CN112877334A

CN112877334A - Portunus trituberculatus fibrinogen related protein PtFREP gene and encoding protein and application thereof

Info

Publication number: CN112877334A
Application number: CN202110155940.8A
Authority: CN
Inventors: 刘媛; 张奥; 李富花
Original assignee: Institute of Oceanology of CAS
Current assignee: Institute of Oceanology of CAS
Priority date: 2021-02-04
Filing date: 2021-02-04
Publication date: 2021-06-01
Anticipated expiration: 2041-02-04
Also published as: CN112877334B

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a Portunus trituberculatus fibrinogen related protein PtFREP gene, and a coded protein and application thereof. The gene PtFREP of the portunus trituberculatus fibrinogen related protein is shown as a base sequence in a sequence table SEQ ID No. 1. The invention lays a foundation for disease control and gene-assisted breeding of the portunus trituberculatus, and has potential application value in the aspects of developing antibacterial drugs, bacterial agglutination preparations, novel immune activators, feed additive production and the like.

Description

Portunus trituberculatus fibrinogen related protein PtFREP gene and encoding protein and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a Portunus trituberculatus fibrinogen related protein PtFREP gene, and a coded protein and application thereof.

Background

Fibrinogen-related proteins (FREPs) are a class of pattern recognition receptors that contain a Fibrinogen-like (FBG) domain at the carboxy terminus. FREPs are widely found in vertebrates and invertebrates, and are a large superfamily, including various family members such as fibrin (ficolin), tenascin (tenascin), interleukin (fibroukin), and lectin (tachylectins).

In vertebrates, fibrinogen is primarily involved in the process of coagulation. Human fibrinogen is synthesized primarily by hepatocytes, the most abundant coagulation factor in plasma, and also the "core" protein in the coagulation system. In invertebrates, FREPs differ widely in their amino-terminal domains and typically contain one or two immunoglobulin superfamily domains (IgSF). The FBG domain of FREPs is mainly involved in sugar recognition, contains specific sugar binding sites, specifically binds N-acetyl-D-galactosamine and N-acetyl-D-glucosamine, and plays an important role in the innate immune defense of invertebrates.

The portunus trituberculatus has the advantages of fast growth, large individual and delicious meat, and becomes an important marine culture variety in China. In recent years, with the continuous expansion of the scale of the cultivation industry, the problem of disease of the portunids becomes more and more serious, and particularly, the healthy development of the portunids cultivation industry is seriously disturbed by vibriosis. Therefore, the research on the disease resistance mechanism of the portunus trituberculatus and the development of novel antibacterial drugs for immune prevention and treatment are urgently needed from the immune defense factors of the portunus trituberculatus. Currently, the research on the fiber protein related protein of the portunus trituberculatus is less, and the influence of the fiber protein related protein on the gene function is not clear.

Disclosure of Invention

The invention aims to provide a Portunus trituberculatus fibrinogen related protein PtFREP gene, and a coding protein and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

a Portunus trituberculatus fibrinogen related protein PtFREP gene is shown as a base sequence in a sequence table SEQ ID No. 1.

The PtFREP gene coding protein is shown as an amino acid sequence in a sequence table SEQ ID No. 2.

The application of the portunus trituberculatus fibrinogen related protein PtFREP gene coded protein is the application of a recombinant expression product of the portunus trituberculatus fibrinogen related protein PtFREP gene coded protein in the preparation of antibacterial drugs, immunoactive agents, bacterial agglutination preparations, bacterial binding preparations, feed additives, preservatives or preservatives.

The recombinant expression product of the portunus trituberculatus fibrinogen related protein PtFREP gene coding protein is applied to the preparation of antibacterial drugs of gram-negative bacteria or gram-positive bacteria.

The gram-negative bacteria are vibrio parahaemolyticus, vibrio alginolyticus or pseudomonas aeruginosa; the gram-positive bacteria is staphylococcus aureus or micrococcus luteus.

The recombinant expression product of the portunus trituberculatus fibrinogen related protein PtFREP gene coding protein is applied to the preparation of agglutination preparations or combination preparations of gram-negative bacteria, gram-positive bacteria or fungi.

Further, at 10mM Ca²⁺In the presence of the recombinant protein PtFREP, the recombinant protein PtFREP has obvious agglutination effect on gram-negative bacteria, gram-positive bacteria or fungi.

The gram-negative bacteria are vibrio parahaemolyticus, vibrio alginolyticus or pseudomonas aeruginosa; the gram-positive bacteria are staphylococcus aureus or micrococcus luteus; the fungus is Pichia pastoris.

The recombinant expression product of the portunus trituberculatus fibrinogen related protein PtFREP gene coding protein is used for preparing a combined preparation of bacterial or yeast cell wall components.

The main components of the bacterial cell wall are lipopolysaccharide and peptidoglycan; the main component of yeast cell wall is glucan.

The invention has the advantages that:

the invention utilizes unigene and RACE technology obtained by transcriptome sequencing to clone a fibrinogen-related protein PtFREP gene cDNA full-length sequence from blue crabs, amplifies a gene fragment for encoding PtFREP mature peptide by a PCR technology, clones the gene fragment into a pET32a (+) expression vector, and realizes in-vitro recombinant expression in escherichia coli BL21(DE 3). After being purified by a TALON column and dialyzed, the recombinant protein PtFREP has obvious inhibiting effect on gram-negative bacteria (vibrio parahaemolyticus, vibrio alginolyticus and pseudomonas aeruginosa) and gram-positive bacteria (staphylococcus aureus and micrococcus luteus), and the minimum inhibitory concentrations are 0.39-0.79 muM, 0.79-1.56 muM and 0.79-1.56 muM respectively. The recombinant protein PtFREP has obvious agglutination and combination effects on vibrio parahaemolyticus, vibrio alginolyticus, pseudomonas aeruginosa, staphylococcus aureus, micrococcus luteus and pichia pastoris. The recombinant protein has binding activity to lipopolysaccharide and peptidoglycan which are main components of bacterial cell walls and glucan which is a main component of yeast cell walls.

