CN114671936B

CN114671936B - Bungeana grahami defensive peptide Zs-CATH and gene and application thereof

Info

Publication number: CN114671936B
Application number: CN202210228015.8A
Authority: CN
Inventors: 沈艳; 武静; 杨海龙; 木丽仙; 米鍇; 边慧; 刘彤; 吕靖
Original assignee: Kunming Medical University
Current assignee: Kunming Medical University
Priority date: 2022-03-08
Filing date: 2022-03-08
Publication date: 2023-09-01
Anticipated expiration: 2042-03-08
Also published as: CN114671936A

Abstract

The invention belongs to the technical field of polypeptide application, and particularly provides a rana grahami defensive peptide Zs-CATH, a gene and application thereof, wherein the defensive peptide Zs-CATH is a cyclic polypeptide coded by a rana grahami defensive peptide gene of China amphibious type, has a molecular weight of 3602.47 daltons, an isoelectric point of 10.49, and an amino acid sequence of the defensive peptide is shown as SEQ ID NO. 1. The gene for coding the defending peptide Zs-CATH precursor consists of 628 nucleotides, the nucleotide sequence of which is shown as SEQ ID NO.2, wherein the 352 th to 450 th nucleotides are the coding genes of the mature big frog defending peptide Zs-CATH. The rana grahami defensive peptide Zs-CATH has broad-spectrum antimicrobial property, has remarkable effect of inhibiting bacterial and fungal growth on infectious diseases caused by escherichia coli, staphylococcus aureus and alcaligenes faecalis, and can be used as an antibacterial agent and a medical instrument coating medicament.

Description

Bungeana grahami defensive peptide Zs-CATH and gene and application thereof

Technical Field

The invention belongs to the technical field of polypeptide application, and particularly relates to a rana grahami defensive peptide Zs-CATH, and a gene and application thereof.

Background

Recent studies have shown that antimicrobial peptides are an important component of the defense system of all animals, plants and microorganisms. The polypeptide is a kind of small molecular active polypeptide produced by organism for resisting pathogenic microorganism invasion, and has the characteristics of small molecular weight, good stability, rapid sterilization, difficult generation of drug resistance and the like. The characteristics lead the antibacterial peptide to have very wide application prospect and be hopeful to replace the traditional antibiotic medicament which is easy to generate drug resistance clinically at present. The reason why the antibacterial peptide is not easy to generate drug resistance is that the sterilization mechanism is different from that of the traditional antibiotics. They act mainly on the cell membrane of bacteria, destroying its stability and thus altering the permeability of the cell membrane, so that the content leaks out, leading to cell death. The structural form and biological activity of antimicrobial peptides from different species sources or in the same species are widely different. Researchers at home and abroad are constantly striving to discover antibacterial peptides with novel structures from different species, and directly use or use the antibacterial peptides as templates for molecular transformation. At present, antibacterial peptides have been used in seafood preservation and animal feed additives. The development of antimicrobial peptides with potential medicinal value from various natural resources by combining ecological environment is a hot spot for the research of new polypeptide drugs at present.

Amphibious animals are the most important resources in the resource discovery of antibacterial peptides, the skin of the amphibious animals is exposed, and the amphibious animals can secrete various antibacterial peptides with novel molecular structures and complex and various functions in order to resist the invasion of various microorganisms in the environment. The active polypeptide is widely involved in various physiological activities of organisms, and has various pharmacological activities such as antimicrobial, antitumor, antioxidant, immunoregulatory, wound repair, analgesic and the like. The tree frog is significantly different from other amphibians in that most of their time spent on the tree and most of the other amphibians are in water or on land. The specific sapling life makes the natural enemy faced by the sapling life and the external microorganism different from other amphibians, and the sapling life has an inherent immune defense system with self advantages after adaptive evolution.

According to the reports of domestic and foreign documents, different active polypeptides have been isolated from various biological sources, and some have entered a clinical stage. Although China has a long history of application to amphibians, the amphibians are integrally used as medicines, and the active ingredients and pharmacological properties of the amphibians are relatively weak to study. The big white frog is mainly distributed in Yunnan, tibet and other places, and is one of the characteristic animals in China, but few antibacterial peptides of cathelicidin family in skin secretion are reported.

