CN108424916B

CN108424916B - Lateolabrax japonicus interleukin IL-12p40 gene and application thereof

Info

Publication number: CN108424916B
Application number: CN201810273601.8A
Authority: CN
Inventors: 邱丽华; 王鹏飞; 赵超; 闫路路; 范嗣刚; 陈祥
Original assignee: South China Sea Fisheries Research Institute Chinese Academy Fishery Sciences
Current assignee: South China Sea Fisheries Research Institute Chinese Academy Fishery Sciences
Priority date: 2018-03-29
Filing date: 2018-03-29
Publication date: 2021-04-30
Anticipated expiration: 2038-03-29
Also published as: CN108424916A

Abstract

The invention discloses cDNA of an interleukins IL-12p40 gene of lateolabrax japonicus, the nucleic acid sequence of which is shown in SEQ ID NO. 1. Also discloses the coding protein of the weever interleukin IL-12p40 gene, the amino acid sequence of which is shown in SEQ ID NO.2, and the invention further discloses an expression vector comprising the cDNA. And the application of the coding protein or recombinant protein of the lateolabrax japonicus interleukin IL-12p40 gene in the preparation of medicaments with the effects of resisting bacteria and enhancing the immunity of fish.

Description

Lateolabrax japonicus interleukin IL-12p40 gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a lateolabrax japonicus interleukin IL-12p40 gene and application thereof.

Background

Lateolabrax japonicus can only rely on innate immunity as the first line of defense to prevent invasion of pathogenic microorganisms and environmental toxic stress. Cytokines play an important role in almost all aspects of pro-inflammatory and immune responses. Interleukins (ILs) are a class of cytokines with complex immunoregulatory functions, which are involved in regulating cell proliferation, maturation, migration and adhesion, and differentiation and activation of immune cells. Interleukins have pro-inflammatory and anti-inflammatory functions, some of which can act as chemoattractants for T-helper cells, function like chemokines, others which participate in the antiviral cellular response process, and function like interferons.

Interleukin 12 is a class of cytokines produced primarily by inflammatory cells and has a wide range of biological activities. Meanwhile, IL-12 is a unique heterodimeric cytokine. It is composed of subunits alpha-chain (p35) and beta-chain (IL-12p40) through disulfide bond to form heterodimer, IL-12p40 can form IL-12 and IL-23 with p35 and p19 respectively, and can form homodimer p80 by itself, at the same time, p80 plays an important role in various research phyla.

However, in summary, IL-12p40 has been studied less and less extensively in fish, particularly in lateolabrax japonicus.

Disclosure of Invention

The first purpose of the invention is to provide cDNA of lateolabrax japonicus interleukin IL-12p40 gene and amino acid of the coding protein thereof.

The second object of the present invention is to provide an expression vector containing cDNA of the interleukin IL-12p40 gene of Japanese sea Perch.

The third purpose of the invention is to provide a preparation method of weever interleukin IL-12p40 recombinant protein and weever interleukin IL-12p40 recombinant protein prepared by the method.

The last purpose of the invention is to provide the application of the coding protein and the recombinant protein of the weever interleukin IL-12p40 gene in the preparation of drugs with the effects of inhibiting and killing bacteria.

Therefore, the invention mainly relates to a cDNA sequence of a lateolabrax japonicus interleukin 12 subunit IL-12p40 (hereinafter referred to as IL-12p40) gene, an amino acid sequence of a coding protein, a space-time expression map, a preparation method of an in vitro recombinant protein, the recombinant protein, a function verification method and an application approach of the gene.

The first object of the present invention is achieved by the following technical solutions: the cDNA of lateolabrax japonicus interleukin IL-12p40 gene has the nucleic acid sequence shown in SEQ ID No. 1.

The amino acid sequence of the coding protein of the weever interleukin IL-12p40 gene is shown in SEQ ID NO. 2.

The cDNA of the weever interleukin IL-12p40 gene is obtained by screening a weever transcriptome to obtain a partial sequence of the weever interleukin IL-12p40, and a PCR primer is designed after the partial sequence of a target gene is verified. The full-length sequence of lateolabrax japonicus IL-12p40 gene is obtained by PCR Amplification of 3 'and 5' ends of target gene by using Rapid Amplification of CDNA Ends (RACE) technology.

The full-length cDNA sequence of interleukin IL-12p40 gene from Lateolabrax japonicus is 1386bp, contains 966bp Open Reading Frame (ORF), comprises 82bp 5 '-non-coding region (5' -UTR), starts from ATG, 338bp 3 '-non-coding region (3' -UTR), ends at TAG, and has poly (A) tail.

The gene codes 321 amino acids, the molecular weight is 34.6kDa, and the PI is 6.0.