The PtFREP gene and the recombinant protein thereof can be used for producing bacteriostatic drugs, bacterium agglutination preparations, bacterium combination preparations and the like, are applied to the treatment of related shrimp and crab diseases in the aquaculture process, or are used for producing feed additives, preservatives or preservatives and the like, can provide a basis for further researching the immune defense mechanism of the portunus trituberculatus, and provide a reference for disease control and gene-assisted breeding of the portunus trituberculatus.

Drawings

FIG. 1 shows the nucleotide and amino acid sequences of the PtFREP gene of the Portunus trituberculatus fibrinogen-related protein provided by the example of the present invention (red underline: start codon and stop codon; red frame portion: signal peptide; shaded portion: FBG domain; black underline: coiled-coil domain; wave underline: sugar binding site).

FIG. 2 shows the purified gene amplification product of Portunus trituberculatus fibrinogen-related protein PtFREP encoding mature peptide (wherein M: DNA marker, 1, 2: gene amplification product of mature peptide) provided by the embodiment of the invention.

FIG. 3 shows an induced and purified Portunus trituberculatus fibrinogen-related protein PtFREP recombinant protein (in FIG. 3a, M: protein marker; 1: protein expressed in uninduced PtFREP thallus; 2: protein expressed in PtFREP after IPTG induction; 3: purified PtFREP recombinant protein; in FIG. 3b, M: protein marker; 1: protein expressed in uninduced pET-32a thallus; 3: protein expressed in pET-32a after IPTG induction; and 4: purified pET-32a recombinant protein) provided by the embodiment of the invention.

FIG. 4 is a graph showing the agglutination activity of the recombinant protein PtFREP for bacteria and fungi (detecting fluorescently labeled Vibrio parahaemolyticus, Vibrio alginolyticus, Pseudomonas aeruginosa, Micrococcus luteus, Staphylococcus aureus and Pichia pastoris, rPtFREP, rTrx, CaCl) of the fibrinogen-related protein of Portunus trituberculatus according to the example of the present invention₂And EDTA 25nmol L each^-1、25nmol L^-1、10mmol L^-1And 10mmol L^-1PBS and rTrx served as blank and negative controls, respectively).

FIG. 5 shows the binding activity of recombinant protein PtFREP of Portunus trituberculatus fibrinogen-related protein on bacteria and fungi (wherein M is protein marker, E is eluent of microorganism and PtFREP recombinant protein, S is supernatant after microorganism and recombinant protein are incubated and centrifuged, and W is washing liquid after microorganism and PtFREP recombinant protein are incubated).

FIG. 6 is a diagram showing ELISA analysis of recombinant PtFREP protein of Portunus trituberculatus fibrinogen-related protein on three PAMPs (LPS, PGN and GLU) molecules according to the example of the present invention. ELISA Index (EI) >1.0 showed positive, i.e. binding activity.

Detailed Description

The following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are for purposes of illustration and explanation only and are not intended to limit the invention.

The invention utilizes unigene and RACE technology obtained by transcriptome sequencing to amplify PtFREP gene cDNA from blue crab, and finds that the recombinant PtFREP protein has obvious bacteriostatic, bacterium agglutination and bacterium removing activities. RecombinationThe protein PtFREP has obvious inhibition effect on gram-negative bacteria (Vibrio parahaemolyticus, Vibrio alginolyticus and Pseudomonas aeruginosa) and gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus), and the minimum inhibition concentrations are 0.39-0.79 MuM, 0.79-1.56 MuM and 0.79-1.56 MuM respectively. In Ca²⁺In the presence of the recombinant protein PtFREP, the recombinant protein PtFREP has obvious agglutination effect on vibrio parahaemolyticus, vibrio alginolyticus, pseudomonas aeruginosa, staphylococcus aureus, micrococcus luteus and pichia pastoris. Meanwhile, the recombinant protein PtFREP has binding activity to vibrio parahaemolyticus, vibrio alginolyticus, pseudomonas aeruginosa, staphylococcus aureus, micrococcus luteus and pichia pastoris, and also has binding activity to lipopolysaccharide and peptidoglycan which are main components of bacterial cell walls and glucan which is a main component of yeast cell walls. The invention lays a foundation for disease control and gene-assisted breeding of the portunus trituberculatus, and has potential application value in the aspects of developing antibacterial drugs, bacterial agglutination preparations, novel immune activators, feed additive production and the like.

The cDNA sequence cloning of the portunus trituberculatus fibrinogen related protein PtFREP comprises the following steps:

a) extracting total RNA of the portunus trituberculatus and detecting mRNA;

b) constructing a portunus trituberculatus cDNA library;

c) sequencing and analyzing portunus trituberculatus transcriptome;

d) carrying out homology analysis on the blue crab unigene sequence and screening a PtFREP gene fragment;

e) and obtaining a full-length sequence of the PtFREP by RACE amplification and verifying the full-length sequence.

Example 1.

The gene PtFREP of the portunus trituberculatus fibrinogen related protein is a base sequence shown in SEQ ID No. 1.

