阅读说明：本技术 一种胰蛋白酶样丝氨酸蛋白酶基因、编码的蛋白质和应用 (Trypsin-like serine protease gene, encoded protein and application ) 是由 梁海鹰 何军军 申铖皓 房晓宸 卢金昭 于 2020-06-24 设计创作，主要内容包括：本发明提供了一种胰蛋白酶样丝氨酸蛋白酶基因、编码的蛋白质及其获取方法和应用,属于基因工程技术领域,所述胰蛋白酶样丝氨酸蛋白酶基因的核苷酸序列如SEQ ID No.1所示,所述胰蛋白酶样丝氨酸蛋白酶基因编码的蛋白的氨基酸序列如SEQ ID No.2所示。本发明提供的基因编码的蛋白能够抑制铜绿假单胞菌、嗜水气单胞菌、副溶血弧菌和哈维氏弧菌。(The invention provides a trypsin-like serine protease gene, a coded protein, an acquisition method and application thereof, and belongs to the technical field of genetic engineering, wherein the nucleotide sequence of the trypsin-like serine protease gene is shown as SEQ ID No.1, and the amino acid sequence of the protein coded by the trypsin-like serine protease gene is shown as SEQ ID No. 2. The gene coded protein provided by the invention can inhibit pseudomonas aeruginosa, aeromonas hydrophila, vibrio parahaemolyticus and vibrio harveyi.)

1. A trypsin-like serine protease gene, characterized in that the nucleotide sequence of the trypsin-like serine protease gene is shown in SEQ ID No. 1.

2. The protein encoded by the trypsin-like serine protease gene of claim 1, wherein the amino acid sequence of said protein is as set forth in SEQ ID No. 2.

3. Use of a protein according to claim 2 for the preparation of a medicament for inhibiting a microorganism.

4. The use according to claim 3, wherein the microorganisms comprise Pseudomonas aeruginosa, Aeromonas hydrophila, Vibrio parahaemolyticus and Vibrio harveyi.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a trypsin-like serine protease gene, a coded protein and application.

Background

In recent years, due to serious marine environmental pollution and frequent diseases of aquaculture animals, the abuse of traditional antibiotics causes more and more serious drug resistance of pathogenic microorganisms, great threat is brought to the health of the aquaculture animals and human beings, and the development of new antibacterial drugs is urgently needed. The sea is the most abundant gathering place of species in the world, and substances with antibacterial activity are searched from marine invertebrates, so that the marine invertebrates have great potential. In order to solve the problems of antibiotic abuse and drug resistance, from aquaculture animals, biological antibiotics close to natural antibiotics are researched, and the antibacterial broad spectrum and the drug resistance are improved.

Disclosure of Invention

In view of the above, the present invention aims to provide a protein encoded by trypsin-like serine protease gene and its application, wherein the protein encoded by the gene provided by the present invention can inhibit four microorganisms.

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

the invention provides a trypsin-like serine protease gene, the nucleotide sequence of which is shown in SEQ ID No. 1.

The invention also provides a protein encoded by the trypsin-like serine protease gene in the technical scheme, and the amino acid sequence of the protein is shown as SEQ ID No. 2.

The invention also provides application of the protein in the technical scheme in preparation of a medicament for inhibiting microorganisms.

Preferably, the microorganisms include Pseudomonas aeruginosa, Aeromonas hydrophila, Vibrio parahaemolyticus, and Vibrio harveyi.

The invention provides a trypsin-like serine protease gene, which is characterized in that the nucleotide sequence of the trypsin-like serine protease gene is shown as SEQ ID No. 1. The amino acid sequence of the protein encoded by the gene provided by the invention is shown in SEQ ID No.2, and the protein can inhibit pseudomonas aeruginosa, aeromonas hydrophila, vibrio parahaemolyticus and vibrio harveyi.

Drawings

FIG. 1 is a schematic diagram of the pMD19-T Vector cloning site;

FIG. 2 is a PmTLS domain prediction;

FIG. 3 is a PmTLS homology alignment;

FIG. 4 is a PmTLS phylogenetic tree construction;

FIG. 5 shows the expression distribution of PmTLS gene in various tissues of Pinctada martensii, M: mantle of the mantle, B: blood cells, Gi: gill, Go: gonad, a: adductor muscle, He: the liver pancreas;

FIG. 6 is a time-series expression of PmTLS in blood cells following PAMPs stimulation;

FIG. 7 is a time-series expression of PmTLS in blood cells following PAMPs stimulation;

FIG. 8 is a time-series expression of PmTLS in blood cells following PAMPs stimulation;

FIG. 9 shows verification of PmTLS prokaryotic expression recombinant plasmid;