The lateolabrax japonicus interleukin IL-12p40 gene was detected universally in all experimental tissues, and was expressed universally and highly in immune tissues. Indicating a sustained effect in physiological processes. The inventor selects the head kidney, spleen and liver as target tissues and treats the target tissues with bacterial stimulation (Vibrio harveyi and Streptococcus agalactiae), and the result shows that the expression level of the gene in immune organs is obviously increased. The gene is highly expressed in immune organs, has certain inhibiting and eliminating effects in the bacterial infection period, and is predicted to play an important role in the antibacterial physiological process.

The second objective of the present invention is achieved by the following technical solutions: comprises the expression vector of cDNA of the weever interleukin IL-12p40 gene.

Obviously, the expression vector also comprises the ORF sequence of the interleukin IL-12p40 gene of the lateolabrax japonicus.

The third object of the present invention is achieved by the following technical solutions: a preparation method of weever interleukin IL-12p40 recombinant protein comprises the steps of transforming host cells by using the expression vector, and culturing a transformant to obtain the weever interleukin IL-12p40 recombinant protein.

Among them, the host cell of the present invention is preferably a prokaryotic or eukaryotic cell.

Further preferred is Escherichia coli BL21(DE 3).

The invention provides a weever interleukin IL-12p40 recombinant protein, which is prepared by a method comprising the steps of transforming host cells by using the expression vector, culturing transformants and obtaining the weever interleukin IL-12p40 recombinant protein from the cultures.

The fourth object of the present invention is achieved by the following technical solutions: the application of the coding protein of the weever interleukin IL-12p40 gene in the preparation of the drugs with the efficacies of resisting bacteria and enhancing the immunity of fish.

Furthermore, the application of the weever interleukin IL-12p40 recombinant protein in preparing medicines with the effects of resisting bacteria and enhancing the immunity of the fish.

According to the invention, through a solid plate bacteriostatic circle experiment, a circular paper sheet is soaked in the encoded protein and recombinant protein of the purified lateolabrax japonicus interleukin IL-12p40 gene, and the paper sheet is placed on a plate fully paved with bacteria, and the specific diameter of the bacteriostatic circle is calculated. The recombinant protein was injected into the culture medium of the bacteria by a liquid bacteria inhibition test, and the inhibitory effect was observed over time. The results show that: the coding protein of the weever interleukin IL-12p40 gene and the weever interleukin IL-12p40 recombinant protein have the antibacterial effect, and the antibacterial effect of the weever interleukin IL-12p40 recombinant protein is superior to that of the weever interleukin IL-12p40 protein.

The invention further injects or does not inject Lateolabrax japonicus interleukin IL-12p40 protein and recombinant Lateolabrax japonicus interleukin IL-12p40 protein, then injects bacteria (Vibrio alginolyticus and aeromonas hydrophila) and then carries out mortality statistics, and determines the actual effect of the bacteria in the antibacterial process. The results show that: the death rate of the experimental group of the coding protein of the weever interleukin IL-12p40 gene and the weever interleukin IL-12p40 recombinant protein is lower than that of the control group, and the death rate of the experimental group of the weever interleukin IL-12p40 recombinant protein is lower than that of the coding protein of the weever interleukin IL-12p40 gene.

Therefore, the cDNA of the weever interleukin IL-12p40 gene is a sequence isolated from the weever. The cDNA of lateolabrax japonicus IL-12p40 encodes 321 amino acid polypeptide, and has 1386 base pairs (bp). The protein encoded by the translation has the conserved structural domain characteristic of IL-12p40c of leukocyte family. From phylogenetic analyses and sequence charts, it can be seen that Lateolabrax japonicus IL-12p40 belongs to a typical IL-12/IL-6 family member in the Lateolabrax japonicus interleukin family. Has strong connection with the immune system. At the transcriptional level, mRNA from Lateolabrax japonicus IL-12p40 was detected ubiquitously in various tissues, suggesting that it plays a key role in the physiological processes of Lateolabrax japonicus. Scientific studies have also shown that lateolabrax japonicus IL-12p40 plays a key role in eliminating immune and inflammatory processes mediated by viral or bacterial infection through bacterial stress experiments. However, IL-12p40 has been studied less and less extensively in fish, especially flowers.

The research provides the useful information that the lateolabrax japonicus IL-12p40 gene has the effect of coping with various toxic factors in prawns, is favorable for further exploring the functional mechanism of the lateolabrax japonicus IL-12p40, can be used as a novel molecular biological standard substance applied to ecotoxicology, or can be used as a medicine for resisting diseases of fishes or enhancing the immunity of the fishes, and is favorable for understanding various physiological mechanisms.