Referring to fig. 1, SEQ ID No.1 of the sequence listing is:

GGTAGTAAAGGAGAGCGCAGAGGGAGGAGAGGGAGAAGAAGAGGAGGTGAAGCTGTGACGCGCC CTACACGCCCTTCAGGTCCTATGTAGCTTCACCAGGGTCCCTCCGTGGTGCTGAACGACGAAGT GGCGGCAGGGACATGGCGGGCGGGACGGCGGTGGTAGTGGCGGTGCTGGGGGTGCTGGTGGCCG TGAACAGCCAGCAGGAAACAATCCCAGAGGACCCGACCACTGAGTATTACATGCGGCTGCTCAG ATCCCTCTTAGCAAACACGACATACGCCCTACATAACGCCCTCACCCGCCTGCAGGAGAACACA GCGCAGGAGACGAGTGTGAGGAGCCTGCAGGAGTCTTTGAGGGACGTGAAGGAGTCTGTGGGCA GTTTGGAGAGGAGCATTGATAGGGTTCGTAACAGTGTTGATAATGGAGTGGACAGGACGCGCGT GGAGATTCGCACCTTCCGTAACCGCGTGTCCGGGGAGCTGGCAGACCTGAGGCACCTGGTGGAG GACAAGACCGGGAGCGTCAACCACAAGGTGGCGGAGATAGTAGCGGGGGACTGCCAGGATCTGT ACGACCTGGGACACAACGCCAGCGGAGTATACCACCTGGCGAGGCACGGGCGTGATGTCCTGTG TGAGATGGAAGGCGGCGACGGAGGCTGGCTGGTGGTGCAGAGGCGGGCGAGGGTGGCTGAGCAG GTAGACTTCAACTTGGGCTGGGACGAGTACAAGAAGGGTTTTGGGGACCTGGAGACGGAGTTCT GGATTGGCAATGATTTCTTACACGTCCTGACTAACCAGAAGACTTATCAGCTCCGTTTTGACTT CCACGACTACGAGGATGGCCCTTTCTATGCTGCCTACAGCACATTCCGAGTGGGCAACGAGGAA AGTCATTACCCTCTCTACATCGGTGACTTCTCTGGCAATGTCACGGACGCCTTCACGTACCACC ACGGACGCTCCTTCTCCACCAAGGACCGCGACAACGACCTCTACTCCGTCGGGAACTGCGCCGA GGAATTCACAGGTGGCTGGTGGTACGACAGGTGCTACGATGCTCACCTGAACGGCTTGTTTCCC GTCGTGCCGGACCGAGTGGAGGCCAAATACATTACGTGGTGGGCGCACAAAGGGGACAAGAGAG TGCCCTTCGTGCTGGACAAGGTCACCATGAGGATCAAGCCGAAACCAGTATGATCTAAAATGTG ACCCTGACCTTCGTGACCTATTCTGTGTGACCTCCAGTATGACCTTGACCTTCGTGTGACTTTT CAATGTGACCTTCAATTCTTGACCTCCAGTGTGACCCCCAGTGTGAGTGACCTTGACCTCTGTG TGATCCTCAATGTGACCTTCATTTCTTGACCTTAGAAATACCCCTGCCAGGAACTCCCATCTTC ATTCCCTCCTGTTATTAACTTTTTTGGTCACCCTGTACTGGTTTACCGAGAAATTTCCCGCCTT TGTAACACACACACCTGCCTGCACCACCCTGTAAGCTGTACTATTATTGTTATTATTATCATTA TTATTATTACAATCACTATTAATACTTTTTTGCTACTTGCTAATCCTATTGCTAACTGATTTGT GACCCTTTCAGTATTGGGACGCATTTTTACTTTGGGTTTTGTGTACCAGAAGACCATTTTACTG ACATTAAGAAGGGTATGGAGGTTAGAAGATTAATAGCCACAGACTTGACTATTTTAATCCCCAC GTGAGTTTGTGAAGGTGTGTAAAATCACCAAATAGTAAGCAGAATGAAGATGAAGATGCGTCAT AGTACTAAAGGGATGAAAGCATTTAGAGCAATTCCCTTCATTTAGTATCTCAGTTGTTTTTCTC CGTTCATTTCTTCCACGGCCTCCCTCACAGACCGCCTTCAGACCGCCTGCCACTTTCTCTCTCT CTCTCTCTCTCTCTCTCTCATTTGATTGTTTGCACTGGTATATATATTGTCACTCTCTTTGGTA TGGTTTAAGTAAGTAGTTGCATTTGTCAAGCCTCAGATTCCTTCATAGTTTTCACTCTCTGGCT CTTTCTCCAATGCTGTGAGTGAATCGATCGACCCATGTATTCATTTTATTTCATTTTTATTTTA TTCTTACCTTTTATTTTCAGTTTATCTTATCTTAGGCGCTGTTTTTTTTTATTTGATTAACTTT GTAAACGAAAAGATCGACTTGGTATCGATCTTTGACTTTCTTGATCGTATTTTGCAACTGTTTC TTACTTTACTTGGTGTTTTAGTTAATGTTCCTCTACGTAAATAATGAGAAGATTTGTCTAATTT TCAGCTTATTTTCATCTTATCTTGATTGTGTTTGGATTGCATGTACACCTTTTAGTCACTGTCT CATCAAGCTATATGAATGAAGAGATTTGTCCCAAAAAAAAAAAAAAAAAAAAAAAAAAAA

(a) sequence characterization

Length: 2428bp (effective length 141 to 1205bp)

Type: base sequence

Chain type: single strand

Topology: linearity

(b) Molecular type: double-stranded DNA

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: portunus trituberculatus (Portugulus trituberculatus)

(f) Specific name: CDS

The construction specific operation is as follows:

1. extracting total RNA of the portunus trituberculatus and purifying mRNA: total RNA of adult tissues of blue crab was extracted using Trizol reagent of Invitrogen corporation. Agarose gel electrophoresis was used to analyze the degree of RNA degradation and whether there was contamination, Nanodrop was used to detect the purity of RNA, Qubit was used to quantify the RNA concentration accurately, and Agilent 2100 was used to detect the integrity of RNA accurately.