Disclosure of Invention

The invention aims to solve the problem of weak research on the polypeptide secreted by the tree frog in the prior art.

Therefore, the invention provides a rana grahami defensive peptide Zs-CATH, which is a cyclic polypeptide coded by a rana grahami defensive peptide gene of China amphibious class, has a molecular weight of 3602.47 daltons and an isoelectric point of 10.49, and an amino acid sequence of the rana grahami defensive peptide is shown as SEQ ID No.1 (ASKKGKCNFMCKVKQKLRAIGSKTVIGTVVHKI).

SEQ ID NO.1：

The seventh cysteine and the eleventh cysteine form a pair of intramolecular disulfide bonds.

The coding gene of the precursor of the rana grahami defensive peptide consists of 628 nucleotides (SEQ ID NO: 2), and the sequence from the 5 'end to the 3' end is shown as SEQ ID NO. 2.

SEQ ID NO.2：

Wherein, the 352 th-450 th nucleotide is the coding gene of mature rana grahami defensive peptide Zs-CATH.

The rana grahami defensive peptide Zs-CATH provided by the invention can be used for preparing a broad-spectrum antimicrobial (comprising gram-negative bacteria and gram-positive bacteria) reagent, and has remarkable treatment effect on infectious diseases caused by escherichia coli, staphylococcus aureus and alcaligenes faecalis.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the novel broad-spectrum antimicrobial big tree frog defensive peptide Zs-CATH provided by the invention has remarkable effect of inhibiting the growth of bacteria and fungi on infectious diseases caused by escherichia coli, staphylococcus aureus and alcaligenes faecalis, and can be used as an antimicrobial agent and a medical instrument coating medicament.

The present invention will be described in further detail with reference to the accompanying drawings.

Drawings

FIG. 1 is a schematic diagram showing Circular Dichroism (CD) spectrum analysis of the rana grahami defensive peptide Zs-CATH in SDS solutions with different concentrations.

FIG. 2 is a schematic diagram showing Circular Dichroism (CD) spectrum analysis of the Butterflybush defensive peptide Zs-CATH in LPS solutions with different concentrations.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in the following examples, and it is obvious that the described examples are only some examples of the present invention, but not all examples. Although representative embodiments of the present invention have been described in detail, those skilled in the art to which the invention pertains will appreciate that various modifications and changes can be made without departing from the scope of the invention. Accordingly, the scope of the invention should not be limited to the embodiments, but should be defined by the appended claims and equivalents thereof.

Example 1: preparation of rana grahami defensive peptide Zs-CATH and amino acid sequence determination

1. Extracting total RNA of the skin of the rana grahami:

the living big tree frog is washed with water, put into liquid nitrogen for quick freezing for 10 hours, 300mg skin tissue is taken, 3ml Trizol solution is added, and homogenized in a 20ml glass homogenizer for 30 minutes. An equal volume of phenol/chloroform solution was added, vigorously mixed, left at room temperature for 10 minutes, centrifuged at 12000rpm for 10 minutes at 4℃and the precipitate discarded. Adding isopropyl alcohol with the same volume into the supernatant, standing at room temperature for 10 minutes, centrifuging at 4 ℃ and 12000rpm for 10 minutes, washing the precipitate with 75% ethanol once, and airing, wherein the precipitate at the bottom of the tube is the total RNA of the skin of the bullfrog.

2. Purification of the skin mRNA of the Bungeana Juz:

mRNA was isolated and purified by PROMEGA, inc. of AmericamRNA Isolation Systems kit.