The invention has the beneficial effects that: the invention obtains a new Lateolabrax japonicus IL-12p40 cDNA sequence from Lateolabrax japonicus transcriptome, discovers that the sequence can be expressed with high efficiency in eukaryotic or prokaryotic cells by exploring the function of the Lateolabrax japonicus IL-12p40 by a biological gene engineering technology; the invention also clones the cDNA sequence of the lateolabrax japonicus IL-12p40 to a prokaryotic or eukaryotic expression vector, transforms an escherichia coli competent cell BL21(DE3), obtains the recombinant protein thereof through the positive induction expression of a positive clone, and researches the property of the recombinant protein to discover that the lateolabrax japonicus IL-12p40 gene is specifically and highly expressed in immune tissues, the recombinant and purified protein thereof has the functions of inhibiting growth and sterilizing bacteria, and the death rate reduction phenomenon caused by harmful stimulation can be reduced in a live injection experiment of the lateolabrax japonicus.

Drawings

FIG. 1 is a diagram showing in a preferred embodiment the blastP results of lateolabrax japonicus IL-12p40, containing the IL12p40c subfamily domain of the Ig subfamily family;

FIG. 2 is a diagram showing the detection of the relative expression level of lateolabrax japonicus IL-12p40 in different tissues by real-time quantitative PCR, wherein beta-actin is selected as an internal reference gene;

FIG. 3A is a graph showing the distribution of Lateolabrax IL-12P40 expression in brain following bacterial stimulation (Vibrio harveyi and Streptococcus agalactiae) in one embodiment, beta-actin was selected as an internal reference gene, representing P <0.05, and P < 0.01.

FIG. 3B is a graph showing the distribution of Lateolabrax IL-12P40 expression in the spleen following bacterial stimulation (Vibrio harveyi and Streptococcus agalactiae) in one embodiment, beta-actin was selected as an internal reference gene, representing P <0.05, representing P < 0.01;

FIG. 3C is a graph showing the distribution of Lateolabrax IL-12P40 expression in the liver following bacterial stimulation (Vibrio harveyi and Streptococcus agalactiae) in one embodiment, beta-actin was selected as an internal reference gene, representing P <0.05, representing P < 0.01;

FIG. 4A is the fusion expression protein of lateolabrax japonicus IL-12p40 gene in Escherichia coli, wherein: m: protein marker; 1-2. E.coli BL21 strain of pGEX4T-1 empty vector, IPTG induced expression, induction time is respectively: 0 hour, 24 hours; and 3-5. E.coli BL21 recombinant strain of pGEX4T-1-LmIL-12p40, and IPTG induced expression for the following induction times: 0 hour, 6 hours, 24 hours; supernatant of E.coli BL21 recombinant protein of pGEX4T-1-LmIL-12p 40;

FIG. 4B is the fusion expression protein of lateolabrax japonicus IL-12p40 gene in Escherichia coli, wherein: m: protein marker; 1-2. E.coli BL21 strain with pET28a empty vector, IPTG induced expression, induction time is respectively: 0 hour, 24 hours; 3-5. E.coli BL21 recombinant strain of pET28a-LmIL-12p40, IPTG induced expression, the induction time is respectively: 0 hour, 6 hours, 24 hours; supernatant of E.coli BL21 recombinant protein of pET28a-LmIL-12p 40;

FIG. 5 is a graph showing the protein interaction verification of lateolabrax japonicus IL-12p40 in the embodiment, wherein (A) is a Pull-down experiment; His-LmIL-12p40 fusion protein and GST fixed on magnetic beads and GST-LmIL-12p40 protein are incubated, denatured and eluted, protein complexes are dissociated from the magnetic beads, and the His-LmIL-12p40 fusion protein is verified by Western blotting with an anti-His antibody; (B) the figure is added to the reaction mixture of His-LmIL-12p40 protein by using anti His antibody immunoblot analysis detection; (C) the figure is bound to magnetic beads GST or GST-LmIL-12p40 protein through the use of anti GST antibody western blot analysis detection, synthesis of LmIL-12p 80;

FIG. 6 shows the Ni-NTA resin affinity purified lateolabrax japonicus IL-12p40 prokaryotic expression protein, wherein M: protein marker; Ni-NTA resin affinity purified pET28a-LmIL-12p40, 2-3, rLmIL-12p80 first collection tube, second collection tube respectively;

FIG. 7 is a test of the antibacterial effect of the lateolabrax japonicus rLmIL-12p40 and rLmIL-12p80 in the in vitro solid experiment on bacteria, wherein A-H respectively represent the antibacterial effects of Vibrio vulnificus, Aeromonas hydrophila, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio anguillarum, Vibrio alginolyticus, Staphylococcus aureus and Streptococcus agalactiae, and a-e respectively represent PBS, unloaded pET28a, unloaded pGEX4T-1, rLmIL-12p40 protein and rLmIL-12p80 protein;

FIG. 8 is a test of the antibacterial effect of the rLmIL-12p40 and rLmIL-12p80 on bacteria in an in vitro liquid assay of lateolabrax japonicus in accordance with an embodiment;

FIG. 9A is a test of the antibacterial effect of rLmIL-12p40 and rLmIL-12p80 on Vibrio alginolyticus in an in vivo experiment of lateolabrax japonicus in accordance with an embodiment;

FIG. 9B is a test of the antibacterial effect on Aeromonas hydrophila in vivo experiments with rLmIL-12p40 and rLmIL-12p80 of weever in a specific embodiment.