2. Constructing a portunus trituberculatus cDNA library: after the mRNA sample obtained in the above step is qualified, mRNA is enriched by magnetic beads with oligo (dT). Then fragmentation buffer is added to break mRNA into short segments, the mRNA is used as a template, six-base random primers are used for synthesizing single-strand cDNA, buffer solution, dNTPs, DNA polymerase I and RNase H are added to synthesize double-strand cDNA, and AMPure XP beads are used for purifying double-strand cDNA. The purified double-stranded cDNA is subjected to end repair, A tail is added and a sequencing adaptor is connected, and then AMPure XP beads are used for fragment size selection. Finally, PCR amplification is carried out, and the PCR product is purified by using AMPure XP beads to obtain a final library (constructed and synthesized by Beijing Nuo grass genesis science and technology Co., Ltd.).

3. Transcriptome sequencing and analysis: and after the cDNA library is qualified, carrying out Illumina HiSeq/MiSeq sequencing on different libraries according to effective concentration and the requirement of target off-machine data volume. For the transcriptome analysis of the species without the reference genome, the sequence obtained by sequencing is spliced into a transcript, and the transcript is taken as a reference sequence for subsequent analysis. The original image Data file obtained by high-throughput sequencing is converted into an original sequencing sequence through CASAVA base recognition analysis, and the original sequencing sequence is called Raw Data or Raw Reads. However, the original sequencing sequence contained low quality reads with a linker. In order to ensure the information analysis quality, raw reads must be filtered to obtain clean reads, and subsequent analysis is based on the clean reads. After clean reads are obtained, splicing the clean reads by using Trinity transcriptome splicing software. The longest transcript in each gene was taken as unigene for subsequent analysis. Subsequent analyses included gene function annotation (gene function annotation databases include Nr, Nt, Pfam, KOG/COG, Swiss-prot, KEGG, GO), CDS prediction, gene expression level analysis, orthologous gene screening, and other bioinformatics analyses.

4. Homology analysis of blue crab unigene sequence and screening of PtFREP gene fragment: 1 FREP-related unigene (c93184_ g1) was obtained from the Portunus trituberculatus transcriptome and subjected to BLASTn and BLASTx analysis in the database, which revealed that the sequence had partial similarity to procambarus clarkii tachylectin and Penaeus vannamei fibleukin and was determined to be the unigene sequence of the PtFREP gene of Portunus trituberculatus.

5. Cloning of PtFREP gene cDNA ORF sequence of blue crab: specific primers F (5'CACGCCCTTCAGGTCCTATGTAGCTT 3') and R (5'TCAGTTAGCAATAGGATTAGCAAGTAG 3') are designed according to unigene sequences homologous to the PtFREP gene, and the ORF of the PtFREP is amplified by using a blue crab cDNA template. Detecting the PCR product by using 1% agarose gel electrophoresis, recovering and purifying the PCR product by using an Axygen gel recovery kit, connecting the PCR product with a pMD-19T vector (Dalianbao bioengineering Co., Ltd.), then transforming escherichia coli competence DH5 alpha (Beijing holotype gold biotechnology Co., Ltd.), selecting a vector primer M13 for positive cloning to perform sequencing, splicing the obtained result by Seqman software, and obtaining the PtFREP gene ORF cDNA sequence of the blue crab, which is shown in SEQ ID No. 1.

6. Amplification of the full-length cDNA sequence of the PtFREP gene of the portunus trituberculatus: two specific primers 3P1(5 'CTACGATGCTCACCTGAACGGCTTGT 3') and 3P2(5 'GGAACTCCCATCTTCATTCCCTCCTG 3') at the 3 'end are designed and amplified on the ORF sequence of the PtFREP gene spliced by sequencing in the step 5, the specific primer 3P1 and 100 xUPM (5' CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT 3') are respectively subjected to first amplification at the 3' end, and the Portunus trituberculatus cDNA is used as a template for full-length amplification. The specific primer 3P2 was amplified for the second time at the 3' end with Nup (5' AAGCAGTGGTAACAACGCAGAGT 3') and the full-length amplification was performed using the result of the first amplification as a template. Sequencing and analysis were as in 5.

ORF amplification reaction system and reaction conditions:

25 μ L reaction:

the reaction was carried out in a TaKaRa PCR Thermal Cycler Dice Model TP600(Takara Bio Inc.): denaturation at 94 deg.C for 3 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 50s, extension at 72 ℃ for 2min, and 35 cycles; extension at 72 ℃ for 10 min.

3' RACE first amplification reaction system and reaction conditions:

25 μ L reaction:

the reaction was carried out in a TaKaRa PCR Thermal Cycler Dice Model TP600(Takara Bio Inc.): denaturation at 98 deg.C for 2 min; denaturation at 98 ℃ for 20s, annealing at 64 ℃ for 30s, extension at 72 ℃ for 2min, and 5 cycles; denaturation at 98 ℃ for 20s, annealing at 61 ℃ for 30s, extension at 72 ℃ for 2min, and 8 cycles; denaturation at 98 ℃ for 20s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 2min, and 25 cycles; extension at 72 ℃ for 10 min.

3' RACE second amplification reaction system and reaction conditions:

50 μ L reaction:

The sequence table SEQ ID No.1 is cloned to PtFREP gene cDNA total length 2428bp from blue crab, wherein, an open reading frame is 1065bp, a 5 'untranslated region is 140bp, a 3' untranslated region is 1223 bp, and a polyadenylic acid tailing signal (AATAAA) and a polyadenylic acid tail are arranged.

