阅读说明：本技术 一种马氏珠母贝Kunitz型丝氨酸蛋白酶抑制剂基因、编码的蛋白质和应用 (Pinctada martensii Kunitz type serine protease inhibitor 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：本发明提供了一种马氏珠母贝Kunitz型丝氨酸蛋白酶抑制剂基因、编码的蛋白质和应用,属于蛋白用途技术领域,所述马氏珠母贝Kunitz型丝氨酸蛋白酶抑制剂基因的核苷酸序列如SEQ ID No.1所示,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对大肠杆菌、铜绿假单胞菌、嗜水气单胞菌、副溶血弧菌和哈维氏弧菌具有抑制作用。(The invention provides a Pinctada martensii Kunitz type serine protease inhibitor gene, a coded protein and application, and belongs to the technical field of protein application, wherein the nucleotide sequence of the Pinctada martensii Kunitz type serine protease inhibitor gene is shown as SEQ ID No.1, and the amino acid sequence of the coded protein is shown as SEQ ID No. 2. The gene coded protein provided by the invention has an inhibiting effect on escherichia coli, pseudomonas aeruginosa, aeromonas hydrophila, vibrio parahaemolyticus and vibrio harveyi.)

1. The nucleotide sequence of the Pinctada martensii Kunitz type serine protease inhibitor gene is shown as SEQ ID No. 1.

2. The protein encoded by the pinctada martensii Kunitz-type serine protease inhibitor gene of claim 1, wherein the amino acid sequence of the protein is shown as SEQ ID No. 2.

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

4. Use according to claim 3, wherein the microorganisms comprise gram-negative bacteria.

5. The use according to claim 4, wherein the gram-negative bacteria comprise Escherichia coli, 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 pinctada martensii Kunitz type serine protease inhibitor gene, encoded protein and application.

Background

In recent years, shellfish breeding species frequently develop various infectious diseases caused by bacteria, viruses and the like due to high-density breeding, eutrophication of breeding water areas, deterioration of ecological environment and the like. In the prevention and treatment process, a large amount of traditional medicines such as antibiotics are used, the micro-ecological balance of the water environment is destroyed, certain pathogens generate drug resistance to the medicines, and the occurrence of diseases cannot be effectively controlled, so that the pinctada martensii dies massively die due to the diseases, and huge economic loss is caused to the pearl industry in China. Therefore, understanding the immune mechanism of Pinctada martensii and improving the stress resistance of Pinctada martensii on the basis of the immune mechanism are problems to be solved urgently at present. The antibacterial peptide is the most important link in a shellfish biological immune mechanism, is also the most important defense line of shellfish against pathogen invasion, and has important significance for developing novel biological antibiotics from the antibacterial peptide of the pinctada martensii by knowing the composition and characteristics of various antibacterial peptide molecules in the pinctada martensii, but the reports of the antibacterial peptide of the pinctada martensii are few at present, and the antibacterial peptide sequence of the pinctada martensii is not reported in a database.

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 pinctada martensii Kunitz type serine protease inhibitor gene, a coded protein and an application thereof, wherein the protein coded by the gene provided by the invention has an inhibitory effect on escherichia coli, pseudomonas aeruginosa, aeromonas hydrophila, vibrio parahaemolyticus and vibrio harveyi.

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

the invention provides a Pinctada martensii Kunitz type serine protease inhibitor gene, wherein the nucleotide sequence of the Pinctada martensii Kunitz type serine protease inhibitor gene is shown as SEQ ID No. 1.

The invention also provides a protein encoded by the Pinctada martensii Kunitz type serine protease inhibitor 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 microorganism comprises a gram-negative bacterium.

Preferably, the gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila, Vibrio parahaemolyticus, and Vibrio harveyi.

The invention provides a Pinctada martensii Kunitz type serine protease inhibitor gene, wherein the nucleotide sequence of the Pinctada martensii Kunitz type serine protease inhibitor gene is shown as SEQ ID No.1, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 2. The gene coded protein provided by the invention has an inhibiting effect on escherichia coli, 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 transmembrane domain prediction of Pinctada martensii PmKuPI;

FIG. 3 is the PmKuPI domain prediction;

FIG. 4 is a PmKuPI homology alignment;

FIG. 5 shows a phylogenetic tree construction (Neighbor-join method);

FIG. 6 shows the expression distribution of the PmKuPI gene in each tissue of Pinctada martensii, M: mantle of the mantle, B: blood cells, Gi: gill, Go: gonad, a: adductor muscle, He: liver pancreas;

FIG. 7 is the verification of PmKuPI prokaryotic expression recombinant plasmid;

fig. 8 is induced expression of rpmwkupi (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. 9 shows the expression induction of rPmKuPI (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 ℃; NC 2: no induced precipitation; 5: inducing precipitation at 15 ℃; 6: inducing precipitation at 37 ℃;

fig. 10 is a rpmpkupi purification assay, a: 1: BSA (2 μ g); m1: marker; 2: renatured protein of interest (2. mu.g); b: m2: marker; 3: the renatured target protein;

FIG. 11 is the antimicrobial activity assay of rPmKuPI (E.coli);

FIG. 12 is an antimicrobial activity assay for rPmKuPI (Pseudomonas aeruginosa);

FIG. 13 is an antibacterial activity assay (Aeromonas hydrophila) for rPmKuPI;

FIG. 14 is the measurement of the antibacterial activity of rPmKuPI (Vibrio parahaemolyticus);

FIG. 15 is the antibacterial activity assay of rPmKuPI (Vibrio harveyi);

fig. 16 is a graph of the morphological changes of rpmpkupi on bacteria: a and E are PBS control groups of pseudomonas aeruginosa and vibrio parahaemolyticus respectively, B, C, D is an experimental group of rPmKuPI acting on pseudomonas aeruginosa, and F, G, H is an experimental group of rPmKuPI acting on vibrio parahaemolyticus.

