CN107893059B - Preparation and application of tilapia disease-resistant immune gene recombinant protein - Google Patents

Abstract

The invention provides a tilapia disease-resistant immune gene recombinant protein, the amino acid sequence of which is SEQ ID NO. 1; one of the nucleotide sequences of the gene is SEQ ID NO. 2. The recombinant protein of the invention is used for preparing biological products for inhibiting microorganisms. The tilapia pik3r3b gene is used as a material to construct a prokaryotic expression vector of fish phosphatidylinositol 3 kinase regulatory subunit genes for the first time and develop recombinant protein, the expression vector is stably expressed in escherichia coli, and the recombinant protein has obvious bacteriostatic activity on various gram negative bacteria and gram positive bacteria and can be applied to disease control of aquatic animals such as fish and the like. The technical method of the invention can also be popularized and applied to other fishes.

Description

Preparation and application of tilapia disease-resistant immune gene recombinant protein

Technical Field

The invention belongs to the field of fish genetic engineering in the field of biotechnology, and relates to a fish immune gene capable of being applied to prevention and treatment of aquatic diseases, and a preparation method of a prokaryotic expression vector and a recombinant protein of the gene.

Background

Tilapia is an important freshwater aquaculture fish in the world and is praised as one of the main sources of future animal protein. In recent years, the tilapia mossambica breeding industry in China is rapidly developed, the breeding scale is continuously enlarged, the breeding variety is continuously increased, the breeding yield is also increased year by year, and the tilapia mossambica is one of the leading aquaculture varieties in China, so that the tilapia mossambica becomes the largest tilapia export country in China. However, with the rapid development of the tilapia aquaculture industry, more and more problems begin to emerge. The most serious of them is that the high-density cultivation brings frequent outbreaks of diseases, which causes serious economic loss. The most serious of them is bacterial disease caused by streptococcus, often resulting in 30-60% mortality of tilapia. Although the medicine can prevent and treat diseases to a certain extent in a short time, the medicine also brings disadvantages, for example, the long-term frequent use of antibiotics and other chemical medicines can cause pathogenic substances to generate drug resistance, the medicine is remained in fish bodies and pollutes water environment, and the like, and potential threats can be generated to the living environment and health of human beings. The breeding of disease-resistant varieties by means of molecular breeding technology has been reported in recent years and has made some progress. Although the method meets the requirement of pollution-free and green development of aquaculture, the urgent need of disease control in the tilapia culture industry is difficult to meet due to the long breeding period. Therefore, the screening of fish disease-resistant immune genes and the development of recombinant disease-resistant proteins through a genetic engineering technology are very important. Firstly, obtaining immune genes, then obtaining a recombinant protein capable of effectively inhibiting the growth of pathogenic bacteria by using an in vitro recombination technology, and providing genetic materials and methods for research on fish disease resistance mechanisms and disease control.

Tilapia is an important cultivated fish in the world, so the research on immune related genes of tilapia has been widely carried out, so far, the research on the cloning of some immune related genes on tilapia is carried out, and the research comprises the molecular cloning, the gene structure, the expression and the polymorphism analysis of major histocompatibility antigen (MHC) IIA and IIB genes, the molecular cloning and the expression analysis of CD59 genes and the relation with streptococcus agalactiae immunoreaction; cloning and expression analysis of CD2BP2 gene, and molecular cloning and functional analysis of CC1 chemokine. However, the genes are obtained by a homologous cloning method according to homologous sequences of other fish immune related genes, the screening of disease-resistant immune genes of tilapia is more important, and the recombinant protein of the genes is more suitable for the requirements of disease prevention and disease resistance of tilapia. Research on cloning, expression analysis, preparation of recombinant protein and antibacterial activity analysis of tilapia phosphatidylinositol 3 kinase PI3K gene, which is an important disease-resistant immune gene, is not reported at present. The invention firstly researches the space-time expression characteristics of tilapia PI3K gene and the change before and after pathogenic bacteria infection, constructs the recombinant expression vector of the gene, and establishes the methods for preparing recombinant protein and analyzing antibacterial activity.

Disclosure of Invention

The invention aims to provide a preparation and application method of tilapia disease-resistant immune gene recombinant protein, wherein the recombinant protein has obvious bacteriostatic activity and provides a gene sequence and a method for research of fish disease-resistant mechanism and development of disease-resistant feed additives.

