A polypeptide for preventing and / or treating sepsis

Abstract

The present application relates to the technical field of biotechnology, and particularly discloses a kind of polypeptide for preventing and / or treating sepsis, which is a polypeptide with at least 97% homology with SEQ ID NO:1 in amino acid sequence.By test, the polypeptide of the present application can reduce inflammatory factors in macrophages, improve lung injury caused by sepsis, and prolong survival period, whether by artificial synthesis or prokaryotic expression and purification.In terms of preventing and treating sepsis, the present application has remarkable effect, and has great potential in preventing and treating sepsis as a pharmaceutical preparation, and has practical application and popularization value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a polypeptide for the prevention and / or treatment of sepsis. Background Technology

[0002] Sepsis is a systemic immune response following infection, which can progress to septic shock, multiple organ failure, and even death in later stages. Clinically, sepsis is a serious complication in critically ill patients with trauma, burns, shock, or infection. Due to its rapid onset and progression, and high mortality rate, it poses significant challenges to clinical treatment. Studies show that in 2017, there were approximately 4,890 cases of sepsis worldwide, with about 11 million deaths, accounting for 19.7% of all deaths globally. Despite continuous advancements in medical technology, the clinical cure rate for sepsis has not significantly improved, and the global mortality rate remains high, placing a tremendous physical, psychological, and economic burden on patients. There is an urgent need for the development of an effective new treatment.

[0003] With the increasing maturity of biotechnology and peptide synthesis technology, more and more peptide drugs are being developed and applied clinically. Due to their broad indications, high safety, and significant efficacy, peptide drugs are currently widely used in the prevention and treatment of diseases such as cancer, hepatitis, diabetes, and AIDS. Patent CN111484544A discloses a peptide for treating sepsis, which can increase the survival rate of sepsis model mice to 67%, and its efficacy has room for further improvement to benefit sepsis patients. Summary of the Invention

[0004] In view of this, the present invention aims to provide a polypeptide for the prevention and / or treatment of sepsis, which is a polypeptide whose amino acid sequence has at least 97% homology with 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, the nucleotide sequence of which is shown in SEQ ID NO:4.

[0007] The present invention also provides the use of the aforementioned polypeptide in the preparation of a medicament for the prevention and / or treatment of 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] Further, the drug is a drug for improving lung tissue damage.

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

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

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

[0014] Further, it is an injection preparation.

[0015] The amino acid sequence of the polypeptide of the present application is shown in SEQ ID NO: 1 or SEQ ID NO: 2, which has been proved by experiments that the polypeptide can reduce inflammatory factors in macrophages, improve lung damage caused by sepsis, and prolong survival period, whether it is synthesized by artificial synthesis or purified by prokaryotic expression. In the prevention and treatment of sepsis, the effect is remarkable, and it has great potential to prevent and treat sepsis as a pharmaceutical preparation, and has practical application and popularization value.

[0016] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modifications, substitutions or changes can be made without departing from the above basic technical idea of the present application.

[0017] The above content of the present application will be further described in detail through the specific embodiments below. However, this should not be understood as limiting the scope of the above subject matter of the present application to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Synthetic polypeptide effectively inhibits inflammatory response

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

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

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

[0022] Figure 5 Mutated polypeptide has similar effect as original polypeptide DETAILED DESCRIPTION

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

[0024] Preparation of an injection preparation containing the polypeptide of the present application

[0025] The polypeptide having the amino acid sequence shown in SEQ ID NO: 1 is mixed with water for injection, filtered and sterilized to prepare an injection preparation.

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

[0027] TSKCRKAGCVYFGTPENKGFCTLCFIEYRENK

[0028] Preparation of an injection preparation containing the polypeptide of the present application

[0029] The polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is mixed with water for injection, filtered and sterilized to prepare an injection preparation.

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

[0031] TSKCRKAGCMYFGTPENKGFCTLCFIEYRENK

[0032] Preparation of an injection preparation containing the polypeptide of the present application

[0033] The polypeptide having the amino acid sequence shown in SEQ ID NO: 1 is mixed with water for injection, filtered and sterilized, and freeze-dried to prepare an injection preparation.

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

[0035] TSKCRKAGCVYFGTPENKGFCTLCFIEYRENK

[0036] Preparation of an injection preparation containing the polypeptide of the present application

[0037] The polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is mixed with water for injection, filtered and sterilized, and freeze-dried to prepare an injection preparation.

