阅读说明：本技术 制备东方火腹蟾蜍抗菌肽bombinin的方法及其应用 (Method for preparing toad antibacterial peptide bombinin and application thereof ) 是由 张小蒙 张莉 周敏 于 2019-10-09 设计创作，主要内容包括：本发明属于基因工程技术领域,涉及一种制备东方火腹蟾蜍抗菌肽bombinin的方法及其应用。该方法包括以下步骤：(1)获取东方火腹蟾蜍抗菌肽bombinin的基因,所述基因的核苷酸序列如SEQ ID No.1所示；(2)利用所述基因构建pBKPU01-Bombinin重组表达载体；(3)将所述载体转化到宿主菌中构建工程菌；(4)利用所述工程菌表达所述东方火腹蟾蜍抗菌肽bombinin,获取所述东方火腹蟾蜍抗菌肽bombinin。该方法能够获得大量的东方火腹蟾蜍抗菌肽bombinin。(The invention belongs to the technical field of genetic engineering, and relates to a method for preparing toad venom antibacterial peptide bombinin and application thereof. The method comprises the following steps: (1) obtaining a gene of toad venom antibacterial peptide bombinin, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 1; (2) constructing a pBKPU01-Bombinin recombinant expression vector by using the gene; (3) transforming the vector into host bacteria to construct engineering bacteria; (4) and expressing the toad-fire-belly toad antibacterial peptide bombinin by using the engineering bacteria to obtain the toad-fire-belly toad antibacterial peptide bombinin. The method can obtain large amount of Bufo siccus antibacterial peptide bombinin.)

1. A method for preparing toad venom antibacterial peptide bombinin, which comprises the following steps:

(1) obtaining a gene of toad venom antibacterial peptide bombinin, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 1;

(2) constructing a pBKPU01-Bombinin recombinant expression vector by using the gene;

(3) transforming the vector into host bacteria to construct engineering bacteria;

(4) and expressing the toad-fire-belly toad antibacterial peptide bombinin by using the engineering bacteria to obtain the toad-fire-belly toad antibacterial peptide bombinin.

2. The method of claim 1, wherein step (2) comprises the steps of:

connecting the gene with a pBKPU01 expression vector by using T4DNA ligase to form a pBKPU01-Bombinin recombinant plasmid;

transferring the pBKPU01-Bombinin recombinant plasmid into escherichia coli DH5 alpha for culture;

extracting the pBKPU01-Bombinin recombinant plasmid from the cultured transferred escherichia coli DH5 alpha, and carrying out PCR and enzyme digestion identification to obtain the pBKPU01-Bombinin recombinant expression vector.

3. The method according to claim 1, wherein in step (3), the host bacterium is Pichia pastoris.

4. The method of claim 3, wherein step (4) comprises the steps of:

inoculating the engineering bacteria into an MD liquid culture medium, culturing for 20-48 hours at 25-32 ℃ under the condition of 100-130r/min, then transferring into a fermentation tank containing a buffer complex culture medium containing methanol for fermenting for 18-36 hours, centrifuging, and taking the supernatant, thereby obtaining the toad bombinin.

5. The method according to any one of claims 1-4, further comprising: (5) and detecting the toad venom antibacterial peptide bombinin.

6. The method according to any one of claims 1 to 4, wherein the amino acid sequence of said Oriental fire-belly toad antibacterial peptide bombinin is shown in SEQ ID No. 2.

7. Use of the toad venom antibacterial peptide bombinin prepared by the method of any one of claims 1 to 6 in the preparation of a preparation for inhibiting and/or killing bacterial strains.

8. The use according to claim 7, wherein the strain is at least one of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Candida krusei.

9. Use of said toad venom peptide bombinin prepared by the method of any one of claims 1 to 6 in animal feed additives.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for preparing toad venom antibacterial peptide bombinin and application thereof.

Background

In recent years, the massive abuse of antibiotics in animal breeding industry causes the problems of continuous emergence of antibiotic resistant strains, drug residues, environmental pollution, ecological destruction and the like, and seriously threatens the development of human health and animal husbandry. Therefore, the search for a pharmaceutical preparation to replace antibiotics is urgent.

The antibacterial peptide is a basic polypeptide which is generated in an insect body, has a molecular weight of about 2000-7000 and consists of 20-60 amino acid residues and has antibacterial activity. Most of the antibacterial peptides have the advantages of strong basicity, broad-spectrum antibacterial property, good thermal stability, no toxicity, no residue, no side effect, safety, high efficiency, no pollution to the environment, no easy generation of bacterial drug resistance and the like.

The Bufo siccus antibacterial peptide Bombinin is an alkaline small molecular polypeptide produced by Bufo siccus, has a cDNA total length of 60bp and a molecular weight of 2382Da, and encodes 20 amino acids. Researches show that the antibacterial peptide depends on charge effect to sterilize and also has the functions of immune activation, regulation and the like.

Therefore, a method for preparing the toad venom peptide bombinin is urgently needed.

Disclosure of Invention

The invention aims to provide a method for preparing toad-fire-belly antibacterial peptide bombinin and application thereof, so as to improve the yield of the toad-fire-belly antibacterial peptide bombinin and reduce the production cost.

In order to achieve the above objects, the present invention provides, in a first aspect, a method for preparing toad venom peptide bombinin. The method comprises the following steps:

(1) obtaining a gene of toad venom antibacterial peptide bombinin, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 1;

(2) constructing a pBKPU01-Bombinin recombinant expression vector by using the gene;

(3) transforming the vector into host bacteria to construct engineering bacteria;

(4) and expressing the toad-fire-belly toad antibacterial peptide bombinin by using the engineering bacteria to obtain the toad-fire-belly toad antibacterial peptide bombinin.