The invention firstly provides a disease-resistant immune gene screened from tilapia, the amino acid sequence of the disease-resistant immune gene is SEQ ID NO. 1;

one of the nucleotide sequences of the gene is SEQ ID NO. 2;

a recombinant expression vector for recombinant expression of the disease-resistant immune gene protein;

the recombinant expression vector is a recombinant prokaryotic expression vector;

the invention also provides the recombinant protein of the disease-resistant immune gene, which is prepared by transferring the recombinant expression vector into host bacteria for expression;

the recombinant protein is used for preparing a biological product for inhibiting microorganisms;

the microorganism is gram-positive bacteria (staphylococcus aureus and streptococcus agalactiae) or gram-negative bacteria (escherichia coli, pseudomonas and parahemolytic bacteria);

one of the products is a feed additive;

the invention clones pik3r3b gene for the first time by using tilapia as a material, constructs a prokaryotic expression vector of fish phosphatidylinositol 3 kinase regulatory subunit gene, develops recombinant protein, has stable expression in escherichia coli, has obvious bacteriostatic activity of the recombinant protein on various gram negative bacteria and gram positive bacteria, and can be applied to disease control of aquatic animals such as fish and the like. The technical method of the invention can also be popularized and applied to other fishes.

Drawings

FIG. 1 shows the cDNA and protein sequence of tilapia pik3r3b gene,

FIG. 2: the pik3r3b gene has a tissue distribution map in Nile tilapia,

FIG. 3: analysis of expression levels in immune tissues of pik3r3b gene after infection with Streptococcus agalactiae,

FIG. 4: a recombinant nile tilapia pik3r3b gene escherichia coli expression vector diagram,

FIG. 5: SDS-PAGE electrophoresis picture, protein purification picture and purified protein verification picture of the recombinant protein after induction, wherein M represents protein marker; supernatant 1, 2: electrophoresis results of the supernatant after the transformation and induction of the recombinant vector; and (3) precipitation: performing electrophoresis on a sediment part in the bacteria liquid after induction expression; blank control: obtaining a result after the induction expression of the empty vector; and (3) whole bacteria: and (5) performing transformation induction on the recombinant vector.

FIG. 6: FIG. 2 is a graph of the results of protein western blot assays, wherein the precipitates: performing electrophoresis on a sediment part in the bacteria liquid after induction expression; supernatant fluid: electrophoresis results of the supernatant after the transformation and induction of the recombinant vector; and (3) whole bacteria: and (5) performing transformation induction on the recombinant vector.

FIG. 7: FIG. 3 is a diagram showing the bacteriostatic activity of recombinant protein against E.coli, wherein the values in the diagram are protein concentrations, and the growth state of the E.coli colonies is shown when the concentrations are respectively 0.1mg/ml to 0.5 mg/ml.

FIG. 8: the bacteriostatic effect of the recombinant protein on the parahaemolyticus is shown in the figure, wherein the numerical value in the figure is a colony growth state diagram when the protein concentration is 0.1mg/ml-0.5mg/ml, respectively.

FIG. 9: the bacterial inhibition effect of the recombinant protein on pseudomonas is a colony growth state diagram, wherein the numerical values in the diagram are protein concentrations which are respectively 0.1mg/ml-0.5mg/ml recombinant protein concentrations.

FIG. 10: the bacteriostatic effect of the recombinant protein on staphylococcus aureus is shown in the figure, wherein the numerical value in the figure is a colony growth state graph when the protein concentration is 0.1-0.5 mg/ml of the recombinant protein concentration respectively.

FIG. 11: a diagram of the bacteriostatic effect of the recombinant protein on streptococcus agalactiae, wherein A: a positive control group of ampicillin; b: high concentration group, recombinant protein stock solution; c: low concentration group, which is recombinant protein stock solution diluted to 50%; d: and the negative control group is PBS.

FIG. 12: the bacteriostatic effect graph of the recombinant protein on Shewanella is shown in the specification, wherein A: a positive control group of ampicillin; b: high concentration group, recombinant protein stock solution; c: low concentration group, which is recombinant protein stock solution diluted to 50%; d: and the negative control group is PBS.

Detailed Description

The following describes embodiments of the present invention in detail, taking cloning of tilapia pik3r3b gene and construction of expression vector as examples, with reference to the accompanying drawings. The method comprises the steps of amplifying an open reading frame ORF region of tilapia pik3r3b gene by adopting a PCR technology, cloning the ORF region to an E1 expression vector, constructing a prokaryotic expression vector of the tilapia pik3r3b gene, converting the expression vector into escherichia coli BL21, carrying out in-vitro recombinant expression of the gene to obtain a recombinant expression product, purifying the recombinant expression product by a GE His-tag affinity chromatographic column to obtain purified tilapia pik3r3b gene recombinant protein, wherein the molecular weight of the recombinant protein is about 65kD, carrying out antibacterial experiments on 3 gram negative bacteria and 1 gram positive bacteria by adopting the recombinant protein to show obvious antibacterial activity, and prompting that the recombinant protein has important application potential and prospect in aquaculture as a fish feed additive.