The extracted total RNA of the skin of the Bungeana Juz is dissolved in 500 mu l DEPC water, put in 65 ℃ water bath for 10 minutes, added with 3 mu l Oligo (dT) probe and 13 mu l 20 XSSC solution, mixed evenly, and then placed at room temperature for cooling, and called A solution. The beads (SA-PMP) were flicked and mixed until the magnetic rack adsorbed for 30 seconds, the supernatant was discarded, 0.3m1 of 0.5 XSSC was added until the magnetic rack adsorbed for 30 seconds, and finally 0.1ml of 0.5 XSSC was added to suspend the mixture, which was called as solution B. Adding solution A into solution B, standing at room temperature for 10 min, adsorbing with a magnetic rack for 30s, discarding supernatant, washing with 0.1 XSSC for 4 times, discarding supernatant, adding 0.1ml DEPC water for suspension, adsorbing on the magnetic rack for 30s, transferring supernatant to a new test tube, adding 0.15m1 DEPC water for resuspension, adsorbing on the magnetic rack for 30s, and transferring supernatant to the test tube to obtain purified skin mRNA of Butyrospermum grahami. 1/10 volume of 3M sodium acetate, pH5.2, equal volume of isopropanol was added, left at-70℃for 30 min, centrifuged at 12000rpm for 10 min at 4℃and the supernatant discarded and the pellet was dissolved in 10. Mu.l = DEPC water.

3. Construction of a skin cDNA library of the rana grahami:

by using a Creator from CLONTECH company ^TM SMART ^TM cDNA Library ConstructionKit plasmid cDNA library construction kit.

(a) First strand cDNA Synthesis (mRNA reverse transcription):

mu.l of the skin mRNA of the Bungeana Juz, 1. Mu.l of SMART IV oligonucleotide, 1. Mu.l of CDS III/3' PCR primer and 2. Mu.l of deionized water were added to a sterile centrifuge tube of 0.5ml to bring the total volume to 5. Mu.l. The reagents in the centrifuge tube were mixed and centrifuged at 12000rpm for 15 seconds and incubated at 72℃for 2 minutes. The tubes were incubated on ice for 2 minutes. Into the centrifuge tube were added 2.0. Mu.l of 5 Xfirst strand buffer, 1.0. Mu.l of 20mM dithiothreitol, 1.0. Mu.l of 10mM dNTP mix, 1.0. Mu.l of PowerScript reverse transcriptase. The reagents in the centrifuge tubes were mixed and centrifuged at 12000rpm for 15 seconds and incubated at 42℃for 1 hour. Place the centrifuge tube on ice to stop first strand synthesis. Mu.l of the first strand of cDNA synthesized was removed from the tube and used.

(b) Amplification of the second strand by Long terminal polymerase chain reaction (LD-PCR) method

The PCR instrument was pre-warmed at 95℃and 2. Mu.l of cDNA first strand (mRNA reverse transcribed), 80. Mu.l of deionized water, 10. Mu.l of 10 XAdvantage 2PCR buffer, 2. Mu.l of 50 XdNTP mix, 2. Mu.l of 5'PCR primer, 2. Mu.l of CDS III/3' PCR primer, and 2. Mu.l of E.coli polymerase centrifuge tube were reacted. Amplification in a PCR instrument was performed as follows: 95 ℃ for 20 seconds; 22 cycles (95 ℃,5 seconds; 68 ℃,6 minutes). After the cycle was completed, cDNA double strand synthesized in the centrifuge tube was recovered.

(c) And (3) PCR product recovery:

by PROMEGA CoSV Gel and PCR Clean-Up System kit is extracted and recovered, and the steps are as follows:

adding the cDNA double chain obtained by PCR into an equal volume of membrane binding buffer, mixing, transferring the mixed solution into a centrifugal purification column, standing for 5 minutes at room temperature, and making the DNA fully bind with the silica gel membrane. Centrifuge at 12000rpm for 30 seconds, pour the waste liquid from the collection tube. Mu.l of the eluate (containing ethanol) was added to the centrifugal purification column, and the column was centrifuged at 12000rpm for 30 seconds to discard the waste liquid in the collection tube. And (5) repeating the step 2. Centrifuge at 12000rpm for 5 minutes and place the spin purification column into a new centrifuge tube. 30. Mu.l of ultrapure water was added thereto, and the mixture was allowed to stand at room temperature for 5 minutes. Centrifuging at 12000rpm for 30 seconds, and obtaining the bottom solution which is the purified cDNA double strand.

