Polypeptide for preventing and / or treating septicopyemia

Abstract

The invention relates to the technical field of biology, and particularly discloses a polypeptide for preventing and / or treating septicopyemia, which is a polypeptide with an amino acid sequence having at least 97% homology with SEQ ID NO: 1. Tests prove that no matter through artificial synthesis or prokaryotic expression purification, the polypeptide can reduce inflammatory factors in macrophages, improve lung injury caused by septicopyemia and prolong the lifetime. The compound has a remarkable effect in the aspect of preventing and treating septicopyemia, has a huge potential of preventing and treating septicopyemia when being prepared into a pharmaceutical preparation, and has practical application and popularization values.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a polypeptide for preventing and / or treating sepsis. Background Art

[0002] Sepsis is a systemic immune response caused by infection. In the later stage, it can develop into septic shock, multiple organ failure and even death. Clinically, sepsis is one of the serious complications of critically ill patients such as trauma, burns, shock and infection. Due to its fierce onset, rapid progression and high mortality rate, it brings great difficulties to clinical treatment. Studies have shown that in 2017, there were about 4,890 patients with sepsis worldwide, of which about 11 million died, accounting for 19.7% of the total global deaths. Despite the continuous improvement of medical standards, the clinical cure rate of sepsis has not been greatly improved, and the global mortality rate remains high, causing great physical, psychological and economic burdens on patients. There is an urgent need for the emergence of an effective new therapeutic drug.

[0003] As biotechnology and peptide synthesis technology mature, more and more peptide drugs are being developed and used in clinical practice. Due to their wide range of indications, high safety and significant efficacy, peptide drugs are currently widely used in the prevention and treatment of diseases such as tumors, hepatitis, diabetes, and AIDS. Patent CN111484544A discloses a peptide for treating sepsis, which can increase the survival rate of sepsis model mice to 67%. There is still room for further improvement in its efficacy to benefit sepsis patients. Summary of the invention

[0004] In view of this, an object of the present invention is to provide a polypeptide for preventing and / or treating sepsis, which is a polypeptide having an amino acid sequence with at least 97% homology to SEQ ID NO:1.

[0005] Furthermore, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0006] The present invention also provides a gene fragment encoding the aforementioned polypeptide, and its nucleotide sequence is shown in SEQ ID NO:4.

[0007] The present invention also provides a use of the above polypeptide in preparing a drug for preventing and / or treating sepsis.

[0008] Furthermore, the drug is a drug that downregulates inflammatory factors in macrophages.

[0009] Furthermore, the inflammatory factors include IL-1β and IL-6.

[0010] Furthermore, the drug is a drug for improving lung tissue damage.

[0011] The present invention also provides a drug for preventing and / or treating sepsis, comprising a polypeptide having an amino acid sequence with at least 97% homology to SEQ ID NO: 1 and a pharmaceutically acceptable excipient.

[0012] Furthermore, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0013] Furthermore, it is an injection preparation or an oral preparation.

[0014] Furthermore, it is an injectable preparation.

[0015] The amino acid sequence of the polypeptide of the present invention is shown in SEQ ID NO: 1 or SEQ ID NO: 2. Experiments have shown that the polypeptide, whether artificially synthesized or purified by prokaryotic expression, can reduce inflammatory factors in macrophages, improve lung damage caused by sepsis, and prolong survival. It has a significant effect in preventing and treating sepsis, and its preparation into a pharmaceutical preparation has great potential for preventing and treating sepsis, and has practical application and promotion value.

[0016] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0017] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Synthetic peptides effectively inhibit inflammatory responses

[0019] Figure 2 Purified peptide effectively prevents sepsis in mice

[0020] Figure 3 Synthetic peptide effectively prevents sepsis in mice

[0021] Figure 4 Peptide can effectively treat sepsis in mice

[0022] Figure 5 The mutant peptide has similar effects to the original peptide DETAILED DESCRIPTION

[0023] The raw materials and equipment used in the specific embodiments of the present invention are all known products, which are obtained by purchasing commercially available products. The preparation process of the injection prepared by the polypeptide of the present invention is mature and can be prepared by referring to the conventional methods in the prior art.