Detailed Description

The invention is further illustrated below by the following specific embodiments, to which, however, the invention is not at all restricted.

1. Extraction of Total RNA and full-Length cDNA library construction

1.1 extraction of Total RNA

After fresh and alive healthy lateolabrax japonicus (weight about 300g) is temporarily cultured in a Zhuhai experimental base of a south sea aquatic research institute for 4 days (water temperature is about 26 +/-2 ℃, air pump is filled, salinity is 10), dissecting shrimp bodies, taking out about 500mg of each tissue, immediately putting into liquid nitrogen, and purifying total RNA from the dissected tissue (about 50 mg). Cryo-mill disruption was performed, total RNA samples were extracted according to Trizol reagent, and the genome was removed using DNase digestion.

1.2 extraction of Total RNA, preparation of full-Length cDNA template

First cDNA was synthesized from 1. mu.g of total RNA according to the instructions of PrimeScript TM Reverse Transcriptase Transcriptase kit (TaKaRa, Dalian, China) and reacted at 42 ℃ for 2 minutes. Taking the reaction solution for amplification reaction, carrying out amplification reaction at 37 ℃, 15 minutes and 85 ℃ for 5 seconds, taking out the mother solution, storing the mother solution at-80 ℃ for later use, keeping the concentration of a common PCR working solution at 70 ng/mu L for later use, keeping the concentration of a quantitative PCR working solution at 30 ng/mu L for later use, and placing the mother solution at-20 ℃ for use.

2. Cloning of complete sequence of lateolabrax japonicus IL-12p40 gene cDNA

2.1 sequence verification

From the cDNA library constructed before, screening Lateolabrax japonicus IL-12p40 partial sequence. Partial sequences were verified using LmIL-12p 40-F5'-ATGCGTACAGTGTTCCTTGTGG-3' and LmIL-12p 40-R5'-CTAGCAGTTCACGTTTTGTCTGTT-3' primers.

Using the synthesized cDNA as a template to perform PCR amplification, wherein the reaction system is as follows: 17. mu.L of ultrapure water, 2.5. mu.L of 10 XExtaq buffer, 2. mu.L of 10mmol/L dNTP, 1. mu.L of cDNA template, 1. mu.L of each of 10nmol/L primers F and R, and 0.5. mu.L of Extaq enzyme, for a total of 25. mu.L.

The reaction conditions are as follows: denaturation at 94 deg.C for 3 min; 35 cycles: high temperature denaturation at 94 ℃ for 30s, low temperature annealing at 58 ℃ for 30s, and extension at 72 ℃ for 100 s; extending for 10min at 72 ℃; keeping the temperature at 4 ℃.

The amplified PCR product was detected by electrophoresis on a 1.5% agarose gel, and the objective product was recovered and purified from the gel. The purified PCR product was then cloned into pUC-18T vector, E.coli DH-5. alpha. competent cells were transformed, single strains of positive clones were picked, shaken at 220rpm for 6h, the plasmids DNA with inserts were purified and sequenced with M13 universal primers.

2.2 obtaining of full-Length cDNA of Lateolabrax japonicus IL-12p40

Specific primers were designed based on the verified sequence fragments. The 3' end of the target gene is subjected to PCR Amplification by using a Rapid Amplification of CDNA Ends (RACE) technique.

3' -RACE primers were synthesized according to sequence: an amplification primer 5'-ACCACCGAGGACACCAAGTA-3' and an amplification primer 5'-ACTCTATTCCTTTATGCCCTAACT-3', adopting the synthesized full-length cDNA as a template, and adopting touchdown PCR and nest PCR methods according to the following steps

RACE5'/3' kit (TaKaRa, Japan) was used for 3' RACE PCR amplification.

The first reaction volume is 25 mu L, and the reaction condition is 94 ℃ denaturation for 3 min; 9 cycles: denaturation at 94 ℃ for 30s, annealing at 72 ℃ (1 ℃ for 30s, extension at 72 ℃ for 50s, and cooling at 1 ℃ every cycle; 24 cycles: denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, and extension at 72 ℃ for 50 s; extending for 10min at 72 ℃; keeping the temperature at 4 ℃.

2, keeping the amplification system unchanged, and carrying out denaturation at 94 ℃ for 3min under the reaction condition; 35 cycles: denaturation at 94 ℃ for 30s, annealing at 66-60 ℃ (gradient reaction) for 30s, and extension at 72 ℃ for 50 s; extending for 10min at 72 ℃; keeping the temperature at 4 ℃.