Example 2.

The base sequence of the PtFREP sequence table SEQ ID No.1 of the portunus trituberculatus, and the amino acid sequence of the PtFREP sequence table SEQ ID No. 2.

SEQ ID No.2 of the sequence table is:

MAGGTAVVVAVLGVLVAVNSQQETIPEDPTTEYYMRLLRSLLANTTYALHNALTRLQ ENTAQETSVRSLQESLRDVKESVGSLERSIDRVRNSVDNGVDRTRVEIRTFRNRVSGELAD LRHLVEDKTGSVNHKVAEIVAGDCQDLYDLGHNASGVYHLARHGRDVLCEMEGGDGG WLVVQRRARVAEQVDFNLGWDEYKKGFGDLETEFWIGNDFLHVLTNQKTYQLRFDFHD YEDGPFYAAYSTFRVGNEESHYPLYIGDFSGNVTDAFTYHHGRSFSTKDRDNDLYSVGNCA EEFTGGWWYDRCYDAHLNGLFPVVPDRVEAKYITWWAHKGDKRVPFVLDKVTMRIKPK PV

it has a complete coding protein containing 354 amino acids, a signal peptide (1-20) in the coding sequence, a predicted molecular weight of 40.32kDa and an isoelectric point of 5.48. The mature peptide contains 334 amino acids with a typical FBG domain (137-352), where two carbohydrate binding sites NASG and NVTD are predicted.

The method for obtaining the portunus trituberculatus fibrinogen related protein PtFREP recombinant protein comprises the following specific operations:

designing specific primer PtFREP-QF (5' CGC) containing restriction enzyme BamHI and XhoI cutting sites according to cDNA sequence corresponding to SEQ ID No.2GGATCCACCACTGAGTATTACATGCGGCTG 3') and PtFREP-QR (5' CCG)CTCGAGTGGTTTCGGCTTGATCCTCAT 3') by PCR amplification of the gene fragment encoding the mature peptide of PtFREP (see FIG. 2) in TaKaRa PCR Thermal Cycler Dice Model TP600(Takara Bio Inc..) under the following conditions: denaturation at 94 deg.C for 3 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 50s, extension at 72 ℃ for 2min, and 35 cycles; finally, extension is carried out for 10min at 72 ℃. Then cloning the gene into a pET32a (+) expression vector by enzyme digestion, transforming the gene into escherichia coli BL21(DE3), inoculating a positive clone into an LB culture medium after confirming the correct expression frame by sequencing, and carrying out shake culture at 37 ℃ until OD is reached_600nmWhen the concentration is 0.4-0.6, IPTG is added to the final concentration of 1mM, induction is carried out for 4 hours, and then the thalli are collected by centrifugation. The cells were treated with ultrasonic waves at 180W for 45min (2 s each time at 2s intervals) under ice bath conditions, and the supernatant was centrifuged off to collect the precipitate. After the precipitate was dissolved in 8M urea, the recombinant product was purified by a TALON column from Clontech. The purified recombinant protein was transferred to a dialysis bag, dialyzed at 4 ℃ against a dialysis renaturation solution (pH 8.0) containing 2mM reduced glutathione, 0.2mM oxidized glutathione, 1mM EDTA, 50mM Tris-HCl, 50mM NaCl, 10% glycerol and 1% glycine and a gradient of urea (6, 5, 4, 3, 2, 1, 0M) to renature the recombinant protein, and finally dialyzed 2 times against 50mM Tris-HCl (pH 8.0) buffer to remove other components in the solution. The recombinant protein after dialysis and renaturation is concentrated by a Microsep Advance ultrafiltration centrifugal concentration tube of PALL company, and the concentration of the recombinant protein PtFREP related to the blue crab fibrinogen protein is 2.5mg/mL (see figure 3) measured by a BCA protein concentration measuring kit of Biyunshi company.

Example 3.

1. In vitro bacteriostasis test of portunus trituberculatus fibrinogen related protein PtFREP recombinant protein

Culturing and preparing microorganisms: culturing Vibrio parahaemolyticus at 28 deg.C with TSB culture medium, culturing Vibrio alginolyticus at 28 deg.C with TSB culture medium, culturing Pseudomonas aeruginosa at 37 deg.C with TSB culture medium, culturing Staphylococcus aureus at 37 deg.C with LB culture medium, culturing Micrococcus luteus at 37 deg.C with LB culture medium, culturing Pichia pastoris at 28 deg.C with YPD culture medium, culturing the above strains at 220rpm/min of shaking table to logarithmic phase, diluting with 50mM Tris-HCl (pH 8.0) buffer solution to obtain bacterial colony number of about 1 × 10 per ml bacterial liquid³And (4) respectively.

Determination of the bacteriostatic activity of recombinant protein fibrinogen-related protein PtFREP: after the recombinant protein PtFREP obtained in the above example was diluted with a gradient of Tris-HCl (50mM, pH 8.0) (1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64), 50 μ L of the recombinant protein diluted to different concentrations was added to a sterile flat-bottomed 96-well plate (Costar, Fisher) and 50 μ L of Tris-HCl (50mM, pH 8.0) was used as a control, 50 μ L of the bacterial suspension was added, and a well to which only 50 μ L of the bacterial suspension was added was used as a blank well. After incubation of the 96-well plate for 2 hours at the culture temperature of the bacterial solution, 150. mu.L of the corresponding medium was added, and the blank wells were incubated overnight with medium up to 200. mu.L. And reading a 96-well plate added with vibrio parahaemolyticus, vibrio alginolyticus, pseudomonas aeruginosa and pichia pastoris at a wavelength of 560nm to measure a light absorption value, and measuring the 96-well plate added with staphylococcus aureus and micrococcus luteus at a wavelength of 600 nm. The minimum inhibitory concentration is the range between the maximum recombinant protein concentration at which the microorganism can grow and the minimum concentration at which the microorganism is completely inhibited from growing. The recombinant protein PtFREP of the above example is found to have obvious inhibitory action on gram-negative bacteria of vibrio parahaemolyticus, vibrio alginolyticus and pseudomonas aeruginosa, gram-positive bacteria of staphylococcus aureus and micrococcus luteus, the minimum inhibitory concentrations are respectively 0.39-0.79 muM, 0.79-1.56 muM and 0.79-1.56 muM, and the recombinant protein PtFREP has no obvious inhibitory action on the fungus pichia pastoris.