Example 1: cloning and structural analysis of full-Length cDNA sequence

1. Cloning and analysis of full-length cDNA sequence: according to the sequencing result of the tilapia spleen tissue transcriptome, finding out an mRNA sequence of the tilapia pik3r3b gene. Firstly, a pair of primers (On-pik 3-S: 5'-CTC ATC AGC CAT TAC AGA CA-3'; On pik 3-A: 5'-TAC AGA GCA GGC ATA GCA C-3') is designed according to the sequence, PCR amplification and sequencing verification are carried out On the core sequence of the coding region of the pik3r3b gene, and the length of the amplified fragment is 780 bp. Then, 4 RACE primers (Pik-GSP5-1: 5'-GTT TCT GGC GAA CTC CTT TAT GA-3'; Pik-GSP5-2: 5'-GAC TCA TGT CTG TAA TGG CTG A-3'; Pik-GSP3-1: 5'-GAA CAG CCT AAA GCC TGA TCT CAT A-3'; Pik-GSP3-2: 5'-GGG TGC TAT GCC TGC TCT G-3') are designed according to the verified sequences, Nile tilapia cDNA is used as a template, the 5 'end and the 3' end of the gene are amplified by RACE technology to obtain 667bp and 647bp amplified fragments respectively, and the full-length cDNA sequence (SEQ ID NO:2) of the gene is obtained by splicing by a SeqMan program in a DNAStar software package (figure 1). The full-length cDNA sequence of the gene contains 2018 bases, including a 171 base 5 'untranslated region, a 1422 base open reading frame (SEQ ID NO:3) and a 425 base 3' untranslated region. The open reading frame of the gene encodes a 473 amino acid protein (SEQ ID NO:1) with a theoretical protein molecular weight of 55.2kD and a theoretical isoelectric point of about 6.25, which does not contain a signal peptide (FIG. 1).

Example 2: expression pattern of pik3r3b gene in tissue distribution of tilapia and immune tissue after streptococcus agalactiae infection

Detecting the relative expression quantity of pik3r3b gene in heart, liver, spleen, kidney, small intestine, brain, gill, skin and blood of healthy nile tilapia by using a real-time quantitative PCR method, and researching the tissue distribution of the gene; the relative expression quantity of pik3r3b gene in 4 immune related tissues such as liver, spleen, kidney, gill and the like after streptococcus agalactiae infection is detected by a real-time quantitative PCR method, and the relation between the gene and the immune response of nile tilapia is analyzed.

Firstly, 9 tissues of healthy tilapia (with the weight of about 300g) are taken, and are respectively as follows: heart, liver, spleen, kidney, small intestine, brain, gill, skin and blood. The total RNA of these tissues was extracted by Trizol method and reverse transcribed into cDNA using Takara reverse transcription kit. Gene-specific primers (pik-RT-S: 5'-GGGGAAGAAGTTGCAGGAATAC-3' and pik-RT-A: 5'-TGAAGGCTTCAATAGCGGTTC-3') were designed based on the sequence of pik3r3b gene, and the relative mutexpression levels of pik3r3b gene in the above 9 tissues were determined by real-time quantitative PCR. The reaction conditions are as follows: firstly, denaturation is carried out at 95 ℃ for 30 s; then 95 ℃ for 5s, 60 ℃ for 34s,40 cycles. The results show that: the pik3r3b gene is expressed in all tissues except the skin of tilapia, wherein the expression level of the pik3r3b gene is higher in 4 immune-related tissues such as liver, spleen, kidney and gill, and the expression level of the pik3r3b gene is highest in the kidney; the expression level in the remaining tissues was low (FIG. 2).

The tilapia (weighing about 300g) in the infected group and the control group was treated with an equal amount of Streptococcus agalactiae (0.2mL, titer about 1X 10)⁷CFU/mL) and sterile PBS buffer solution, and taking 4 immune-related tissues of liver, spleen, kidney, gill and the like of each experimental group at seven time points of 0h, 6h, 12h, 24h, 48h, 72h and 96h after injection. The relative expression level of pik3r3b gene in the above 4 tissues was determined by real-time quantitative PCR. The primers, reagents and experimental methods used were as described in the previous step. The results show that: the expression level of pik3r3b gene in the liver, spleen, kidney and gill of tilapia is increased to different degrees 12h after injection of streptococcus agalactiae, wherein the increase in the kidney is most significant; at a later time, the expression level began to gradually decrease until 96h, and was substantially restored to the original level. The above results indicate that pik3r3b gene is involved in immune response of tilapia (fig. 3).

Example 3: construction method of tilapia pik3r3b gene recombinant expression vector

According to the obtained sequence of the tilapia pik3r3b gene, a specific primer is designed, kidney cDNA is used as a template, and the full-length protein coding sequence of the gene is amplified by PCR. And connecting to a commercial pEAZY-E1 expression vector to construct an Escherichia coli expression vector of the gene. Transferring the constructed recombinant expression vector into an expression strain BL21(DE3) competent cell, coating the competent cell on an LB plate culture medium containing ampicillin for culturing for 12 hours, screening antibiotic and detecting PCR (polymerase chain reaction) of the selected monoclonal, screening positive clones containing the recombinant expression vector, and storing the positive clones for later use.