In the present invention, the nucleotide sequence shown by SEQ ID No.1 is 5'-CGCGAAGCCCGACUGAACUUCUCAGCCGGACGGGAGCAGCGCGUGAAGGCAAAG-3'. The sequence may be a synthetic sequence.

In one embodiment of the present invention, the step (2) comprises the steps of:

connecting the gene with a pBKPU01 expression vector by using T4DNA ligase to form a pBKPU01-Bombinin recombinant plasmid;

transferring the pBKPU01-Bombinin recombinant plasmid into escherichia coli DH5 alpha for culture;

extracting the pBKPU01-Bombinin recombinant plasmid from the cultured transferred escherichia coli DH5 alpha, and carrying out PCR and enzyme digestion identification to obtain the pBKPU01-Bombinin recombinant expression vector. The pBKPU01-Bombinin recombinant expression vector can be obtained through the step (2), and prokaryotic expression identification can also be carried out.

The skilled person can select the host bacteria in step (3) according to actual conditions to construct the engineering bacteria. Preferably, in step (3), the host bacterium is pichia pastoris. The vector can be transformed into a host bacterium by means of electrotransformation.

In one embodiment of the present invention, the step (4) comprises the steps of:

inoculating the engineering bacteria into an MD liquid culture medium, culturing for 20-48 hours under the conditions of 25-32 ℃ and 100-130r/min, then transferring the engineering bacteria into a fermentation tank of a Buffered methane-complex medium (BMMY culture medium for short) containing Methanol for fermenting for 18-36 hours, centrifuging, and taking the supernatant to obtain the toadfoo antibacterial peptide bombinin. The centrifugation condition can be 10000r/min, and the centrifugation time can be 5 min.

In a preferred embodiment of the present invention, the method further comprises: (5) and detecting the toad venom antibacterial peptide bombinin. Specifically, the method is to detect the bacteriostatic activity of the antibacterial peptide bombinin by adopting an agar diffusion method. For example, the bacteriostatic activity of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, or Candida krusei was examined.

The amino acid sequence of the toad venom antibacterial peptide bombinin is shown in SEQ ID No. 2. The nucleotide sequence of SEQID No.2 is 5 '-REARLKFSAGREQRVKAK-3'.

In a second aspect, the invention provides an application of the toad venom antibacterial peptide bombinin prepared by the method in preparation of a preparation for inhibiting and/or killing bacterial strains.

Preferably, the strain is at least one of staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, acinetobacter baumannii and candida krusei.

The third aspect of the invention provides an application of the toad antibacterial peptide bombinin prepared by the method in animal feed additives.

The method for preparing the toad antibacterial peptide bombinin can obtain a large amount of the toad antibacterial peptide bombinin.

The method for preparing the toad venom antibacterial peptide Bombin provided by the invention can be used for obtaining a bacterial strain which is highly expressed in pichia pastoris and has actual production capacity and can produce the antibacterial peptide Bombin.

The method for preparing the toad venom antibacterial peptide bombinin can be applied to actual production.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.

FIG. 1 shows gel electrophoresis of PCR amplified peptide Bombinin gene of Bufo siccus in Oriental fire belly. In the figure, M represents 5000bp DNA marker; 1 represents a PCR product.

FIG. 2 shows the hemolysis experiment result of the antibacterial peptide Bombinin.

FIG. 3 is a graph showing the results of the antimicrobial peptide Bombin acting on Staphylococcus aureus and Escherichia coli, respectively, after different temperature treatments. In the figure, A represents Staphylococcus aureus; b represents Escherichia coli.

FIG. 4 is a graph showing the results of the antimicrobial peptide Bombin acting on Staphylococcus aureus and Escherichia coli, respectively, after treatment at different pH values. In the figure, A represents Staphylococcus aureus; b represents Escherichia coli.

FIG. 5 is a graph showing the results of different concentrations of trypsin-treated antimicrobial peptide Bombinin acting on Staphylococcus aureus and Escherichia coli, respectively. In the figure, A represents Staphylococcus aureus; b represents Escherichia coli.

Detailed Description

Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.

The materials used in the following examples and test examples are as follows:

plasmids and strains

Pichia pastoris was purchased from Invitrogen; the pBKPU01 expression vector is constructed and stored in the laboratory; microorganisms such as staphylococcus aureus, escherichia coli and the like are purchased from China center for culture collection and management of industrial microorganisms.

Primary reagents and instruments

Plasmid extraction kit, gel recovery kit, protein small molecular weight Marker and the like are purchased from bio-engineering (Shanghai) corporation; EcoR V enzymes, trypsin, and the like, available from Takara bioengineering (Dalian) Inc.; culture medium, blood agar, etc. were purchased from Jiangsu Kai base biotech, Inc.; other chemical reagents are domestic analytical purifiers.

PCR instrument (Thermo Scientific, USA); clean bench (Shanghai Boxun medical Bioinstrumentation, Inc.); gel imager (Bio-Rad, USA); a small vertical protein electrophoresis apparatus (six biotech ltd, beijing); constant temperature shaker (shanghai jinpeng analysis instruments ltd); a constant temperature incubator and a constant temperature incubator shaker (Shanghai and Shikusho instruments Co., Ltd.).