2. FITC staining of in vitro microorganism agglutination experiment FITC of Portunus trituberculatus fibrinogen related protein PtFREP recombinant protein: collecting 1.0mL of the above bacteria and fungi cultured to logarithmic phase, centrifuging at 4 deg.C and 4000rpm for 5min, collecting thallus, discarding cultureAfter that, the cells were washed 3 times with PBS. Adding to the mixture to a final concentration of 0.1mg mL^-1FITC, slow shake staining overnight in the dark. The cells were collected by centrifugation at 4000rpm for 5min at 4 ℃ and washed 3 times with PBS after discarding the medium to remove the remaining FITC.

And (3) bacteria coagulation experiment: FITC-labeled bacteria were resuspended in sterile PBS and the concentration adjusted to 2.5X 10⁹Each mL^-1. In the experimental group, 25 mu L of PtFREP recombinant protein and 20 mu L of FITC labeled bacterial suspension are uniformly mixed in a 1.5mL centrifuge tube; in the control group, 25. mu.L of rTrx was mixed with 20. mu.L of FITC-labeled bacterial suspension. To observe Ca²⁺Whether or not it has an influence on the agglutination activity, 10mM CaCl was added to each of the experimental and control groups₂And chelation of Ca with 10mM EDTA²⁺. The sample was incubated in the dark at 28 ℃ for 2h with slow shaking and 5. mu.L was taken and observed under a fluorescent microscope (see FIG. 4). The recombinant protein PtFREP of the above example was found in Ca²⁺In the presence of the compound has obvious agglutination effect on gram negative bacteria of vibrio parahaemolyticus, vibrio alginolyticus and pseudomonas aeruginosa, gram positive bacteria of staphylococcus aureus and micrococcus luteus and fungus pichia pastoris.

3. In vitro microorganism binding experiment of Portunus trituberculatus fibrinogen related protein PtFREP recombinant protein

Respectively taking 1.0mL of the bacteria and the fungi which are cultured to logarithmic phase, centrifuging at 10000rpm for 1 min, and collecting thalli; the collected cells were fixed in 1.0mL of 37% formaldehyde and shaken gently at 37 ℃ for 1 hour. Centrifuging bacteria and fungi at 4 deg.C and 4000rpm for 10min, and collecting thallus; washing the thallus with 1.0mL of Tris-HCl buffer solution for 2 times, and finally resuspending the thallus with 1.0mL of Tris-HCl buffer solution; respectively mixing 0.5mL of the bacterial suspension with 0.5mL of PtFREP recombinant protein, shaking gently at 4 ℃ for 30min, and then centrifuging at 4 ℃ and 4000rpm for 5 min; reserving the supernatant to a new centrifuge tube, washing the precipitate for 2 times by using 1.0mL of Tris-HCl buffer solution, and reserving the washing solution for the 1 st time to the new centrifuge tube; eluting the recombinant thallus protein conjugate with 50 μ L of 1 xSDS-PAGE loading buffer solution, and respectively mixing 50 μ L of supernatant and 50.0 μ L of washing solution with 50 μ L of 5 xSDS-PAGE loading buffer solution; the eluate, supernatant and washing solution were subjected to 15% SDS-PAGE, respectively (see FIG. 5). As can be seen from FIG. 5, the recombinant protein PtFREP of the above example has binding activity to the gram-negative bacteria Vibrio alginolyticus, Vibrio parahaemolyticus and Pseudomonas aeruginosa, gram-positive bacteria Staphylococcus aureus and Micrococcus luteus, and the fungus Pichia pastoris.

4. Portunus trituberculatus fibrinogen-related protein PtFREP recombinant protein PAMPs combination experiment

Detection of pathogen association of recombinant proteins by enzyme-linked immunosorbent assayMoleculeThe binding activity of PAMPs (pathogenic-associated molecular patterns, some shared highly conserved molecular structures on the surface of pathogenic microorganisms) is modeled. The main components of Lipopolysaccharide (LPS) of gram-negative bacteria cell wall, Peptidoglycan (PGN) of gram-negative bacteria and gram-positive bacteria cell wall, and dextran (GLU) of yeast cell wall are selected as research objects.

