CN115724937B

CN115724937B - Repeated motif of Sericiin 3 protein with antibacterial activity and application thereof

Info

Publication number: CN115724937B
Application number: CN202211030502.XA
Authority: CN
Inventors: 赵萍; 郭凯雨; 张晓璐; 夏庆友
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2022-08-26
Filing date: 2022-08-26
Publication date: 2023-09-15
Anticipated expiration: 2042-08-26
Also published as: CN115724937A

Abstract

The invention discloses a repeated motif of a Sericiin 3 protein with antibacterial activity and application thereof, wherein the amino acid sequence of the repeated motif of the Sericiin 3 protein is shown as SEQ ID NO.9, and a polypeptide with a molecular size of 5kDa has antibacterial activity on escherichia coli, staphylococcus aureus and micrococcus luteus.

Description

Repeated motif of Sericiin 3 protein with antibacterial activity and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a repetitive motif of a Sericiin 3 protein with antibacterial activity, and also relates to application of the repetitive motif.

Background

Silk is the earliest and most widely used natural animal protein fiber for humans. The silk cocoon silk is composed of two parts of sericin and silk fibroin. Silk is the central component of silk and accounts for about 70-80% of the weight of silk. Sericin is a gelatinous protein, which is present at the periphery of silk fibroin, and occupies about 20-30% of the total silk. The current study found and identified 5 Sericin proteins altogether and were named Sericin1, sericin2, sericin3, sericin4 and Sericin5, respectively. Of these, sericin1 and Sericin3 are the main Sericin proteins in silk, while Sericin2, sericin4 and Sericin5 are present in non-silk secreted in the young silkworm. In silk cocoon silk, sericin3 and Sericin1 are sequentially arranged from the outer layer to the inner layer, and in non-cocoon silk, sericin mainly comprises Sericin2, sericin5 and Sericin4 from the outer layer to the inner layer. In recent years, research shows that the sericin has better antibacterial property and is an excellent natural biological material. In 2003, sarovart et al found that sericin had an inhibitory effect on micrococcus growth. In 2010, khalid et al found that sericin has good antibacterial properties and can inhibit the growth of pathogenic bacteria such as pseudomonas aeruginosa, staphylococcus aureus, escherichia coli and the like. In 2012, chen Zhongmin et al studied small molecule sericin peptides prepared by extracting sericin from cocoon shells and subjecting the cocoon shells sericin to enzymolysis, and found that both the extracted sericin and the sericin peptides have remarkable inhibitory effects on staphylococcus aureus and escherichia coli.

The sericin has antibacterial activity, so that the sericin can be used as a natural antibacterial agent, and has potential application value in various fields of agriculture, medicine, food, beauty cosmetics and the like. At present, many reports on the aspects of sericin antibacterial research exist, but the sericin antibacterial research is performed based on sericin mixtures extracted from silkworm cocoons, and the specific sericin has no antibacterial effect. In addition, sericin has the characteristics of large molecular weight and highly repeated sequence, and is difficult to separate and express. The method for preparing sericin by mass synthesis is a primary premise for realizing large-scale application of sericin. In recent years, a great deal of research has shown that the highly repetitive motifs in spider silk proteins and fibroin determine their biological functions. Sericin amino acid repetitive motifs account for more than 50% of the full-length sequence, and researchers have obtained a Sericin1 repetitive motif comprising single or multiple repetitive units by polypeptide synthesis and prokaryotic expression, and found that it has anti-apoptotic and fibroblast properties. Therefore, research on sericin repeated motif with biological activity is of great importance for developing sericin antibacterial products.

Disclosure of Invention

Accordingly, it is an object of the present invention to provide a repetitive motif of a Sericiin 3 protein having antibacterial activity; it is a further object of the present invention to provide the use of a repetitive motif of a Sericin3 protein having antibacterial activity for the preparation of an antibacterial agent.

In order to achieve the above purpose, the present invention provides the following technical solutions:

1. the amino acid sequence of the repeated motif of the Sericiin 3 protein with antibacterial activity is shown as SEQ ID NO. 9.

2. Use of a repeated motif of a Sericin3 protein having antibacterial activity in the preparation of an antibacterial formulation.

Preferably, the antibacterial agent is an antibacterial agent against gram-positive or gram-negative bacteria.