PCR products are separated by 1.5 percent agarose gel electrophoresis, a PUC-18T vector is subcloned, T7 and SP6 are subjected to sequencing reaction to obtain a nucleic acid sequence of 338bp of a 3' RACE fragment, and the full-length cDNA obtained after splicing is 1386 bp.

2.3 bioinformatic analysis of Lateolabrax japonicus IL-12p40

TABLE 1 blast analysis results of Lateolabrax japonicus IL-12p40

Homology analysis was performed using Blast (http:// www.ncbi.nim.nih.gov /), and as a result, as shown in Table 1 above, the gene has high homology with IL-12p40 proteins of large yellow croaker, yellowtail, and the like, and it was revealed that the gene was the IL-12p40 gene.

Bioinformatic analysis using DNAstar et al revealed that the initiation codon ATG was located at nucleotides 83-85, the stop codon was located at nucleotides 1046-1048, and the open reading frame was 966 nucleotides, presumably encoding a protein of 321 amino acids. The 5' UTR is 82bp, the 3' UTR is 338bp, and a typical tailing signal AATAAA exists in the 3' UTR. The predicted molecular mass was 34.6kDa and the theoretical PI value was 6.0.

The nucleotide and deduced amino acid sequence of lateolabrax japonicus IL-12p40 are shown above; wherein each row of numbers represents a nucleotide or amino acid position, the initiation codon (ATG) and the terminator codon (TAA) are boxed; domains are shaded; WDYPD is a class I cytokine profile; the seven active cysteines are double underlined, the three glycosylation sites are underlined, the polyadenylation signal sequence (AATAAA) is in bold, and the poly (A) signal sequence is in italics.

The protein is analyzed by ExPASy software and SignalIP software, the Lateolabrax japonicus IL-12p40 protein sequence belongs to the interleukin 12 family, the protein is analyzed to be a signal peptide sequence at 1-16 amino acids, and has two conserved structural domains of IL-12p40, IGc2 and IL-12p40C are respectively positioned at 46-92 and 127-202 amino acids, and the C end of the protein has a WDYPD structural domain, so that the protein is disclosed to be a type I cytokine receptor and is extremely conserved.

The comparison of the nucleotide and amino acid sequences of the lateolabrax japonicus interleukin IL-12p40 gene is shown in figure 1.

3, detecting the distribution of the lateolabrax japonicus IL-12p40 in different tissues of the lateolabrax japonicus by PCR

RNA of different tissues (including gill, brain, liver, heart, spleen, intestine, muscle and head kidney) of the male and female weever is extracted. The method for extracting total RNA is described above.

The PCR reaction used quantitative primers qLmIL-12p40-F (5'-TGGTGAAGGCAGAGCGTCATTTGG-3') and qLmIL-12p40-R (5'-TGGTGAGGGAGATGCGGTGTTGT-3') to amplify lateolabrax japonicus IL-12p40 gene, and housekeeping gene beta-actin (GenBank: HE577671.1) (beta-actin-F: 5'-CAACTGGGATGACATGGAGAAG-3', beta-actin-R: 5'-TTGGCTTTGGGGTTCAGG-3') as internal reference, and the above synthesized cDNA as template.

The reaction total was 12.5. mu.L, containing 6.25. mu.L of 2 XSSYBR Pre-Mix ExTaq (TaKaRa, Dalian, China), 1. mu.L of cDNA template, 0.5. mu.L of 30mmol/L of each of qLmIL-12p40-F and qLmIL-12p40-R, and 4.25. mu.L of deionized water.

PCR reaction conditions were set at 94 ℃, 30s, then 40 cycles: 94 ℃, 5s, 60 ℃, 30s, 72 ℃, 30 s. The dissolution curve analysis was 65-95 ℃ to ensure amplification of each single product.

Through 2^-ΔΔCtThe method analyzes the relative expression level of the lateolabrax japonicus IL-12p 40. The results of qRT-PCR are shown in FIG. 2, and IL-12p40 was expressed in each tissue, expressed in high abundance in immune tissues such as the head kidney, intestine and spleen, and expressed the highest in the head kidney.

PCR detection of expression of lateolabrax japonicus IL-12p40 mRNA under different immune stimulation conditions

The Chinese lateolabrax japonicus breeding industry has a lot of difficulties and problems in the development process, wherein diseases seriously threaten the development of the global lateolabrax japonicus breeding industry and cause huge economic loss. These include Vibrio harveyi and Streptococcus agalactiae. Bacterial infestation or environmental stress usually causes the activation of the innate immune system of marine organisms, wherein IL-12p40 plays a key role in protection, p40 and p80 are genes with immune function, and genes with p40 induced expression are often considered to participate in the natural immune process of host antibiotics. To confirm whether lateolabrax japonicus p40 was involved in this process, Vibrio harveyi and Streptococcus agalactiae stimulated expression experiments were performed.