(d) Preparation of E.coli DH 5. Alpha. Competent cells:

single DH5 alpha colonies are picked up, inoculated into 3ml of Luria-Bertani (LB) medium without ampicillin, cultured overnight at 37 ℃, the bacterial liquid is taken out the next day according to the proportion of 1:100, and then inoculated into 50ml of LB medium, and oscillated for 2 hours at 37 ℃. When OD is ₆₀₀ When the value reached 0.35, bacterial cultures were harvested. Bacteria were transferred to a sterile, single-use, 50m1 polypropylene tube pre-chilled with ice, and the culture was allowed to cool to 0 ℃ for 10 minutes above ice. Cells were recovered by centrifugation at 4100rpm for 10 minutes at 4 ℃. The broth was decanted and the tube inverted for l min to drain out the last traces of broth. 0.1mol/LCaCl pre-chilled per 50ml initial broth and 30ml ₂ -MgCl ₂ Solution (80 mmol/L MgCl) ₂ ,20mmol/L CaCl ₂ ) Each cell pellet was resuspended. Cells were recovered by centrifugation at 4100rpm for 10 minutes at 4 ℃. The broth was decanted and the tube was inverted for l minutes to drain the last traces of broth. 0.1mol/L CaCl pre-chilled with ice with 2m1 per 50m1 initial culture ₂ And re-suspending each cell sediment, and sub-packaging for later use.

4. Clone screening of the rana grahami defensive peptide genes:

cathelicidins are one of the most characteristic antimicrobial peptides, and since Cathelicidins encoded by genes are very conserved in vertebrates, they consist of four exons, the first one encoding an N-terminal signal peptide of 29-30 amino acid residues in size; the second and third exons encode a highly conserved "cathelin" region consisting of 99-114 amino acids; the fourth exon encodes a mature peptide whose antibacterial domain consists of 12-100 amino acids, and thus, it is possible to confirm whether the cathelicidins-encoding gene is present in the mRNA by designing primers for cathelicidins. The sequence of the 3' -end reverse primer Zs-CATH-R1 designed according to the reported conserved region of the amphibian cathelicidins is as follows: 5'- (A/T) (G/C) C (A/G) CAG (A/G) (C/T) CTTCACCTCC-3', the other 5 'end amplification primer is a 5' primer self-contained in CLONTECH SMART TM cDNA Library Construction Kit, the sequence is 5'-AAGCAGTGGTATCAACGCAGAGT-3', and PCR amplification is carried out by taking the skin two-chain cDNA of the rana grahami as a template. The PCR reaction was performed under the following conditions: 94 ℃ for 30s;52 ℃,45s; at 72℃for 2min, 35 cycles were total.

5. Determination of the sequence of the rana grahami defensive peptide gene:

after amplification, the nucleic acid electrophoresis detection shows that a band of about 500bp exists, and the product is sent to Shanghai biochemical engineering for sequencing. According to the 5 'end sequence obtained by sequencing, designing a 5' end primer Zs-CATH-F1, wherein the sequence is as follows: CTGGCAGTGTTTTCTATGGCTC the sequence of the 3' primer contained in the library kit is 5'-ATTCTAGAGGCCGAGGCGGCCG-3', and the two-strand cDNA of the skin of the tree frog is used as a template for PCR amplification, and the reaction conditions are as described above. After the amplification is completed, the target fragment is recovered by using a DNA gel recovery kit, the recovered fragment is connected with a pMD19-T vector, and the connection product is transformed into E.coli DH5 alpha competent cells. After transformation, the cDNA library construction was completed. The band of about 500bp is selected and sent to Shanghai worker for sequencing.

The gene sequencing result shows that the gene for encoding the precursor of the rana grahami defensive peptide Zs-CATH consists of 628 nucleotides (GenBank accession number: OM 365993), and the sequence from the 5 'end to the 3' end is shown as SEQ ID NO. 2.

SEQ ID NO.2：

Example 2: synthesis and structural identification of Zs-CATH

According to the deduced amino acid sequence of the gene encoding the rana grahami defensive peptide Zs-CATH, the complete sequence is synthesized by an automatic polypeptide synthesizer (433A,Applied Biosystems) as shown in SEQ ID NO.1 (ASKKGKCNFMCKVKQKLRAIGSKTVIGTVVHKI).

SEQ ID NO.1：

In this example, the Bungeana Juz defensive peptide Zs-CATH was synthesized by Shanghai Jie peptide biosciences, inc., and desalted and purified by HPLC reverse column chromatography, and the purity was determined to be more than 98%. All polypeptides were dissolved in ultrapure water for activity determination at a stock concentration of 2mg/ml and stored in aliquots at-20 ℃.