Preferably, the antibacterial agent is an antibacterial agent against Escherichia coli, staphylococcus aureus or Micrococcus luteus.

The invention has the beneficial effects that: the invention provides a repeated motif of a Sericiin 3 protein with antibacterial activity, which is obtained by truncating a repeated unit of the Sericiin protein, and the polypeptide with the size of 5kDa has antibacterial activity on escherichia coli, staphylococcus aureus and micrococcus luteus.

Drawings

In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:

FIG. 1 shows the relative increase rates of E.coli, staphylococcus aureus, bacillus putida, micrococcus luteus, pseudomonas aeruginosa after incubation with the 1 st repeat unit peptide fragment of Serucin 1 (Serucin 1-rp 1);

FIG. 2 shows the relative increase rates of E.coli, staphylococcus aureus, bacillus putida, micrococcus luteus, pseudomonas aeruginosa after incubation with the 2 nd repeat unit peptide fragment of Serucin 1 (Serucin 1-rp 2);

FIG. 3 shows the relative increase rates of E.coli, staphylococcus aureus, bacillus putida, micrococcus luteus, pseudomonas aeruginosa after incubation with Serlicin 2 repeat unit peptide (Serlicin 2-rp);

FIG. 4 shows the relative increase rates of E.coli, staphylococcus aureus, bacillus putida, micrococcus luteus, pseudomonas aeruginosa after incubation with the 2 nd repeat unit peptide fragment of Serucin 3 (Serucin 3-rp 2);

FIG. 5 shows the relative increase rate of E.coli, staphylococcus aureus, bacillus putida, micrococcus luteus after incubation with the 1 st repeat peptide of Serucin 4 (Serucin 4-rp 1);

FIG. 6 shows the relative increase rate of E.coli, staphylococcus aureus, bacillus putida, micrococcus luteus after incubation with the 2 nd repeat peptide fragment of Serucin 4 (Serucin 4-rp 2);

FIG. 7 shows the relative increase rate of E.coli, staphylococcus aureus, bacillus putida, micrococcus luteus after incubation of Pseudomonas aeruginosa with the Serialin 5 repeat unit peptide fragment (Serialin 5-rp);

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.

Example 1, sericiin protein bioinformatics analysis

Nucleotide and amino acid sequences of Sericin1 (gene number gi| 1938996292) (SEQ ID NO. 1), sericin2 (gene number gi| 289803015) (SEQ ID NO. 2), sericin3 (gene number gi| 167860163) (SEQ ID NO. 3), sericin4 (gene number gi| 1938997474) (SEQ ID NO. 4), sericin5 (gene number gi| 1938997822) (SEQ ID NO. 5) downloaded based on NCBI database (https:// www.ncbi.nlm.nih.gov /). Sequence analysis was performed using the following online software and biological software:

hydrophilic-hydrophobic and isoelectric point prediction of proteins: expASY (http:// www.expasy.org/tools /);

based on the previous results, 5 silkworm Sericin protein repeat motifs were analyzed and the results are shown in table 1.

TABLE 1 repeated sequence characterization of silkworm Sericiin proteins

Example 2 Synthesis of Sericiin protein repeat units

The single or multiple repeat units of the Serin protein are obtained by chemical synthesis and are designated as Serin 1-rp1, serin 1-rp2, serin 2-rp, serin 3-rp2, serin 4-rp1, serin 4-rp2 and Serin 5-rp, respectively. The sequence 1-rp1 (Ser 1-rp 1) contains 2 minimum repeating units, the sequence 1-rp2 (Ser 1-rp 2) contains 2 minimum repeating units, the sequence 2-rp (Ser 2-rp) contains 3 minimum repeating units, the sequence 3-rp2 (Ser 3-rp 2) contains 6 minimum repeating units, the sequence 4-rp1 (Ser 4-rp 1) contains 1 minimum repeating unit, the sequence 4-rp2 (Ser 4-rp 2) contains 1 minimum repeating unit, and the sequence 5-rp (Ser 5-rp) contains 1 minimum repeating unit, as shown in Table 2.