Healthy lateolabrax japonicus according to 400 mu L1X 10⁸CFU/mL density Vibrio harveyi, Streptococcus agalactiae, control group injected with 400. mu.L PBS buffer (NaCl 137mM, KCl 2.7mM, Na)₂HPO₄ 10mM，KH₂PO₄2mM, pH7.4) intraperitoneal injection. The head kidney, spleen and liver were harvested at 0,6,12,24,48,72 and 96h post injection, minced, stored in liquid nitrogen and taken back to the laboratory for analysis.

The total RNA extraction method and the cDNA reverse transcription method are described above. The fluorescent quantitative PCR primer is designed according to the whole cDNA of the Lateolabrax japonicus IL-12p40 gene, and beta-actin is selected as an internal reference gene. The quantitative system and the reaction procedure were as described above. The results are shown in the attached figures 3A-3C, the lateolabrax japonicus IL-12p40 has obvious up-regulation or down-regulation tendency in head kidney, spleen and liver of immune tissues after injection, the expression level is obviously higher than that of a control group, and the fact that the lateolabrax japonicus IL-12p40 participates in the immune process after immune stimulation is shown, and the lateolabrax japonicus IL-12p40 is an immune response expression protein.

5. Preparation of rLmIL-12p40 fusion protein of lateolabrax japonicus

The entire lateolabrax japonicus IL-12p40 coding region was amplified by PCR (rLmIL12p 40-F15 ' -GATCTGGTTCCGCGTGGATCCATGCGTACAGTGTTCCTTGTG-3 ', rLmIL12p 40-R15 ' -GTCACGATGCGGCCGCTCGAGCTAGCAGTTCACGTTTTGTC-3;) and (rLmIL12p 40-F25 ' -CAGCAAATGGGTCGCGGATCCATGCGTACAGTGTTCCTTGTG-3 ', rLmIL12p 40-R25 ' -GTGGTGGTGGTGGTGCTCGAGGCAGTTCACGTTTTGTC-3 ') and cloned into the pET28a plasmid and pGEX 4T-1.

The recombinant plasmids pET28a-LmIL-12p40 and pGEX4T-1-LmIL-12p40 were transformed into E.coli BL21(DE3) strain. Two kinds of E.coli BL21 cells were cultured in Luria-Bertani (LB) medium containing 100. mu.g/mL ampicillin and 50. mu.g/mL kanamycin until OD600 of E.coli BL21 cell solution reached 0.4-0.6. As shown in FIGS. 4A-4B, two proteins, pET28a-LmIL-12p40(rLmIL-12p40) and pGEX4T-1-LmIL-12p40, were successfully induced and expressed by prokaryotic expression.

When 1mM isopropyl-. beta. -D-thiogalactopyranoside (IPTG) was added to LB medium at a final concentration of 1mol/L, pET28a-LmIL-12p40 induced cells for 6h at 37 ℃ and 220rpm, and pGEX4T-1-LmIL-12p40 induced cells for 24h at 16 ℃ and 100 rpm.

The recombinant lateolabrax japonicus IL-12p40 protein was purified by Ni-NTA affinity chromatography according to the His-binding Purification Kit (Novagen). The purified protein was verified by SDS-PAGE and Western blot, and the 6 XHis tag fusion protein added by the vector was removed and was consistent with the predicted protein size results. The concentration of the obtained lateolabrax japonicus protein was measured using a modified BCA protein assay kit (Sangon Biotech, Shanghai, China).

Purified recombinant lateolabrax japonicus pET28a-LmIL-12p40(rLmIL-12p40) and pGEX4T-1-LmIL-12p40 proteins were stored at-80 ℃ for further use, as shown in FIG. 5, where there was an interaction between rLmIL-12p40, rLmIL-12p80 can be synthesized.

6. Interaction of the bass rLmIL-12p40 and synthesis of rLmIL-12p80

GST pull-down experiment is an effective in vitro test technology for verifying protein interaction, and is mainly characterized in that target protein-GST (Glutathione-S-transferase Glutathione sulfydryl transferase) fusion protein is subjected to affinity solidification on Glutathione affinity resin and serves as a support for affinity with target protein, the target protein solution passes through a column, and can capture 'capture protein' (target protein) interacted with the target protein from the column, after the conjugate is eluted, SDS-PAGE electrophoresis analysis is carried out, so that the interaction between the two proteins or the corresponding target protein is screened, and the 'bait protein' and the 'capture protein' can be obtained through methods such as cell lysate, purified protein, an expression system, an in vitro transcription and translation system and the like.

The bait protein pGEX4T-1-LmIL-12p40 and the preprotein rLmIL-12p40 were obtained from the above experiment, and rLmIL-12p80 was synthesized by pull down experiment (FIG. 6).

7. In vitro antibacterial function research of Lateolabrax japonicus IL-12p40 and IL-12p80 recombinant protein

Experiments show that the LmIL-12p40 and LmIL-12p80 recombinant proteins of the lateolabrax japonicus have the antibacterial effect on vibrio vulnificus, aeromonas hydrophila, vibrio harveyi, vibrio parahaemolyticus, vibrio anguillarum, vibrio alginolyticus, staphylococcus aureus and streptococcus agalactiae in living bodies from two angles of solid and liquid.

