Motilin analog peptide TCL1 specifically includes the following steps: (1) Add the artificial peptide GTFKFIQWLVTSPP to the end of the amino acid sequence of wild-type Motilin to obtain the amino acid sequence of the Motilin analog peptide TCL1, as shown in SEQ ID NO.1; (2) Based on the amino acid sequence of the Motilin analog peptide TCL1, the gene sequence was reverse-engineered and the codons were optimized to obtain the nucleotide sequence encoding the Motilin analog peptide TCL1, as shown in SEQ ID NO.2; (3) Based on the nucleotide sequence obtained in step (2), design upstream and downstream primers to amplify the corresponding nucleotide sequence of the Motilin analog peptide TCL1, and connect the nucleotide sequence of the Motilin analog peptide TCL1 amplified by the primers to the pET30a vector to construct a recombinant plasmid. (4) Transform the recombinant plasmid constructed in step (3) into competent cells to construct a recombinant strain; after IPTG induction, obtain bacterial cell precipitate; (5) The bacterial precipitate obtained in step (4) is separated and purified to obtain the Motilin analog peptide TCL1.

3. The protein composition according to claim 2, characterized in that, The upstream primer sequence of step (3) is shown in SEQ ID NO.3; the downstream primer sequence is shown in SEQ ID NO.

4.

4. The protein composition according to claim 2, characterized in that, The competent cells in step (4) are E. coli BL21 (DE3) competent cells.

5. The protein composition according to claim 2, characterized in that, In step (4), the concentration of IPTG-induced expression is 0.3-0.6 mmol / L, and the IPTG-induced expression time is 6-10 h.

6. A method for preparing a protein composition according to any one of claims 1-5, characterized in that, Specifically, the following steps are included: Weigh out the autologous blood immunostimulant and Motilin analog peptide TCL1 according to the stated weight proportions, stir and mix evenly to obtain the final product.

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