阅读说明：本技术 一种天蚕素f蛋白抗菌肽及其应用 (Cecropin F protein antibacterial peptide and application thereof ) 是由 程功 肖小平 陈依玲 于 2020-03-20 设计创作，主要内容包括：本发明公开了一种天蚕素F蛋白抗菌肽及其应用,所述天蚕素F蛋白抗菌肽的氨基酸序列如SEQ ID NO.1所示,其对应的基因的核酸序列如SEQ ID NO.2所示。本发明所述的天蚕素F蛋白抗菌肽经过试验验证对大肠杆菌和绿脓杆菌具有很强的抑制作用,可用于制备预防和抵抗细菌感染的伤口的修复的药物使用、以及美容相关行业中的预防和抵抗细菌感染的产品使用,还可以作为食品行业中抑制细菌的保鲜剂使用。(The invention discloses cecropin F protein antibacterial peptide and application thereof, wherein the amino acid sequence of the cecropin F protein antibacterial peptide is shown as SEQ ID NO.1, and the nucleic acid sequence of the corresponding gene is shown as SEQ ID NO. 2. The cecropin F protein antibacterial peptide disclosed by the invention has a strong inhibiting effect on escherichia coli and pseudomonas aeruginosa through experimental verification, can be used for preparing medicaments for preventing and resisting repair of wounds caused by bacterial infection and products for preventing and resisting bacterial infection in related beauty treatment industries, and can also be used as a preservative for inhibiting bacteria in food industries.)

1. An cecropin F protein antibacterial peptide is characterized in that the amino acid sequence of the cecropin F protein antibacterial peptide is shown as SEQ ID No.1, and the nucleic acid sequence of the gene is shown as SEQ ID No. 2.

2. Use of cecropin F protein antibacterial peptide according to claim 1 for the preparation of a medicament for the prevention and treatment of diseases of bacterial infection.

3. Use of cecropin F protein antimicrobial peptide according to claim 1 for the preparation of a medicament for the prevention and repair of wounds against bacterial infection.

4. Use of cecropin F protein antimicrobial peptide according to claim 2 or 3 in the manufacture of a medicament, wherein the bacteria comprise escherichia coli and pseudomonas aeruginosa.

5. Use of cecropin F protein antimicrobial peptide according to claim 1 for the preparation of a cosmetic product for the prevention and resistance of bacterial infections.

6. The use of cecropin F protein antimicrobial peptide according to claim 1 in the preparation of food preservation products for inhibiting bacteria.

[ technical field ] A method for producing a semiconductor device

The invention belongs to the technical field of biological medicines, and particularly relates to cecropin F protein antibacterial peptide and application thereof.

[ background of the invention ]

Previous research shows that the antibacterial peptide has inhibition effect on bacteria, protozoa and viruses. The antibacterial peptide plays an antibacterial role by acting on the cell membrane of bacteria, and the target strain is not easy to mutate by a special mechanism that charge adsorption acts on the cell membrane of the bacteria, so the antibacterial peptide has wide application prospect in the medical industry.

Currently, the antibacterial peptides found in nature are mainly classified into defensins (defensins in) and cecropin (cecropin). Cecropin was first discovered from the drosophila genome and later subsequently discovered in many species in succession. Cecropin generally contains 37-39 amino acid residues and does not contain cysteine, the N-terminal region of cecropin has strong basicity and can form an almost perfect amphiphilic spiral structure, a hydrophobic spiral can be formed in the C-terminal region of cecropin, a hinge region formed by glycine and proline is arranged between cecropin and hydrophobic spiral, and the C-terminal of most polypeptides is amidated, and the amidation plays an important role in the antibacterial activity of most polypeptides. Different sources of cecropin have different corresponding antibacterial spectra.

In recent years, chronic wound infections have placed tremendous strain on patients and society, and bacterial infections can further delay wound healing and can severely cause sepsis. Due to the emergence of drug-resistant bacteria, the treatment effect of antibacterial drugs on wound infection is poor, so that the search for novel wound anti-infective drugs is urgent. The cecropin antibacterial peptide is expected to become one of the medicines for treating wound infection due to the advantages of unique action mechanism, difficult generation of drug resistance and the like.

[ summary of the invention ]

In order to solve the above problems, the present invention provides a cecropin F protein antimicrobial peptide, and the object of the present invention is achieved by:

the Cecropin F protein (Cecropin F) is derived from the genome of Aedes aegypti, is numbered AAEL000625, is called CecF for short, and has the total length of 59 amino acidsThe amino acid sequence is shown as follows:

the underlined part is a signal peptide part, and the inventors cut the signal peptide part to leave 36 amino acids, thereby obtaining the cecropin F protein antibacterial peptide of the present invention, the amino acid sequence of which is shown below:

the nucleic acid sequence of the gene is shown as follows:

among them, the underlined part is a coding region (ORF), and the other part is an untranslated region.

The invention also provides application of the cecropin F protein antibacterial peptide in preparing a medicament for preventing and resisting diseases caused by bacterial infection.