20mg of LPS, GN and GLU were dissolved in 100mL of 50mM NaCO3-NaHCO3 buffer (pH 9.6), respectively. 100 μ L of LPS, GN and GLU solution (20 μ g) was added to a 96-well plate and coated for 16-18h at 4 ℃. The uncoated PAMPs in the 96-well enzyme label plate are poured out, washed for 3 times with PBS-T, 5min each time, 3% BSA prepared by 200 mu L PBS is added into each well, and the wells are blocked for 1h at 37 ℃. Washing with PBS-T for 5min 3 times, adding 100 μ L of recombinant protein diluted in gradient and 5mM CaCl to each well₂And 0.1mg/mL BSA, incubated at 18 ℃ for 3 h. Thereafter, the cells were washed 3 times with PBS-T for 5min, and 100. mu.L of diluted anti-His-tag antibody (1:1000) was added to each well, followed by incubation at 37 ℃ for 1 h. Then, the cells were washed 3 times with PBS-T for 5min, 100. mu.L of a diluted secondary HRP-labeled goat anti-mouse antibody (1:1000) was added to each well, and incubated at 37 ℃ for 1 h. Washing with PBS-T for 3 times, each time for 5min, preparing color development working solution with TMB color development kit, incubating at room temperature for 30min, and reading at 450nm wavelength. Each experiment was repeated 3 times. ELISA Index (EI) was calculated by the formula, EI ═ OD value of sample/(OD average of negative controls + s.d. value). When EI is>1.0, the recombinant protein is considered to have binding activity to PAMP. As can be seen from FIG. 6, the EI values of the recombinant protein PtFREP of the above examples to LPS, PGN and GLU were all higher than 1.0, indicating that the recombinant protein PtFREP had binding activity to LPS, PGN and GLU.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> oceanographic institute of Chinese academy of sciences

<120> Portunus trituberculatus fibrinogen associated protein PtFREP gene, and coding protein and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2428

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ggtagtaaag gagagcgcag agggaggaga gggagaagaa gaggaggtga agctgtgacg 60

cgccctacac gcccttcagg tcctatgtag cttcaccagg gtccctccgt ggtgctgaac 120

gacgaagtgg cggcagggac atggcgggcg ggacggcggt ggtagtggcg gtgctggggg 180

tgctggtggc cgtgaacagc cagcaggaaa caatcccaga ggacccgacc actgagtatt 240

acatgcggct gctcagatcc ctcttagcaa acacgacata cgccctacat aacgccctca 300

cccgcctgca ggagaacaca gcgcaggaga cgagtgtgag gagcctgcag gagtctttga 360

gggacgtgaa ggagtctgtg ggcagtttgg agaggagcat tgatagggtt cgtaacagtg 420

ttgataatgg agtggacagg acgcgcgtgg agattcgcac cttccgtaac cgcgtgtccg 480

gggagctggc agacctgagg cacctggtgg aggacaagac cgggagcgtc aaccacaagg 540

tggcggagat agtagcgggg gactgccagg atctgtacga cctgggacac aacgccagcg 600

gagtatacca cctggcgagg cacgggcgtg atgtcctgtg tgagatggaa ggcggcgacg 660

gaggctggct ggtggtgcag aggcgggcga gggtggctga gcaggtagac ttcaacttgg 720

gctgggacga gtacaagaag ggttttgggg acctggagac ggagttctgg attggcaatg 780

atttcttaca cgtcctgact aaccagaaga cttatcagct ccgttttgac ttccacgact 840

acgaggatgg ccctttctat gctgcctaca gcacattccg agtgggcaac gaggaaagtc 900

attaccctct ctacatcggt gacttctctg gcaatgtcac ggacgccttc acgtaccacc 960

acggacgctc cttctccacc aaggaccgcg acaacgacct ctactccgtc gggaactgcg 1020

ccgaggaatt cacaggtggc tggtggtacg acaggtgcta cgatgctcac ctgaacggct 1080

tgtttcccgt cgtgccggac cgagtggagg ccaaatacat tacgtggtgg gcgcacaaag 1140

gggacaagag agtgcccttc gtgctggaca aggtcaccat gaggatcaag ccgaaaccag 1200

tatgatctaa aatgtgaccc tgaccttcgt gacctattct gtgtgacctc cagtatgacc 1260

ttgaccttcg tgtgactttt caatgtgacc ttcaattctt gacctccagt gtgaccccca 1320

gtgtgagtga ccttgacctc tgtgtgatcc tcaatgtgac cttcatttct tgaccttaga 1380

aatacccctg ccaggaactc ccatcttcat tccctcctgt tattaacttt tttggtcacc 1440

ctgtactggt ttaccgagaa atttcccgcc tttgtaacac acacacctgc ctgcaccacc 1500

ctgtaagctg tactattatt gttattatta tcattattat tattacaatc actattaata 1560

cttttttgct acttgctaat cctattgcta actgatttgt gaccctttca gtattgggac 1620

gcatttttac tttgggtttt gtgtaccaga agaccatttt actgacatta agaagggtat 1680

ggaggttaga agattaatag ccacagactt gactatttta atccccacgt gagtttgtga 1740

aggtgtgtaa aatcaccaaa tagtaagcag aatgaagatg aagatgcgtc atagtactaa 1800

agggatgaaa gcatttagag caattccctt catttagtat ctcagttgtt tttctccgtt 1860

catttcttcc acggcctccc tcacagaccg ccttcagacc gcctgccact ttctctctct 1920

ctctctctct ctctctctca tttgattgtt tgcactggta tatatattgt cactctcttt 1980

ggtatggttt aagtaagtag ttgcatttgt caagcctcag attccttcat agttttcact 2040

ctctggctct ttctccaatg ctgtgagtga atcgatcgac ccatgtattc attttatttc 2100

atttttattt tattcttacc ttttattttc agtttatctt atcttaggcg ctgttttttt 2160

ttatttgatt aactttgtaa acgaaaagat cgacttggta tcgatctttg actttcttga 2220

tcgtattttg caactgtttc ttactttact tggtgtttta gttaatgttc ctctacgtaa 2280

ataatgagaa gatttgtcta attttcagct tattttcatc ttatcttgat tgtgtttgga 2340

ttgcatgtac accttttagt cactgtctca tcaagctata tgaatgaaga gatttgtccc 2400

aaaaaaaaaa aaaaaaaaaa aaaaaaaa 2428

<210> 2

<211> 354

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ala Gly Gly Thr Ala Val Val Val Ala Val Leu Gly Val Leu Val