[0024] Example 1 Preparation of an aqueous injection preparation containing the polypeptide of the present invention

[0025] Take a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1, add water for injection and mix well, and filter and sterilize to prepare a water for injection preparation;

[0026] Polypeptide sequence (SEQ ID NO.1):

[0027] TSKCRKAGCVYFGTPENKGFCTLCFIEYRENK

[0028] Example 2 Preparation of water for injection containing the polypeptide of the present invention

[0029] A polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 is taken, mixed with water for injection, and filtered and sterilized to obtain a water for injection preparation.

[0030] Polypeptide sequence (SEQ ID NO.2):

[0031] TSKCRKAGCMYFGTPENKGFCTLCFIEYRENK

[0032] Example 3 Preparation of powder for injection containing the polypeptide of the present invention

[0033] A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 is taken, mixed with water for injection, sterilized by filtration, and freeze-dried to obtain a powder for injection.

[0034] Polypeptide sequence (SEQ ID NO.1):

[0035] TSKCRKAGCVYFGTPENKGFCTLCFIEYRENK

[0036] Example 4 Preparation of powder for injection containing the polypeptide of the present invention

[0037] A polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 is taken, mixed with water for injection, sterilized by filtration, and freeze-dried to obtain a powder for injection.

[0038] Polypeptide sequence (SEQ ID NO.2):

[0039] TSKCRKAGCMYFGTPENKGFCTLCFIEYRENK

[0040] The beneficial effects of the present invention are further illustrated by test examples below.

[0041] Experimental Example 1 Study on peptides for treating sepsis

[0042] 1. Experimental methods:

[0043] 1. Construction of peptides

[0044] 1.1 Peptides for Experiment

[0045] Polypeptide sequence (SEQ ID NO.1):

[0046] TSKCRKAGCVYFGTPENKGFCTLCFIEYRENK

[0047] Mutant polypeptide sequence (SEQ ID NO.2):

[0048] TSKCRKAGCMYFGTPENKGFCTLCFIEYRENK

[0049] The mutant polypeptide has a mutation from V to M at the 10th amino acid relative to the original polypeptide, and the amino acid sequence homology reaches 97%.

[0050] 1.2 Peptide construction method

[0051] 1) Obtained by prokaryotic expression and purification: The peptide was obtained by expression and purification of a recombinant plasmid connected with the nucleotide sequence (SEQ ID NO.4): ACAAGCAAGTGCAGGAAAGCTGGCTGCATGTATTTTGGGACTCCA GAAAACAAGGGCTTTTGCACTCTATGTTTCATCGAATACAGAGAAA ATAAG. The peptide was filtered out of endotoxins through a molecular sieve and the endotoxin content was tested to eliminate the effect of endotoxins on the experiment. Finally, the concentration of the peptide was tested. A brief introduction is as follows: a. The N-terminus of the peptide was fused with a GST tag to construct a recombinant plasmid, and protein expression and purification were performed through a prokaryotic expression system; b. The target protein was obtained through plasmid transformation, small-scale amplification, large-scale amplification and protein induction expression; c. The purified target peptide, i.e., purified polypeptide, was obtained through bacterial lysis, GST purification and on-column enzyme digestion.

[0052] 2) Direct synthesis by the company: The peptide is directly synthesized by the company, and the purity of the synthesized peptide is greater than 98%. The synthesized peptide is dissolved in double distilled water.

[0053] 2. Modeling

[0054] 6-8 week old C57 / BL6 male mice were purchased from Vital River Biotechnology. After acclimation for about one week, sepsis models were established using two methods:

[0055] 1) LPS induction: The above-mentioned 6-8 week old C57 / BL6 male mice were selected. When the mice weighed about 20-25 g, LPS was injected intraperitoneally once at a dose of 25 mg / kg (solvent: PBS). The mice were observed for 72 hours of survival, and their lung tissues were collected. The success of the mouse model construction was evaluated by tissue HE staining combined with the mouse survival period.