FIG. 10 shows the induced expression of rPmTLS (SDS-PAGE), PC 1: BSA (1. mu.g); PC 2: BSA (2 μ g); m1: marker; NC: no induced bacteria; 1: inducing the whole strain at 15 ℃; 2: inducing the whole strain at 37 ℃; NC 1: no induction supernatant; 3: inducing the supernatant at 15 ℃; 4: inducing supernatant at 37 ℃; NC 2: no induced precipitation; 5: inducing precipitation at 15 ℃; 6: inducing precipitation at 37 ℃;

FIG. 11 shows the induced expression of rPmTLS (Western Blot), M2: marker; NC: no induced bacteria; 1: inducing the whole strain at 15 ℃; 2: inducing the whole strain at 37 ℃; NC 1: no induction supernatant; 3: inducing the supernatant at 15 ℃; 4: inducing supernatant at 37 ℃; 5: inducing precipitation at 15 ℃; 6: inducing precipitation at 37 ℃;

FIG. 12 is an analysis of rPmTLS purification (SDS-PAGE); m1: marker; 1: BSA (2 μ g); 2: renatured protein of interest (2. mu.g); 3: target protein before renaturation (2. mu.g)

FIG. 13 is an antimicrobial activity assay for rPmTLS (Pseudomonas aeruginosa);

FIG. 14 is an antibacterial activity assay (Aeromonas hydrophila) for rPmTLS;

FIG. 15 is the antibacterial activity assay of rPmTLS (Vibrio parahaemolyticus);

FIG. 16 is an antibacterial activity assay of rPmTLS (Vibrio harveyi);

FIG. 17 shows the morphological changes of rPmTLS on bacteria; A. e, I are PBS control groups of Pseudomonas aeruginosa, Aeromonas hydrophila and Vibrio parahaemolyticus, B, C, D is an experimental group of rPmTLS acting on Pseudomonas aeruginosa, F, G, H is an experimental group of rPmTLS acting on Aeromonas hydrophila, and J and K are experimental groups of rPmTLS acting on Vibrio parahaemolyticus, respectively.

Detailed Description

The invention provides a trypsin-like serine protease gene, the nucleotide sequence of which is shown as SEQ ID No.1 and specifically comprises the following components:

ACATGGGCTAATAAATGGATCGTGACAGCCGCCCACTGTATCGTGCGTTTTCCCGAGAAATTCCACGAATTGTTCCACCCCTCTAAGGTCACCCTTATTATTGGTACAGAGCAGTGTAGCGGATATGACGGCCAAATCGTGGACATCGAGTCATATGTTGTGCATCCTAGATTTGCAGAAAGGGCTCCATACGACCATGATATAGCTTTGATAGAACTTCGTCAAGATTTAAACTTTACAGAACGTGTACAACCAATATGTCTCAAGCAGCCGGATTACGTGAATACTGCTTTCCTTCATCGCAAAGTCGGGCGTAAGGCAGGGAGGGTTGTAGGGTGTGGTCAATTGTATGAAAATGTAGATGCTATACCCACGGAGCTACATGACGTTTTCGTACCAACAGTGACTAGGGAGAAATGTATGGAGGCGGACATAGGGCGAGGAAATT TCACTGACACTATGTTCTGCGCAGGGTATGACAGGGCTTTATTCGGAGATGCTTGTTATGGTGATAGTGGTGGCTC TTTGGCGATGAATGACTCCCCATTTGACCCCTGGGTCCTTGTGGGCGTGGTGTCATGGGGAGTTGGGTGTGACCGA CAAGGACATTATGGATACTATACAAATATAGCTCACTTTTATAACTGGATACAAAATGTCACAAATGTTTTAAATA ATTAGGATTGAAACAATAAAGAGATATAGATCTTAATTTATACTATTGAGACACAATTAAAAAAAGTTTAACCCTAAAAAAAAAAAAAAAAAAAAAAAAAAA are provided. The open reading frame ORF of the trypsin-like serine protease gene is underlined.

The invention also provides a protein encoded by the trypsin-like serine protease gene in the technical scheme, which is called PmTLS for short, and is a pinctada martensii trypsin-like serine protease, and the amino acid sequence of the protein is shown as SEQ ID No.2, and specifically comprises the following components:

MEADIGRGNFTDTMFCAGYDRALFGDACYGDSGGSLAMNDSPFDPWVLVGVVSWGVGCDRQGHYGYYTNIAHFYNWIQNVTNVLNN。

the invention also provides application of the protein in the technical scheme in preparation of a medicament for inhibiting microorganisms. In the present invention, the microorganisms preferably include Pseudomonas aeruginosa, Aeromonas hydrophila, Vibrio parahaemolyticus, and Vibrio harveyi.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.