Solid zone culture, experiments with the full of various bacteria LB plate, rLmIL-12p40 protein, rLmIL-12p80 protein, no load of pET28a, no load of pGEX4T-1 and PBS phosphate buffer 50 u g suction injection on the circular filter paper. Detecting the diameter of the bacteriostatic zone at 28 ℃ for 12-16h, and checking the bacteriostatic effect.

The results show that the lateolabrax japonicus IL-12p40 and IL-12p80 recombinant proteins have bacteriostatic effects, and the bacteriostatic effect of the rLmIL-12p80 is better than that of the rLmIL-12p40 (figure 7).

Liquid bacteriostasis experiment, the concentration of 8 bacteria in 10ml centrifuge tube is 1X 10⁶CFU/mL, rLmIL-12p40 protein, rLmIL-12p80 protein, no load of pET28a, no load of pGEX4T-1 and PBS phosphate buffer 50 u g suction injection in the centrifuge tube. Checking the bacteriostasis effect at 28 ℃ and 180rpm for 3-6 h.

The results show that the lateolabrax japonicus IL-12p40 and IL-12p80 recombinant proteins have bacteriostatic effects, and the bacteriostatic effect of the rLmIL-12p80 is better than that of the rLmIL-12p40 (figure 8).

8. Research on in vivo antibacterial function of LmIL-12p40 and LmIL-12p80 recombinant proteins of lateolabrax japonicus

To further confirm the antibacterial effect of LmIL-12p40 and LmIL-12p80 recombinant proteins of lateolabrax japonicus on Vibrio alginolyticus and Aeromonas hydrophila in living bodies, the following experiment was performed.

The experimental lateolabrax japonicus was divided into five groups of 20 pieces, and the first and second groups were used as control groups to which 100. mu.L of Vibrio alginolyticus suspension (5. about.10. mu.L) was injected 12 hours after the injection of 100. mu.L of sterilized phosphate buffered saline (PBS, pH7.4)⁸CFU/mL) or 100. mu.L of Aeromonas hydrophila suspension (5 x 10)⁸CFU/mL); third group, fourth group as experimental group after injecting 200. mu.g of LmIL-12p40 and LmIL-12p80 recombinant proteins into Lateolabrax japonicus for 12 hours, Vibrio alginolyticus suspension (5 x 10) was injected respectively⁸CFU/mL) or 100. mu.L of Aeromonas hydrophila suspension (5 x 10)⁸CFU/mL). Death was recorded after 24,48 and 72h, respectively.

The results show that the mortality rate of the rLmIL-12p40 and rLmIL-12p80 experimental group is lower than that of the control group, and the mortality rate of the lateolabrax japonicus rLmIL-12p80 experimental group is lower than that of the rLmIL-12p40 (FIG. 9A, FIG. 9B).

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 and are included in the scope of the present invention.

Sequence listing

<110> research institute for aquatic products in south China sea

<120> lateolabrax japonicus interleukin IL-12p40 gene and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1386