Detailed Description

The invention provides a Pinctada martensii Kunitz type serine protease inhibitor gene, wherein the nucleotide sequence of the Pinctada martensii Kunitz type serine protease inhibitor gene is shown as SEQ ID No.1, and specifically comprises the following steps:

AAGCAGTGGTATCAACGCAGAGTACATGGGGGTGTCAATACTCTATTTAGATATTTACGTTATGATGA GCGTGGTTTGTTACTTTACGTTTATTTTAGGTGCCTTTTTGTTCCTGTGTGATCATGGTATTGCACAGTCACCTTT ATCAATATTACCAGTCCCACCCCCTTTCCCAGACAGGGGCCCTTGCTCAGATAGACCGGCTGTAGTAGGGCCATGC AGAGCACGTCTACGCCGTTATACATACAGAAATGGAAGATGCGAAGAATTTTACTATGGAGGTTGTCTTGGTAACA GAAATAATTTCAGAAGTAGGAGAGAGTGTCAAAGGCAATGTGGCGGGGGAGGTGGAGGCGGAGGAGATATTTGCCA GTTGCCTCATGCACAGCCAGGACCGTGTTTAGCGTACATGCCACGTTATACATTCAACTCTAACACAGGCCGGTGT GAGGAGTTTATTTACGGTGGTTGTCAAGGAAATGCAAACAGATTCGAGACCCTGCAGGAGTGTAGGAGGCGCTGTG GAGGAGGGGGGCCACCAAGGGACCGTTGCTTTGAAAGACCAAAAGTCCAAGGACCATGTGAAGCTGCTATACCTAG CTTTTCATACAACCCGAGAACGAGGAGGTGTGAGGAGTTTACATACGGCGGATGTGGAGGAACCCGAAACAGGTTC TCTACGCTCAGAGAATGCCGGGATCGGTGTCAAAGAGGGGGAGGTGGAGGGGGAGGTGTGGACATTTGCGAACTCC CACCCAGGGCCAGTGGACTGTGTTTGGCCTATATCCCTAGCTATTCATACGACTCGGCAAGAGGAGAATGTGTAAG GTTCATTTACGGTGGATGCGGTGGAAATGAAAACAGATTTGGTTCCCTACAGGAGTGTCAGCGACGATGTGGTGGG GGAGGTGGTGGTGGGGGCGGAGACGGAGATGTGAACGATTTATCTATGGTGGATGTCTGGGAACAAACAATAGGTT TCCTTCTCGAAGAGAATGTGAAAGAAGATGTGGAGACGGCGGTCGACCAAGAAGATGCTTACTGCCCAGAGAGACT GGTCCATGTAGAGCTGCCTTTCCAAGATACTACTTTAATAGAGAATCTGGACGTTGTGAGAGATTCATATACGGAG GTTGTCAAGGAAACCAAAATAACTTTAGATCAGCAAATGAGTGTAGACGTGTGTGTAGAAGGCGACGAGGGTAAAA ATGTCTTCGTCTAAAATTGTAAATATGTACTTTAAGATTTTAGTTGCAATTAAGGTGAATTAAAGCACTAATTAATTCGTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAA are provided. The open reading frame of the Pinctada martensii Kunitz-type serine protease inhibitor gene is underlined.

The invention also provides a protein encoded by the Pinctada martensii Kunitz type serine protease inhibitor gene, which is represented by SEQ ID No.2 and specifically comprises the following components:

MGVSILYLDIYVMMSVVCYFTFILGAFLFLCDHGIAQSPLSILPVPPPFPDRGPCSDRPAVVGPCRARLRRYTYRNGRCEEFYYGGCLGNRNNFRSRRECQRQCGGGGGGGGDICQLPHAQPGPCLAYMPRYTFNSNTGRCEEFIYGGCQGNANRFETLQECRRRCGGGGPPRDRCFERPKVQGPCEAAIPSFSYNPRTRRCEEFTYGGCGGTRNRFSTLRECRDRCQRGGGGGGGVDICELPPRASGLCLAYIPSYSYDSARGECVRFIYGGCGGNENRFGSLQECQRRCGGGGGGGGGDGDVNDLSMVDVWEQTIGFLLEENVKEDVETAVDQEDAYCPERLVHVELPFQDTTLIENLDVVRDSYTEVVKETKITLDQQMSVDVCVEGDEGKNVFV。

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 microorganism preferably includes gram-negative bacteria, and the gram-negative bacteria preferably include Escherichia coli, 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.