The theoretical isoelectric point (isoelectric point, pI) and Molecular Weight (MW) were calculated using Bioinformatics Resource Portal tools (http:// www.expasy.org/tools /), which had a molecular weight of 3602.47 daltons and an isoelectric point of 10.49.

The structure was analyzed using circular dichroism spectroscopy, which was collected on a Jasco-810 spectropolarimeter (Jasco, tokyo, japan) having a cell with a 1mm long path at 25℃and 0.2nm spacing (190-260 nm). Zs-CATH is dissolved in H ₂ O、SDS/H ₂ O and LPS/H ₂ In O, the concentration was 0.3mg/ml. Three scans of each spectrum were averaged using the Jasco-810 software. CD data are expressed as average residual ovality (θ) in deg.cm2.dmol-1.

As shown in FIG. 1, zs-CATH is dissolved in H ₂ At O, the CD spectrum has a strong negative peak at 199nm and a small negative peak at 211nm, indicating that it is predominantly in the beta-sheet and random coil conformations. SDS/H of Zs-CATH in membrane simulation ₂ In the O solution environment, there is a small negative peak at 211nm, and two different negative peaks at 208 and 222nm, indicating that Zs-CATH mainly adopts a beta-sheet structure and has a small alpha-helix. Dissolved in 40mM SDS/H ₂ The secondary structure of Zs-CATH in O solution is composed of: 24.6% alpha-helix, 39.4% beta-sheet and 36.0% random coil. Furthermore, as shown in fig. 2, in the LPS solution environment, there is a characteristic peak of α -helix, and the degree of α -helix content increases with increasing LPS concentration.

Example 3: identification of antibacterial activity of rana grahami defensive peptide Zs-CATH

The Luria-Bertani (LB) liquid culture medium is used as a culture medium, and after the tested strain is recovered by the LB solid culture medium, the strain drop is diluted by sterile water to be 2X 10 per milliliter ⁵ Bacterial liquid of each bacterium is reserved. When the minimum inhibitory concentration is measured, double dilution is adoptedThe method for antibacterial detection comprises the following specific steps:

adding 0.01ml sample into 0.19ml culture medium as the first well, mixing, adding 0.1ml sample into 2 nd well (0.1 ml fresh culture medium is added), sequentially diluting by multiple ratio, sucking 0.1ml sample out of 6 th well, discarding, comparing all four wells, adding corrected bacterial liquid (2×10) ⁵ cfu/ml) 0.1ml, incubated at 37℃for 18 hours after mixing, and light absorption measured at a wavelength of 600 nm. The minimum inhibitory concentration is the lowest sample concentration at which no bacterial growth is visible.

Bacterial strains were derived from the university of Kunming medical science, and this test was repeated three times and averaged, with the results shown in Table 1.

Table 1 action of the Bungella baiting defensin peptide Zs-CATH to inhibit bacterial growth:

as can be seen from Table 1, the Bungella baija defensin peptide Zs-CATH has a remarkable effect of inhibiting bacterial growth.

The foregoing examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention, and all designs that are the same or similar to the present invention are within the scope of the present invention.

Claims

1. A rana grahami defensive peptide Zs-CATH is characterized in that: the defensive peptide Zs-CATH is a cyclic polypeptide of which the amino acid sequence is SEQ ID NO.1, and a pair of intramolecular disulfide bonds are formed by seventh cysteine and eleventh cysteine, and has a molecular weight of 3602.47 daltons and an isoelectric point of 10.49, and the amino acid sequence is shown as SEQ ID NO. 1.

2. A gene encoding a precursor of the rana grahami defensin peptide Zs-CATH, characterized in that: the gene GenBank Accession Number OM365993 consists of 628 nucleotides, the nucleotide sequence is shown as SEQ ID NO.2, wherein the 352 th to 450 th nucleotides are encoding genes of mature rana grahami defensive peptide Zs-CATH.

3. The use of the tree frog defensin peptide Zs-CATH according to claim 1 for preparing a therapeutic drug for infectious diseases caused by escherichia coli, staphylococcus aureus and alcaligenes faecalis.