[0056] 2) Cecal ligation (CLP): Select the above-mentioned 6-8 week old C57 / BL6 male mice, and start the experiment when the mice weigh about 25g (fast for 12 hours before the experiment). Depilate the abdomen of the mice, disinfect with alcohol, anesthetize with avertin, and fix the anesthetized mice on the operating board in a supine position. Use a scalpel to make an opening in the abdomen, with an incision of about 2 cm; enter the abdomen through the incision and ligate 1 / 3 of the cecum at the distal end of the cecum with No. 3 line, pierce the ligated section of the cecum with a No. 18 syringe needle, squeeze out a certain amount of feces; suture the wound. Observe the survival of the mice for nearly 7 days, and collect the lung tissue of the mice. The success of the mouse model construction is evaluated by tissue HE staining combined with the survival of the mice.

[0057] 3. Peptide intervention

[0058] 1) Preventive intervention: Mice were purchased as described above, and the peptide was intraperitoneally injected 1 hour before the establishment of the sepsis model, with a dose of 200 μg per mouse (LPS model). Mice injected with PBS were used as controls, and the efficacy of the peptide was evaluated by observing the survival time of the mice and HE staining of the lung and kidney tissues.

[0059] 2) Therapeutic intervention: Mice were purchased as described above, and the peptides were injected intraperitoneally or intravenously within 1 hour after the establishment of the sepsis model, with a dose of 200 μg (LPS model) or 300 μg (CLP model) per mouse. Mice injected intraperitoneally or intravenously with PBS were used as controls, and the efficacy of the peptides was evaluated by observing the survival of the mice.

[0060] 4. HE staining of tissue

[0061] The lung tissue was cut into pieces of about 0.5 cm in size, fixed with 4% paraformaldehyde for 24-72 hours, then rinsed overnight, dehydrated with xylene, embedded in paraffin and sliced, the slice thickness was generally about 5 μm. The slices were dried at 63°C for 2 hours, quickly placed in xylene I and II for 10 minutes each, then dewaxed with 100%, 95%, 80% and 70% alcohol, and stained with eosin and hematoxylin. After returning to blue, the slices were dehydrated, dried and sealed, and the images were taken.

[0062] 5. Survival Observation

[0063] After modeling, the timing starts, and the survival of mice is regularly observed every 6-12 hours. The number and time of death of mice are recorded in time, and finally the survival curve is drawn according to the time point and number of mouse deaths.

[0064] 2. Experimental Results

[0065] 1. Peptides have anti-inflammatory effects

[0066] Immortalized macrophages (iBMDM) were used at a rate of 5×10 5 The cells were plated at a density of 100 μg / ml in a six-well plate. After 12 hours, the cells were treated with synthetic peptides at a final concentration of 100 ng / ml. After 24 hours, LPS (100 μg) or TNFα (20 ng) was added for stimulation for 6 hours, and the changes of inflammatory factors such as IL-1β and IL-6 were detected. Figure 1 It can be seen that compared with the simple stimulation group, the inflammatory factors in the polypeptide treatment group were significantly downregulated.

[0067] 2. Preventive effect of purified peptides on sepsis

[0068] First, the preventive effect of the purified peptide on the mouse sepsis model was tested. The survival time of the mice and the pathological examination of lung tissue were Figure 2 As shown. Figure 2 Figure a shows the changes in the survival of mice under the intervention of purified polypeptides at different concentrations (n=10); Figure b shows the pathological detection of lung tissues of mice under the intervention of purified polypeptides at different concentrations.

[0069] Depend on Figure 2 It can be seen that: compared with the control group (PBS), the survival time of mice was significantly improved after intervention with purified polypeptides, and all mice in the sepsis model survived when the polypeptide concentration was 5 mg / kg; at the same time, the improvement of mouse sepsis by polypeptides was also reflected at the pathological level. Compared with the control group (PBS), the lung tissue of mice tended to be normal after intervention with purified polypeptides.