1. Construction of recombinant expression vectors: the adopted carrier is pEAZY-E1 carrier of Beijing holotype gold company, and the carrier has the advantages that: the method is rapid and efficient, and the N end of the recombinant protein is provided with 6 His tags, so that the recombinant protein is convenient to separate and purify. According to the sequence of the tilapia pik3r3b gene open reading frame, a pair of specific primers are designed

Pik-E-S:5’-ATGTATAACACAGTCTGGACGACCA-3’；

Pik-E-A:5’-TCATCTTCGTCCAGAGGGCA-3’；

The kidney cDNA is taken as a template, the open reading frame region of the gene is amplified by a conventional PCR method, an amplification product is recovered, and the kidney cDNA is connected into a pEAZY-E1 expression vector after the sequencing verification that the amplification sequence is correct. The ligated plasmid was transferred to E.coli TOP10 strain by heat shock, plated on LB plate medium containing 100. mu.g/mL ampicillin, and cultured at 37 ℃ for 12 hours. Selecting a single clone, adding 1mL of liquid LB culture medium, performing shake culture at 37 ℃ for 1 hour, and performing conventional PCR amplification by using a T7 primer by using a bacterial liquid as a template so as to detect a positive clone containing the recombinant plasmid pEAZY-E1-Pik. The primers used were: t7 Promoter primer: 5'-TAA TAC GAC TCA CTA TAG GG-3' and T7 Terminator primer: 5'-GCT AGT TAT TGC TCA GC GG-3'. And carrying out amplification culture on the positive clones, and extracting plasmids to obtain a recombinant escherichia coli expression vector pEAZY-E1-Pik of tilapia Pik3r3b gene (shown in figure 4).

2. Induced expression of recombinant protein and analysis of expression product:

the vector was transformed into E.coli BL21(DE3) plyS competence, positive monoclonals were picked up into 1mL of liquid LB medium containing ampicillin at a final concentration of 50. mu.g/mL, incubated at 37 ℃ with shaking at 200rpm for 8h to logarithmic growth phase, and strain 1: 1000 inoculating LB culture medium 37 deg.C, 200r/min overnight culture, then according to 1: adding the bacterial liquid into LB culture medium at a ratio of 100, expanding culture at 37 ℃ for 200r/min, culturing until OD600 is 0.6-0.8, adding IPTG (isopropyl-beta-D-thiogalactopyranoside) with the final concentration of 1mM, and inducing overnight at 20 ℃. About 3ml of the bacterial solution was centrifuged for 10min to collect the bacterial pellet, washed with 200. mu.L of precooled PBS at 12000rpm for 1min, centrifuged, suspended with 30. mu.L of PBs, added with 10. mu.L of 4 Xprotein loading buffer, boiled for 5min, and cooled for loading. The success of the induction expression was verified by SDS-PAGE vertical electrophoresis detection (FIG. 5), and the electrophoresis voltage was set as: 180V and 1 h. And (4) detecting to obtain a corresponding target band, performing large-scale induction, centrifuging the induced bacterial liquid, and washing with PBS.

3. Recombinant protein isolation, purification and validation

3.1 isolation and purification of recombinant proteins

20mL of lysate (25mmol/L Tris, 5mmol/L EDTA 50mmol/L NaCl; pH7.5) is added to the collected cell pellet, 0.3mg/mL lysozyme and PMSF (phenyl methyl sulfonyl fluoride) with the final concentration of 1mmol/L are added at the same time, the mixture is mixed well, placed on ice for 2h, and ultrasonically crushed for 30min (crushing for 0.5s, interval of 2 s). And after the crushing is finished, centrifuging at 8000rpm for 30min at low temperature, separating the supernatant and the precipitate into different collecting pipes, performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) protein electrophoresis on the supernatant and the precipitate respectively, and detecting that the expressed protein exists in the form of inclusion bodies. The column was washed twice with 20mL of solution A (50mmol/L Tris-HCl, 2mol/L urea, 0.5mmol/L EDTA, 100mmol/L NaCl, 1% Triton X-100; pH 8.0), centrifuged at 12000rpm at 4 ℃ for 30min after each wash, and the supernatant was discarded. It is washed twice with solution B (50mmol/L Tris-HCl, 2mol/L urea, 0.5mmol/L EDTA, 100mmol/L NaCl; pH 8.0) and likewise centrifuged. After washing, the precipitate was dissolved in a pre-cooled lysis solution (100mmol/L Tris, 8mmol/L urea, 10mmol/L imidazole, 500mmol/L NaCl; pH7.4) by magnetic stirring in a refrigerator at 4 ℃ for 2 hours until the solution became transparent, and centrifuged at 12000rpm at 4 ℃ for 30 minutes. The supernatant obtained by centrifugation was sterilized by filtration through a 0.22 μm filter, and then purified using a HisTrap FF column 1ml (GE, USA) according to the following steps:

(1) firstly, adding a proper amount of sterile water to rinse a HisTrap purification column;

(2) adding 10 column volumes of binding buffer (100mmol/L Tris, 8mmol/L urea, 10mmol/L imidazole, 500mmol/L NaCl; pH7.4) to equilibrate the column;

(3) adding 5 column volumes of protein solution to be purified to fully combine the target protein with the purification column;

(4) washing the purification column again by 10 column volumes of binding buffer;

(5) followed by elution with 10 column volumes of eluent (100mmol/LTris, 500mmol/LNaCl, 8mmol/L urea, 250mmol/L imidazole; pH7.4) to collect the protein;

(6) continuously adding 10 column volumes of binding buffer solution to clean the purification column;

(7) finally, the purification column is cleaned by 20 percent ethanol with 10 column volumes, and the air bubbles are removed for sealing and storage. The purified sample is again subjected to SDS-PAGE vertical electrophoresis detection.

Purifying the recombinant protein, detecting the recombinant protein correctly, filling 10mL of recombinant protein solution into a dialysis bag which is boiled, sealing, and placing the dialysis bag in 2L of precooled dialysate for magnetic stirring gradient dialysis at 4 ℃. The dialysate is added with a proper amount of protein protective agent according to the concentration of urea to set 6 concentration gradients, the dialysis time is 12 hours each time, and the formula of the dialysate in each step is as follows:

(1) dialysate I (25mM Tris-Hcl, 200mM NaCl, 1mM EDTA, 5mM DTT, 6 Murea; pH7.4)

(2) Dialysate II (25mM Tris-Hcl, 50mM NaCl, 1mM EDTA, 4M urea, 1mM GssG, 1mM GsH; pH7.4)

(3) Dialysate III (25mM Tris-Hcl, 20mM NaCl, 1mM EDTA, 2M urea, 1mM GssG, 1mM GsH; pH7.4)

(4) Dialysate IV (25mM Tris-Hcl, 10mM NaCl, 1mM EDTA, 1M urea, 1mM DTT, 1% cysteine, 1mM GssG, 1mM GsH; pH7.4)

(5) Dialysate V (25mM Tris-Hcl, 10mM NaCl, 1mM EDTA, 0.5M urea, 1mM DTT, 1% cysteine, 1mM GssG, 1mM GsH; pH7.4)

(6) Dialysate VI (25mM Tris-Hcl, 10mM NaCl; pH7.4)

After dialysis, the protein solution is transferred into a new clean centrifuge tube, centrifuged at 12000rpm at 4 ℃ for 30min, and the supernatant is renatured protein, and the protein concentration detected by the BCA kit is 2.04mg/mL and stored in a refrigerator at-20 ℃.

3.2 Western-blotting validation of recombinant proteins

After the SDS-PAGE discontinuous electrophoresis is finished, transferring the protein onto a PVDF membrane by using an electrotransfer method, wherein the membrane is opposite to the anode and the gel is opposite to the cathode when the protein is transferred onto the membrane, and the parameters are set to be 200mA and 1h when the protein is transferred onto the membrane; after the membrane transfer is finished, sealing the membrane with 5 percent (5g of skimmed milk powder is dissolved in 100ml of 1 XTSST buffer) of skimmed milk powder for 3 hours; using anti-His as a primary antibody (combined overnight), the dilution ratio is 1:7000, using HRP-labeled goat anti-mouse IgG as a secondary antibody (combined for 2h), the dilution ratio is 1:5000, combining with PVDF membrane in turn, and paying attention to that the target membrane should be washed by 1 × TBST buffer solution before and after combining twice; and finally, performing color development by using a DAB color development kit of Beijing Zhongxiu Jinqiao company, and performing operation according to the kit instruction. The results showed a single band for the purified protein (FIG. 6) and fit the predicted size (65 kD). Therefore, the invention firstly develops the recombinant expression protein of the tilapia pik3r3b gene.

Example 4: production of recombinant protein and method for detecting bacteriostatic activity

1. The induction expression method of the recombinant protein comprises the following steps: firstly, the screened positive colonies are subjected to enrichment culture, positive single colonies are selected and inoculated in a liquid LB culture medium for amplification culture. Culturing until OD600 of the bacterial liquid reaches 0.6, adding IPTG for induction, and culturing at 20 deg.C for 12 hr.

2. Separation and purification of the recombinant protein: the bacterial solution was centrifuged, the medium was removed, and lysis buffer was added to resuspend the cells. Under the ice bath condition, the thalli are broken by ultrasonic waves, and the supernatant is collected by centrifugation. Separating, purifying and enriching protein by using affinity chromatography column.

3. The detection method of the in vitro antibacterial activity of the recombinant protein comprises the following steps:

the bacteriostatic activity of the recombinant protein was performed by bacteriostatic experiments using 3 gram-negative bacteria: escherichia coli, pseudomonas, parahaemolytica, and a gram-positive bacterium: staphylococcus aureus.