TABLE 2 amino acid sequence of repeating units of silkworm Sericiin protein

Example 3 verification of bacteriostatic Activity of peptide fragments of the repeated units of the Sericin protein

To evaluate the bacteriostatic activity of peptide fragments of different Sericiin proteins, five bacteria were selected for this experiment: coli (gram-negative bacteria, G-), putida (gram-negative bacteria, G-), pseudomonas aeruginosa (gram-negative bacteria, G-), staphylococcus aureus (gram-positive bacteria, g+), micrococcus luteus (gram-positive bacteria, g+), respectively incubating with each of the serucin protein repeating unit peptide fragments, detecting the growth curve of the bacteria by spectrophotometry, and calculating the bacterial growth rate relative to the control group. The specific method comprises the following steps:

1) Dissolving a sufficient amount of the peptide of the repeated unit of the Sericiin protein to a protein concentration of 0.1mg/mL by using sterile Phosphate Buffer (PBS);

2) Using LB liquid medium, the culture medium was used toBacteria are cultivated to OD ₆₀₀ ＝0.2-0.3；

3) Taking 50 mu L of protein solution and 150 mu L of bacterial liquid, respectively adding the protein solution and the bacterial liquid into each hole of a 96-well plate, fully and uniformly mixing, and taking a sterile PBS solution group without adding protein as a blank control and an ethylenediamine tetraacetic acid (EDTA) solution group with equal amount of protein as a positive control;

4) The 96-well plate was incubated at 37℃and 60rpm for 8 hours and the OD of each well was measured every 1 hour using an ultraviolet spectrophotometer ₆₀₀ Is recorded and recorded;

5) The relative growth rate of the recorded data based on the blank group is calculated according to the following formula:

6) The relative growth rate curves of the groups are plotted according to the processed data, and the results are shown in fig. 1-7.

Experimental results show that the experimental results of the protein of the Serin show that the repeated unit peptide fragments of the proteins of the Serin 1, the Serin 2, the Serin 4 and the Serin 5 are applied to the escherichia coli, the five bacteria of staphylococcus aureus, malodor bacillus, micrococcus luteus and pseudomonas aeruginosa have no inhibition effect, and only the 2 nd repeating unit peptide of the protein of the Sericiin 1 has a slight inhibition effect on micrococcus luteus, but the inhibition effect is poor. The 2 nd repeating unit peptide of the Sericiin 3 protein shows inhibition activity to three bacteria of escherichia coli, staphylococcus aureus and micrococcus luteus, and the specific result is that the maximum inhibition rate of the Sericiin 3 protein repeating unit peptide (Ser 3-rp 2) to escherichia coli (G-); for Staphylococcus aureus (G+), the maximum inhibition rate of the peptide fragment of the repeated unit of the Sericiin 3 protein (Ser 3-rp 2) was about 35.7%; for Micrococcus luteus (G+), the maximum inhibition of the peptide fragment of the repeated unit of the protein Sericiin 3 (Ser 3-rp 2) was about 25.9%. The repeated unit peptide of the Sericiin 3 protein can continuously inhibit bacteria in the experimental process. As in fig. 5.

In summary, the invention obtains peptide fragments of 7 kinds of Sericiin protein repeating units by a polypeptide synthesis mode, and verifies the antibacterial activity of the peptide fragments in vitro. The results of bacteriostasis experiments show that the repeated units of the proteins of the Sericin1, the Sericin2, the Sericin4 and the Sericin5 have no bacteriostasis activity, and the repeated units of the protein of the Sericin3 have better inhibition activity on gram-positive bacteria and gram-negative bacteria. The result of the method determines for the first time that the protein Sericiin 3 has antibacterial activity, and compared with the mode of acquiring the repeated unit of the protein Sericiin by a prokaryotic expression technology reported before, the method disclosed by the invention has the advantages of poor stability, low efficiency, long experimental period and the like. Through chemical synthesis of the repeated units of the Sericiin 3 protein, the experimental cost is further reduced, and the success rate of the experiment is improved.

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims

1. A peptide fragment of the repetitive motif of the protein of Sericin3 having antibacterial activity, characterized in that: the amino acid sequence of the peptide fragment is shown as SEQ ID NO. 9.

2. Use of the peptide according to claim 1 for the preparation of an antibacterial agent against escherichia coli, staphylococcus aureus or micrococcus luteus.