1 5 10 15

Ala Val Asn Ser Gln Gln Glu Thr Ile Pro Glu Asp Pro Thr Thr Glu

20 25 30

Tyr Tyr Met Arg Leu Leu Arg Ser Leu Leu Ala Asn Thr Thr Tyr Ala

35 40 45

Leu His Asn Ala Leu Thr Arg Leu Gln Glu Asn Thr Ala Gln Glu Thr

50 55 60

Ser Val Arg Ser Leu Gln Glu Ser Leu Arg Asp Val Lys Glu Ser Val

65 70 75 80

Gly Ser Leu Glu Arg Ser Ile Asp Arg Val Arg Asn Ser Val Asp Asn

85 90 95

Gly Val Asp Arg Thr Arg Val Glu Ile Arg Thr Phe Arg Asn Arg Val

100 105 110

Ser Gly Glu Leu Ala Asp Leu Arg His Leu Val Glu Asp Lys Thr Gly

115 120 125

Ser Val Asn His Lys Val Ala Glu Ile Val Ala Gly Asp Cys Gln Asp

130 135 140

Leu Tyr Asp Leu Gly His Asn Ala Ser Gly Val Tyr His Leu Ala Arg

145 150 155 160

His Gly Arg Asp Val Leu Cys Glu Met Glu Gly Gly Asp Gly Gly Trp

165 170 175

Leu Val Val Gln Arg Arg Ala Arg Val Ala Glu Gln Val Asp Phe Asn

180 185 190

Leu Gly Trp Asp Glu Tyr Lys Lys Gly Phe Gly Asp Leu Glu Thr Glu

195 200 205

Phe Trp Ile Gly Asn Asp Phe Leu His Val Leu Thr Asn Gln Lys Thr

210 215 220

Tyr Gln Leu Arg Phe Asp Phe His Asp Tyr Glu Asp Gly Pro Phe Tyr

225 230 235 240

Ala Ala Tyr Ser Thr Phe Arg Val Gly Asn Glu Glu Ser His Tyr Pro

245 250 255

Leu Tyr Ile Gly Asp Phe Ser Gly Asn Val Thr Asp Ala Phe Thr Tyr

260 265 270

His His Gly Arg Ser Phe Ser Thr Lys Asp Arg Asp Asn Asp Leu Tyr

275 280 285

Ser Val Gly Asn Cys Ala Glu Glu Phe Thr Gly Gly Trp Trp Tyr Asp

290 295 300

Arg Cys Tyr Asp Ala His Leu Asn Gly Leu Phe Pro Val Val Pro Asp

305 310 315 320

Arg Val Glu Ala Lys Tyr Ile Thr Trp Trp Ala His Lys Gly Asp Lys

325 330 335

Arg Val Pro Phe Val Leu Asp Lys Val Thr Met Arg Ile Lys Pro Lys

340 345 350

Pro Val

Claims

1. A Portunus trituberculatus fibrinogen related protein PtFREP gene is characterized in that: the gene PtFREP of the portunus trituberculatus fibrinogen related protein is shown as a base sequence in a sequence table SEQ ID No. 1.

2. The protein encoded by the Portunus trituberculatus fibrinogen-related protein PtFREP gene of claim 1, which is characterized in that: the PtFREP gene coding protein is shown as an amino acid sequence in a sequence table SEQ ID No. 2.

3. The use of the portunus trituberculatus fibrinogen-related protein PtFREP gene-encoded protein according to claim 2, characterized in that: the recombinant expression product of the portunus trituberculatus fibrinogen related protein PtFREP gene coding protein is applied to the preparation of antibacterial drugs, immunoactive agents, bacteria agglutination preparations, bacteria combination preparations, feed additives, preservatives or preservatives.

4. The use of the portunus trituberculatus fibrinogen related protein PtFREP gene encoding protein of claim 3, characterized in that: the recombinant expression product of the portunus trituberculatus fibrinogen related protein PtFREP gene coding protein is applied to the preparation of antibacterial drugs of gram-negative bacteria or gram-positive bacteria.

5. The use of the portunus trituberculatus fibrinogen related protein PtFREP gene encoding protein according to claim 4, characterized in that: the gram-negative bacteria are vibrio parahaemolyticus, vibrio alginolyticus or pseudomonas aeruginosa; the gram-positive bacteria is staphylococcus aureus or micrococcus luteus.

6. The use of the portunus trituberculatus fibrinogen related protein PtFREP gene encoding protein of claim 3, characterized in that: the recombinant expression product of the portunus trituberculatus fibrinogen related protein PtFREP gene coding protein is applied to the preparation of agglutination preparations or combination preparations of gram-negative bacteria, gram-positive bacteria or fungi.

7. The use of the portunus trituberculatus fibrinogen related protein PtFREP gene encoding protein of claim 6, characterized in that: the gram-negative bacteria are vibrio parahaemolyticus, vibrio alginolyticus or pseudomonas aeruginosa; the gram-positive bacteria are staphylococcus aureus or micrococcus luteus; the fungus is Pichia pastoris.

8. The use of the portunus trituberculatus fibrinogen related protein PtFREP gene encoding protein of claim 3, characterized in that: the recombinant expression product of the portunus trituberculatus fibrinogen related protein PtFREP gene coding protein is used for preparing a combined preparation of bacterial or yeast cell wall components.

9. The use of the portunus trituberculatus fibrinogen related protein PtFREP gene encoding protein of claim 8, characterized in that: the main components of the bacterial cell wall are lipopolysaccharide and peptidoglycan; the main component of yeast cell wall is glucan.