And detecting the antibacterial experiment effect by adopting a colony counting method. Firstly, taking strains stored in a laboratory, and carrying out the following steps: 200, adding corresponding culture medium, performing overnight amplification culture at different temperatures of a 200rpm shaking table for about 12h, detecting the absorbance of the bacterial liquid, and diluting the bacterial liquid to 10 degrees by using PBS buffer solution²-10³CFU/mL. 50. mu.L of the diluted bacterial solution and 50. mu.L of 5-histone solutions (final concentration of 0.1mg/ml, 0.2mg/ml, 0.3mg/ml, 0.4mg/ml and 0.5mg/ml in this order) diluted in a gradient were mixed, respectively. In order to ensure that the bacteriostatic effect is the production of protein per se, the group at 0.1mg/ml was used as a control group. After the bacterial liquids are mixed, the bacterial liquids are coated on plates together, 3 times of experiments are set for each group, and the bacteria are cultured overnight at a proper temperature. And counting and analyzing each group of plate colonies the next day, and calculating the bacteriostasis rate according to a formula. The inhibition rate is (number of negative control colonies-number of experimental group colonies)/number of negative control colonies × 100%. Drawing a bacteriostasis curve graph and analyzing the bacteriostasis effect. The results show that the recombinant proteins are resistant to gram-positive (staphylococcus aureus) and gram-negative (escherichia coli,pseudomonas and parahaemolytica) have bacteriostatic activity.

4. Detection of bacteriostatic activity of recombinant protein

The recombinant protein function identification is carried out by bacteriostatic experiments, and the experiments adopt 4 gram-negative bacteria: escherichia coli, pseudomonas, parahaemolytica and shewanella and 2 gram-positive bacteria: staphylococcus aureus and Streptococcus agalactiae, the culture conditions for the experimental bacteria are shown in Table 1.

Table 1: culture conditions of experimental bacteria

And detecting the antibacterial experiment effect by adopting a colony counting method. Firstly, taking glycerol strain preserved in a laboratory, and carrying out the following steps: 200, adding corresponding culture medium, performing overnight amplification culture at different temperatures of a 200rpm shaking table for about 12h, detecting the absorbance of the bacterial liquid, and diluting the bacterial liquid to 10 degrees by using PBS buffer solution²-10³CFU/mL. 50. mu.L of the diluted bacterial solution and 50. mu.L of 5-histone solutions (final concentration of 0.1mg/ml, 0.2mg/ml, 0.3mg/ml, 0.4mg/ml and 0.5mg/ml in this order) diluted in a gradient were mixed, respectively. In order to ensure that the bacteriostatic effect is the production of protein per se, the group at 0.1mg/ml was used as a control group. After the bacterial liquids are mixed, the bacterial liquids are coated on plates together, 3 times of experiments are set for each group, and the bacteria are cultured overnight at a proper temperature. And counting and analyzing each group of plate colonies the next day, and calculating the bacteriostasis rate according to a formula. The inhibition rate is (number of negative control colonies-number of experimental group colonies)/number of negative control colonies × 100%. And drawing a bacteriostasis curve graph by using an Excel table, and analyzing the bacteriostasis effect. The result shows that the recombinant protein of the tilapia pik3r3b gene has bacteriostatic activity on gram-positive bacteria (staphylococcus aureus) and gram-negative bacteria (escherichia coli, pseudomonas and parahaemolyticus). See fig. 7-10. Meanwhile, the recombinant protein of tilapia pik3r3b gene is adopted to carry out bacteriostatic experiments on streptococcus agalactiae and Shewanella, frozen strains at minus 80 ℃ are taken, 100 mu L of bacterial liquid is taken and added into 10mL of liquid culture medium after being melted on ice, and the bacterial liquid is cultured and activated overnight at 150rpm at the corresponding culture temperature. The next day, according to the activated bacteria liquid: the culture medium is 1:200The ratio of (1), enlarged culture, and the light absorption value OD of the bacteria solution₆₀₀Stopping culturing when the concentration is about 0.2-0.4. Dilute 10 with sterile PBS buffer⁴In each case 200. mu.L of each strain was applied evenly to the corresponding solid medium plate. 4 Oxford cups, marked A, B, C, D, were placed on each plate. 30 mu L of high-concentration protein, medium-concentration protein, low-concentration protein and sterile PBS buffer solution are sequentially dripped into each Oxford cup. The recombinant protein is cultured in a positive way for about 20 hours, the growth condition of the experimental bacteria is observed, and the experimental result shows that the recombinant protein has obvious bacteriostatic activity on streptococcus agalactiae (figure 11) and has no bacteriostatic activity on shewanella (figure 12). Therefore, the invention discovers for the first time that the recombinant protein of the tilapia pik3r3b gene has obvious bacteriostatic activity on 5 microorganisms, and the recombinant protein can be used as a fish feed additive to improve the disease-resistant immunity of cultured fishes, so that the gene and the recombinant protein thereof have important application potential and wide popularization prospect in aquaculture.