The invention also provides application of the cecropin F protein antibacterial peptide in preparing a medicament for preventing and resisting repair of a wound infected by bacteria.

Preferably, the bacteria include escherichia coli and pseudomonas aeruginosa.

The invention also provides application of the cecropin F protein antibacterial peptide in preparing a beauty product for preventing and resisting bacterial infection.

The invention also provides application of the cecropin F protein antibacterial peptide in preparation of food fresh-keeping products for inhibiting bacteria.

The invention also provides a synthetic method of the cecropin F protein antibacterial peptide, which comprises the following steps:

s1, weighing n equivalents of resin, putting the resin into a reactor, adding DCM (dichloromethane) to swell for half an hour, then pumping out DCM, adding 2n equivalents of the first amino acid in the sequence of SEQ ID NO.1, adding 2n equivalents of DIEA, a proper amount of DMF, DCM (the proper amount is that the resin can be fully stirred), DIEA (diisopropylethylamine), DMF (dimethylformamide), DCM, and nitrogen bubbling reaction for 60 min. Then adding about 5n equivalent of methanol, reacting for half an hour, pumping out reaction liquid, and washing with DMF and MEOH;

s2, adding the second amino acid (also 2N equivalent) in the sequence of SEQ ID NO.1, 2N equivalent HBTU (1-hydroxy, benzo, trichloroazol tetramethyl hexafluorophosphate) and DIEA, N2 into the reactor, carrying out bubbling reaction for half an hour, washing off liquid, detecting ninhydrin, and then capping with pyridine and acetic anhydride. Finally, cleaning, adding a proper amount of decapping liquid to remove the Fmoc (9-fluorenylmethyloxycarbonyl) protecting group, cleaning, and detecting ninhydrin;

s3, adding different amino acids in the SEQ ID NO.1 sequence in sequence according to the mode of the step S2, and carrying out various modifications;

s4, drying the resin by using nitrogen, taking the resin out of the reaction column, pouring the resin into a flask, adding a certain amount of cutting fluid (the composition is 95% TFA, 2% ethanedithiol, 2% triisopropylsilane and 1% water) (the ratio of the cutting fluid to the resin is about 10 ml/g) into the flask, shaking, and filtering the resin to obtain a filtrate;

s5, adding a large amount of ether into the filtrate of S4, separating out a crude product, centrifuging, and cleaning to obtain a crude product of the sequence;

s6, purifying the crude product of S5 to 90.07% by high performance liquid chromatography.

S7, putting the liquid purified in the S6 into a freeze dryer for concentration, and freeze-drying the liquid into white powder to obtain the cecropin F protein antibacterial peptide.

Compared with the prior art, the invention has the following beneficial effects: the cecropin F protein antibacterial peptide is cecropin F protein derived from Aedes aegypti genome, and experiments prove that the medicine or preparation containing the cecropin F protein antibacterial peptide has strong inhibition effect on escherichia coli and pseudomonas aeruginosa. Escherichia coli is a clinical opportunistic infection pathogen, is widely distributed in the environment and is easy to infect in an operation; pseudomonas aeruginosa is a clinically serious pathogenic bacterium, and the bacterium is easy to generate drug resistance, multiple drug-resistant bacteria appear clinically, and the bacterium can be suppurative after infection and can possibly die if the treatment is not carried out in time. The medicament utilizing the cecropin F protein antibacterial peptide can prevent and treat infection of escherichia coli and pseudomonas aeruginosa and reduce the use of antibiotics; secondly, the generation of drug-resistant bacteria can be prevented; thirdly, the wound healing can be promoted; fourthly, the product can be used as a product related to beauty and skin care for preventing and resisting bacterial infection; fifthly, the bacteriostatic agent can be used as a fresh-keeping bacteriostatic agent in the food fresh-keeping industry.

[ description of the drawings ]

FIG. 1 shows the inhibitory activity of the CecF antimicrobial peptide solutions of different concentrations of the present invention against E.coli.

FIG. 2 is a graph showing the effect of the cecF antimicrobial peptide of the present invention on the minimum effective concentration for inhibiting the proliferation of E.coli.

FIG. 3 is a graph showing the effect of the CecF antimicrobial peptide of the present invention on the minimum effective concentration for inhibiting the proliferation of Pseudomonas aeruginosa.

FIG. 4 shows the present invention wound production in mice of the test group and the control group subjected to the wound healing test.

FIG. 5 is a graph showing the effect of the cecF antimicrobial peptides of the present invention on wound healing in mice.

FIG. 6 shows the present invention wound production in mice of the test group and the control group tested for wound bacterial infection.

FIG. 7 shows mice from the experimental group and the control group in which the wound of the present invention is infected with bacteria.

FIG. 8 is a graph showing the effect of the CecF antimicrobial peptides of the present invention on the healing of mouse wound-infected bacteria.

[ detailed description ] embodiments

The principles and features of this invention are described in conjunction with the following examples, which are set forth merely to illustrate the invention and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.