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

[0039] TSKCRKAGCMYFGTPENKGFCTLCFIEYRENK

[0040] The advantageous effects of the present application are further illustrated by the following experimental examples

[0041] Experimental Example 1: Study of polypeptide for treating sepsis

[0042] I. Experimental methods

[0043] 1. Construction of polypeptide

[0044] 1.1 Polypeptide for experiment

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

[0046] TSKCRKAGCVYFGTPENKGFCTLCFIEYRENK

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

[0048] TSKCRKAGCMYFGTPENKGFCTLCFIEYRENK

[0049] The amino acid at position 10 of the mutated polypeptide is mutated from V to M, and the amino acid sequence homology reaches 97% compared with the original polypeptide.

[0050] 1.2 Method for constructing polypeptide

[0051] 1) Obtained by prokaryotic expression and purification: the peptide segment was obtained by expression and purification of a recombinant plasmid having a nucleotide sequence (SEQ ID NO. 4): ACAAGCAAGTGCAGGAAAGCTGGCTGCATGTATTTTGGGACTCCAGAAAACAAGGGCTTTTGCACTCTATGTTTCATCGAATACAGAGAAAATAAG, and the obtained peptide segment was filtered through a molecular sieve to remove endotoxin and subjected to endotoxin content detection to eliminate the influence of endotoxin on the experiment. Finally, the concentration of the peptide segment was detected. Briefly introduced as follows: a. The N-terminal of the peptide segment is fused with a GST tag to construct a recombinant plasmid, and the protein is expressed and purified through a prokaryotic expression system; b. The target protein is obtained through plasmid transformation, small-scale amplification, large-scale amplification and protein induction expression; c. The purified target peptide segment, i.e. the purified polypeptide, is obtained through bacterial lysis, GST purification and on-column enzyme digestion.

[0052] 2) Direct synthesis by company: the polypeptide is directly synthesized by the company, and the synthesized polypeptide has a purity of more than 98%. The synthesized polypeptide is dissolved with double-distilled water.

[0053] 2. Modeling

[0054] 6-8 weeks C57 / BL6 male mice were purchased from Vivotecnia Company, and after about one week of adaptation, the sepsis model was constructed by two methods:

[0055] 1) LPS induction: select the above 6-8 weeks C57 / BL6 male mice, mice weighing about 20-25 g, a one-time injection of LPS in the abdominal cavity, the injection dose is 25 mg / Kg (solvent is PBS), observe the survival of mice for 72 hours, and collect the lung tissue of mice, through the HE staining of tissue combined with the survival of mice to evaluate the success of the construction of mouse model.

[0056] 2) Cecal ligation and puncture (CLP): select the above 6-8 weeks C57 / BL6 male mice, mice weighing about 25 g, start the experiment (fast for 12 hours before the experiment). The abdomen of the mouse is depilated and disinfected with alcohol, and the mouse is anesthetized with avertin and fixed on the operation board in a supine position after anesthesia. Make an incision about 2 cm on the abdomen; enter the abdomen through the incision and ligate 1 / 3 of the distal cecum with a No. 3 thread, puncture the ligated cecum with an 18-gauge syringe needle, and squeeze out a certain amount of feces; suture the wound. Observe the survival of mice for nearly 7 days, and collect the lung tissue of mice, through the HE staining of tissue combined with the survival of mice to evaluate the success of the construction of mouse model.

[0057] 3. Polypeptide intervention

[0058] 1) Preventive intervention: as described above, purchase mice, 1 hour before the construction of sepsis model, inject polypeptide into the abdominal cavity, the dose of each mouse is 200 μg (LPS model), at the same time, inject PBS into the abdominal cavity of mice as control, evaluate the efficacy of polypeptide by observing the survival of mice, HE staining of lung and kidney tissues.

[0059] 2) Therapeutic intervention: as described above, purchase mice, within 1 hour after the construction of sepsis model, inject polypeptide into the abdominal cavity or intravenously, the dose of each mouse is 200 μg (LPS model) or 300 μg (CLP model), at the same time, inject PBS into the abdominal cavity or intravenously of mice as control, evaluate the efficacy of polypeptide by observing the survival of mice.

[0060] 4. HE staining of tissue

[0061] Cut the lung tissue into pieces, about 0.5 cm in size, fix with 4% paraformaldehyde for 24-72 hours, then wash overnight, dehydrate with xylene, then paraffin embedding and tissue sectioning, the thickness of the section is generally about 5 μm. 63°C for 2 hours, quickly put the slice into xylene I and II, each for 10 minutes, then deparaffinization treatment with 100%, 95%, 80% and 70% alcohol, and perform eosin and hematoxylin staining, return blue, dehydrate, dry, and mount the slide, and take pictures.

[0062] 5. Survival observation

[0063] After modeling, timing was started, and the survival status of mice was observed regularly every 6-12 hours. The number and time of mouse deaths were recorded in a timely manner. Finally, a survival curve was plotted based on the time and number of mouse deaths.