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

agcagagcca tctcacctct ctggcacgct cattaagtca gctttcagtt gtgaacattt 60

tggatcaagt ccagttctca agatgcgtac agtgttcctt gtggtcctgt atgctgcact 120

gtgctatgcc agctctgaca gcagccaaga aaacattgag actctaatgg ataatgttct 180

ggtcctgagg gtgccttatg gtctgggcac cagggtgaat gttcctctga cctgtggaga 240

agcttatgaa aaccagcccg tgttttggca gaaaaacggc gtggttgttg agccagctct 300

gcaggggaac caggtcaatg ttctggtgga ggagatggat ggaggaaact acacttgtca 360

cctcggccca ggcggcgaat acctcaacca caccgtgatc ctaatccaac tagacccaga 420

caacaggact gtcatactgg aagaaaaatc ccctgcggat ggtcacatcc actgttcagc 480

acccaactat aaaggctctt tccactgcac ttggacaaga acaaggtcca gatccaatgc 540

cgctgtgcta ctggtgaagg cagagcgtca tttggaaaag attccctgtg agctggatgc 600

tgatggatca gggctacact gccaggatgc cagctgccca tacaaagagg aacaacaccg 660

catctccctc accatttaca tacatagcta ctctcgccta gaggcctaca caaaggcttt 720

ctacctgaga gagattgtga ggccagaaaa actccctaac ctgcacatca gtaatgggaa 780

tgtgttcagc tgggactacc ctgactcctg ggagaagccc tgcaccttct tcggcctgca 840

gtttcaggtc aaggtggtcc acagtggaca ttcctgtagc agtgaagaac acgaaataat 900

gcacaccacc accgaggaca ccaagtatga agtcaatgtc aaaaccaaga agtacgtttt 960

ctgtgtgaga gctcaggaca agttcactag cgggccattt agccactgga gcaaatgcat 1020

agtgaacaga caaaacgtga actgctagct tcttcactgg gctgcagcca taaaggaaga 1080

aaggacacaa ggatggaatt tctttttatt ttcatatatt acaaggcaat ctgcatcaac 1140

cacaaaagtg gtgctgcacc ttgggagtga aagtatacct gtacagtatt tattgatcac 1200

tcatttaaag ttaacattat ttgtaacatg attgaaataa attactctat tcctttatgc 1260

cctaactttt attattaact taccattcaa aatatttgtt aaaatgtttc taaacatttc 1320

ataattaatt aaaatgttga cttcttgcta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380

aaaaaa 1386

<210> 2

<211> 321

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Arg Thr Val Phe Leu Val Val Leu Tyr Ala Ala Leu Cys Tyr Ala

1 5 10 15

Ser Ser Asp Ser Ser Gln Glu Asn Ile Glu Thr Leu Met Asp Asn Val

20 25 30

Leu Val Leu Arg Val Pro Tyr Gly Leu Gly Thr Arg Val Asn Val Pro

35 40 45

Leu Thr Cys Gly Glu Ala Tyr Glu Asn Gln Pro Val Phe Trp Gln Lys

50 55 60

Asn Gly Val Val Val Glu Pro Ala Leu Gln Gly Asn Gln Val Asn Val

65 70 75 80

Leu Val Glu Glu Met Asp Gly Gly Asn Tyr Thr Cys His Leu Gly Pro

85 90 95

Gly Gly Glu Tyr Leu Asn His Thr Val Ile Leu Ile Gln Leu Asp Pro

100 105 110

Asp Asn Arg Thr Val Ile Leu Glu Glu Lys Ser Pro Ala Asp Gly His

115 120 125

Ile His Cys Ser Ala Pro Asn Tyr Lys Gly Ser Phe His Cys Thr Trp

130 135 140

Thr Arg Thr Arg Ser Arg Ser Asn Ala Ala Val Leu Leu Val Lys Ala

145 150 155 160

Glu Arg His Leu Glu Lys Ile Pro Cys Glu Leu Asp Ala Asp Gly Ser

165 170 175

Gly Leu His Cys Gln Asp Ala Ser Cys Pro Tyr Lys Glu Glu Gln His

180 185 190

Arg Ile Ser Leu Thr Ile Tyr Ile His Ser Tyr Ser Arg Leu Glu Ala

195 200 205

Tyr Thr Lys Ala Phe Tyr Leu Arg Glu Ile Val Arg Pro Glu Lys Leu

210 215 220

Pro Asn Leu His Ile Ser Asn Gly Asn Val Phe Ser Trp Asp Tyr Pro

225 230 235 240

Asp Ser Trp Glu Lys Pro Cys Thr Phe Phe Gly Leu Gln Phe Gln Val

245 250 255

Lys Val Val His Ser Gly His Ser Cys Ser Ser Glu Glu His Glu Ile

260 265 270

Met His Thr Thr Thr Glu Asp Thr Lys Tyr Glu Val Asn Val Lys Thr

275 280 285

Lys Lys Tyr Val Phe Cys Val Arg Ala Gln Asp Lys Phe Thr Ser Gly

290 295 300

Pro Phe Ser His Trp Ser Lys Cys Ile Val Asn Arg Gln Asn Val Asn

305 310 315 320

Cys

Claims

1. An interleukin IL-12p40 gene of lateolabrax japonicus, which is characterized in that: the cDNA nucleic acid sequence is shown in SEQ ID NO. 1.

2. The protein encoded by the interleukin IL-12p40 gene of claim 1, which is characterized in that: the amino acid sequence of the polypeptide is shown as SEQ ID NO. 2.

3. An expression vector comprising the cDNA of claim 1.

4. A method for preparing recombinant protein of weever interleukin IL-12p40, which comprises transforming host cells by the expression vector of claim 3, and culturing the transformant to obtain recombinant protein of weever interleukin IL-12p 40.

5. The method of claim 4, wherein: the host cell is a prokaryotic cell or a eukaryotic cell.

6. The method of claim 5, wherein: the host cell is escherichia coli BL 21.

7. A recombinant protein of weever interleukin IL-12p40, which is prepared by the method comprising the steps of transforming host cells by the expression vector of claim 3, culturing the transformant and obtaining the recombinant protein of weever interleukin IL-12p40 from the culture.

8. The use of the encoded protein of the interleukin IL-12p40 gene of claim 2 in the preparation of medicaments for resisting bacteria and enhancing the immunity of fish.

9. The use of the recombinant protein of the interleukin IL-12p40 of claim 7 in the preparation of medicaments with antibacterial and fish immunity enhancing effects.