Sequence listing

<110> research institute for aquatic products in yellow sea of China institute for aquatic science

<120> preparation and application of tilapia disease-resistant immune gene recombinant protein

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Gln Pro Ser Ser Val Ala Met Ala Gly Gly Ser Ser Asn Val Ala Lys

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Asp Gly Gly Ala Gly Gly Ser Leu Gln Glu Ala Glu Trp Tyr Trp Gly

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Asp Ile Ser Arg Glu Glu Val Asn Asp Lys Leu Arg Asp Met Pro Asp

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Gly Thr Phe Leu Val Arg Asp Ala Ser Thr Lys Met Gln Gly Asp Tyr

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Thr Leu Thr Leu Arg Lys Gly Gly Asn Asn Lys Leu Ile Lys Ile Tyr

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

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Val Val Glu Leu Ile Ser His Tyr Arg His Glu Ser Leu Ala Gln Tyr

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Asn Thr Lys Leu Asp Val Lys Leu Met Tyr Pro Ile Ser Arg Phe Gln

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Gln Asp Gln Leu Val Lys Glu Asp Asn Ile Asp Ala Val Gly Lys Lys

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Leu Gln Glu Tyr His Asn Gln Tyr Gln Glu Lys Ser Lys Glu Tyr Asp

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Arg Leu Tyr Glu Glu Tyr Thr Lys Thr Ser Gln Glu Ile Gln Met Lys

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Arg Thr Ala Ile Glu Ala Phe Asn Glu Thr Ile Lys Ile Phe Glu Glu

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Gln Cys His Thr Gln Glu Arg Tyr Ser Lys Asp Tyr Ile Glu Arg Phe

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Arg Arg Glu Ser Asn Asp Lys Glu Ile Glu Arg Ile Met Met Asn Tyr

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Leu Glu Gln Asp Leu Lys Thr Gln Ala Met Asp Asn Arg Glu Thr Asp

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Arg Asp Gln Tyr Leu Val Trp Leu Asn His Lys Gly Val Arg Gln Lys

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Arg Ile Asn Asp Trp Leu Gly Ile Lys Asn Glu Asn Thr Asp Glu Gly

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Tyr Phe Val Ser Glu Glu Asp Glu Asn Leu Pro His Tyr Asp Glu Lys

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Leu Gly Lys Pro Asp Gly Ala Phe Leu Ile Arg Glu Ser Ser Lys Lys

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Gly Cys Tyr Ala Cys Ser Val Val Val Glu Gly Glu Val Lys His Cys

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<211> 2018

<212> DNA

<213> Tilapia (Oreochromys mossambicas)