[0064] II. Experimental Results

[0065] 1. Peptides have anti-inflammatory effects.

[0066] Immortalized macrophages (iBMDM) at 5 × 10 5 Cells were seeded in six-well plates at a density of [insert density here], and after 12 hours, synthetic peptides were added to treat the cells 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 changes in 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 peptide treatment group were significantly downregulated.

[0067] 2. The preventive effect of purified polypeptides on sepsis.

[0068] First, the preventive effect of the purified peptide on a mouse model of sepsis was tested. The survival time of the mice and lung tissue pathological examination were as follows: Figure 2 As shown. Among them. Figure 2 In Figure a, the changes in the survival time of mice under different concentrations of purified peptide intervention (n=10) are shown; in Figure b, the pathological examination of mouse lung tissue under different concentrations of purified peptide intervention is shown.

[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 peptides, and all mice in the sepsis model survived when the peptide concentration was 5 mg / kg; 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 purified peptides.

[0070] 3. The preventive effect of synthetic polypeptides on sepsis

[0071] To eliminate the potential influence of residual endotoxins from the purified peptide on the experimental results, the peptide was synthesized in vitro with a purity greater than 98%. The preventive effect of the synthesized peptide on a mouse sepsis model was then examined; mouse survival and lung tissue pathology were measured as follows. Figure 3 As shown. Among them. Figure 3 In Figure a, the changes in the survival time of mice under different concentrations of synthetic peptide intervention (n=10) are shown; in Figure b, the pathological examination of mouse lung tissue under different concentrations of synthetic peptide intervention is shown.

[0072] Depend on Figure 3It can be seen that, compared with the control group (PBS), the survival time of mice is also obviously improved after the intervention of the synthetic polypeptide; at the same time, the improvement of the polypeptide on the mouse sepsis is also reflected on the pathological level, compared with the control group (PBS), the lung tissue of the mouse tends to be normal after the intervention of the synthetic polypeptide.

[0073] 4. Therapeutic effect of polypeptide on sepsis

[0074] Finally, the therapeutic effect of the polypeptide on sepsis is detected, as shown in Figure 4 , wherein Figure 4 a is the therapeutic effect of the purified polypeptide in the LPS model (n=10); b is the therapeutic effect of the synthetic polypeptide in the LPS model (n=10); c is the therapeutic effect of the synthetic polypeptide in the CLP model (n=20), wherein a normal group sham without operation is added as a negative control. It can be seen from the figure that, whether the purified polypeptide or the synthetic polypeptide, both have good therapeutic effect on the mouse sepsis model.

[0075] 5. Mutated polypeptide and original polypeptide have the same effect

[0076] Finally, the preventive effect of the mutated polypeptide on the mouse sepsis model is detected, and Figure 5 it can be seen that: after the intervention of the synthetic mutated polypeptide, it has similar effect as the synthetic original polypeptide group, and the survival time of the mouse is obviously improved.

[0077] Another polypeptide (amino acid sequence SEQ ID NO. 3: CCKLTAKNGRSYEYGTEIFKNRFGPKCVFTEC) is designed in the previous experiment, which is applied to sepsis intervention and treatment, and the experimental results show that there is no effect, and the experimental animals all die in a short period of time, which shows that the polypeptide of the application produces excellent effect in preventing and treating sepsis by specific amino acid composition.

[0078] In summary, the polypeptide of the application can reduce inflammatory factors in macrophages, improve lung injury caused by sepsis, and prolong the survival time, whether it is artificially synthesized or purified by prokaryotic expression. The effect is remarkable in preventing and treating sepsis, and it has great potential in preventing and treating sepsis as a pharmaceutical preparation, and has practical application and popularization value.

Claims

1. A polypeptide for use in the prevention and / or treatment of sepsis, characterized in that, which is an amino acid sequence as shown in SEQ ID NO:

2.

2. A gene fragment encoding the polypeptide of claim 1 as shown in SEQ ID NO: 2, characterized in that: which is an amino acid sequence as shown in SEQ ID NO:

2.

3. Use of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 in the preparation of a medicament for preventing and / or treating sepsis.

4. Use according to claim 3, characterized in that, The medicament is a medicament for down-regulating inflammatory factors in macrophages.

5. Use according to claim 4, characterized in that, The inflammatory factors include IL-1β and IL-6.

6. Use according to claim 4, characterized in that, The medicament is a medicament for improving lung tissue damage.

7. A medicament for preventing and / or treating sepsis, characterized by, which comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 and a pharmaceutically acceptable adjuvant.

8. The medicament according to claim 7, characterized in that, The medicament is an injection preparation or an oral preparation.

9. The medicament according to claim 8, characterized in that: The medicament is an injection preparation.

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