[0070] 3. Preventive effect of synthetic peptides on sepsis

[0071] In order to exclude the influence of potential residual endotoxin in purified peptides on experimental results, peptides were synthesized in vitro with a purity of more than 98%. The preventive effect of the synthesized peptides on the mouse sepsis model was then tested. The survival time of mice and lung tissue pathology tests were as follows: Figure 3 As shown. Figure 3 Figure a shows the changes in the survival period of mice under the intervention of different concentrations of synthetic peptides (n=10); Figure b shows the pathological detection of lung tissue of mice under the intervention of different concentrations of synthetic peptides.

[0072] Depend on Figure 3It can be seen that compared with the control group (PBS), the survival time of mice was also significantly improved after intervention with synthetic peptides; at the same time, the improvement of sepsis in mice by peptides was also reflected at the pathological level. Compared with the control group (PBS), the lung tissue of mice tended to be normal after intervention with synthetic peptides.

[0073] 4. The therapeutic effect of peptides on sepsis

[0074] Finally, the therapeutic effect of the peptide on sepsis was tested. Figure 4 As shown. Figure 4 Figure a shows the therapeutic effect of purified peptides in the LPS model (n=10); b shows the therapeutic effect of synthetic peptides in the LPS model (n=10); c shows the therapeutic effect of synthetic peptides in the CLP model (n=20), in which a normal group of sham mice without surgery was added as a negative control. As can be seen from the figure, both purified and synthetic peptides have a good therapeutic effect on the mouse sepsis model.

[0075] 5. The mutant peptide has the same effect as the original peptide

[0076] Finally, the preventive effect of the mutant peptide on the mouse sepsis model was tested. Figure 5 It can be seen that after the intervention of the synthetic mutant polypeptide, it has a similar effect to the synthetic original polypeptide group, and the survival time of mice is significantly improved.

[0077] In the preliminary experiments, another polypeptide (amino acid sequence SEQ ID NO.3: CCKLTAKNGRSYEYGTEIFKNRFGPKCVFTEC) was designed and applied to the intervention and treatment of sepsis. The experimental results showed no effect and all the experimental animals died within a short period of time. This shows that the polypeptide of the present invention has an excellent effect in preventing and treating sepsis through its specific amino acid composition.

[0078] In summary, the polypeptide of the present invention, whether artificially synthesized or purified by prokaryotic expression, can reduce inflammatory factors in macrophages, improve lung damage caused by sepsis, and prolong survival. It has a significant effect in preventing and treating sepsis, and its preparation into a pharmaceutical preparation has great potential for preventing and treating sepsis, and has practical application and promotion value.

Claims

1. A polypeptide for preventing and / or treating sepsis, characterized in that: It is a polypeptide having an amino acid sequence at least 97% homologous to SEQ ID NO:

1.

2. The polypeptide according to claim 1, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

3. A gene fragment encoding the polypeptide according to claim 1 or 2, characterized in that: Its nucleotide sequence is shown in SEQ ID NO:

4.

4. Use of the polypeptide according to claim 1 or 2 in the preparation of a medicament for preventing and / or treating sepsis.

5. The use according to claim 4, characterized in that The drug is a drug that downregulates inflammatory factors in macrophages.

6. The use according to claim 5, characterized in that The inflammatory factors include IL-1β and IL-6.

7. The use according to claim 5, characterized in that The drug is a drug for improving lung tissue damage.

8. A drug for preventing and / or treating sepsis, characterized in that: The invention comprises a polypeptide having an amino acid sequence with at least 97% homology to SEQ ID NO: 1 and a pharmaceutically acceptable auxiliary material.

9. The drug according to claim 8, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

10. The drug according to claim 8, characterized in that It is an injection preparation or an oral preparation, preferably an injection preparation.

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