<400> 2

ggaattcaga tagaagactt agaagtaaac agatagatta acatcgaggc atatgacagg 60

agcacacgat gattgtttct ttttttcttt tttaaatacg ccggtcattt ttactttgtt 120

taatagcgtt tttctaatac aaagactgcc tggttaagct acaagaaagc aatgtataac 180

acagtctgga cgaccaaaaa agccggcgag gaaggagact ggagggacgt gatgatgccc 240

tactccacag agctgatatt ttatctggaa atggaccaac cgccagccct tcctccaaag 300

ccaaccaagc ctcagcagcc ctcctcggtc gcaatggcag ggggcagcag caatgtcgcc 360

aaggatggag gagcaggagg ctccttacaa gaggccgaat ggtactgggg tgacatatcc 420

agggaggagg tgaatgataa gctcagagac atgcctgatg ggactttcct ggttcgggac 480

gcgtctacta agatgcaagg cgactacacg ctaacgctaa ggaaaggggg caataacaag 540

ctgataaaga tctaccacag agatgggaag tacggctttt ctgaccccct gactttcagc 600

tcagtggtgg agctcatcag ccattacaga catgagtcac tggctcaata caacaccaag 660

ctggatgtca agctcatgta ccccatctcc cgcttccagc aggaccagct ggtgaaggag 720

gacaacatcg atgcagtggg gaagaagttg caggaatacc ataatcagta ccaggagaaa 780

agcaaagaat atgacagact gtatgaagag tacaccaaga catcacagga gatccagatg 840

aagcgaaccg ctattgaagc cttcaatgaa accatcaaga ttttcgagga gcaatgccac 900

acgcaggagc gctacagcaa ggactacatt gagcgtttta ggcgtgagag caatgataaa 960

gagattgagc gcatcatgat gaactatgaa aagcttaaat ctcgcctcgg agagatccac 1020

gacagcaaga tgcggctgga gcaggaccta aagacgcaag ccatggacaa ccgggagacg 1080

gacaaaaaga tgaacagcct aaagcctgat ctcatacagc tccgcaaaat cagggatcag 1140

tatctagtct ggctcaatca taaaggagtt cgccagaaac gaattaatga ttggctggga 1200

atcaagaatg agaacacaga cgaaggttac tttgtgagtg aggaagatga gaacttgcct 1260

cactatgatg agaagaactg gtttgtgggg gatctgaaca ggacacaggc agaggagctg 1320

ctcctgggga agccagatgg agcttttctt atccgagaaa gcagcaaaaa agggtgctat 1380

gcctgctctg tagttgtgga aggggaggtg aagcactgtg tcatctacag tacaccacgt 1440

ggctttggct ttgctgagcc atacaacctc tacagcagcc tgaaggacct ggtgctccac 1500

taccaccaga cctccctggt gcagcacaat gactcactca atgtgcgcct agcctaccct 1560

gtctacgcac agatgccctc tggacgaaga tgaaccccaa ccaacgatgc taaacaacta 1620

cccccagaag gacgacacat tttaccgtct tcaggataaa ggagaaaagg gagtgttatg 1680

aagcttggtg gtggtggtat tggttgaatt acaaaacaac cctgtatcca gacctgatga 1740

acatcgatac aattttaaca tttgctgcaa cacacacatc agccaccttg gagttatata 1800

tttttacaag aacaattttg gagggcattc tttctaaaga ctgcttgatt tgcacaagga 1860

agttttaaat gtttgtgaat gtgaaaacaa gagtgaccga aggaaggaaa tgaagtagga 1920

gacgtcagag tttcaggttg atcccgctgc tacctaaact ccagctcaga ctttaataaa 1980

ccagaaaccc aaaaaaaaaa aaaaaaaaaa aaaaaaaa 2018

<210> 3

<211> 1422

<212> DNA

<213> Tilapia (Oreochromys mossambicas)

<400> 3

atgtataaca cagtctggac gaccaaaaaa gccggcgagg aaggagactg gagggacgtg 60

atgatgccct actccacaga gctgatattt tatctggaaa tggaccaacc gccagccctt 120

cctccaaagc caaccaagcc tcagcagccc tcctcggtcg caatggcagg gggcagcagc 180

aatgtcgcca aggatggagg agcaggaggc tccttacaag aggccgaatg gtactggggt 240

gacatatcca gggaggaggt gaatgataag ctcagagaca tgcctgatgg gactttcctg 300

gttcgggacg cgtctactaa gatgcaaggc gactacacgc taacgctaag gaaagggggc 360

aataacaagc tgataaagat ctaccacaga gatgggaagt acggcttttc tgaccccctg 420

actttcagct cagtggtgga gctcatcagc cattacagac atgagtcact ggctcaatac 480

aacaccaagc tggatgtcaa gctcatgtac cccatctccc gcttccagca ggaccagctg 540

gtgaaggagg acaacatcga tgcagtgggg aagaagttgc aggaatacca taatcagtac 600

caggagaaaa gcaaagaata tgacagactg tatgaagagt acaccaagac atcacaggag 660

atccagatga agcgaaccgc tattgaagcc ttcaatgaaa ccatcaagat tttcgaggag 720

caatgccaca cgcaggagcg ctacagcaag gactacattg agcgttttag gcgtgagagc 780

aatgataaag agattgagcg catcatgatg aactatgaaa agcttaaatc tcgcctcgga 840

gagatccacg acagcaagat gcggctggag caggacctaa agacgcaagc catggacaac 900

cgggagacgg acaaaaagat gaacagccta aagcctgatc tcatacagct ccgcaaaatc 960

agggatcagt atctagtctg gctcaatcat aaaggagttc gccagaaacg aattaatgat 1020

tggctgggaa tcaagaatga gaacacagac gaaggttact ttgtgagtga ggaagatgag 1080

aacttgcctc actatgatga gaagaactgg tttgtggggg atctgaacag gacacaggca 1140

gaggagctgc tcctggggaa gccagatgga gcttttctta tccgagaaag cagcaaaaaa 1200

gggtgctatg cctgctctgt agttgtggaa ggggaggtga agcactgtgt catctacagt 1260

acaccacgtg gctttggctt tgctgagcca tacaacctct acagcagcct gaaggacctg 1320

gtgctccact accaccagac ctccctggtg cagcacaatg actcactcaa tgtgcgccta 1380

gcctaccctg tctacgcaca gatgccctct ggacgaagat ga 1422

Claims (2)

1. The application of disease-resistant immune gene protein screened from tilapia in preparing biological products for inhibiting microorganisms, wherein the microorganisms are staphylococcus aureus, streptococcus agalactiae, escherichia coli, pseudomonas or parahaemolyticus; the amino acid sequence of the disease-resistant immunity gene protein is SEQ ID NO. 1.

2. The use according to claim 1, wherein the product is a feed additive.

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