CN113368218B - Antiviral composition containing lateolabrax japonicus interferon IFNd and receptor thereof and application - Google Patents

Abstract

The invention discloses an antiviral composition containing lateolabrax japonicus interferon IFNd and an acceptor thereof and application thereof. The antiviral composition comprises weever interferon IFNd and weever type I interferon receptors, wherein the weever type I interferon receptors are one or more of CRFB1, CRFB2 and CRFB 5. The application is the application of the antiviral composition containing the lateolabrax japonicus interferon IFNd and the receptor thereof in preparing the antiviral drugs for aquatic animals; or the application in preparing the medicine for enhancing the antiviral immunity of the aquatic animals. The antiviral composition can protect the activity of brain cells of the lateolabrax japonicus, and can reduce the copy number of ISKNV in the head kidney cells of the lateolabrax japonicus after the infectious spleen and kidney necrosis virus is attacked. Has better effect on the induction of downstream antiviral genes Mx, viperin, ISG15 and PKR.

Description

Antiviral composition containing lateolabrax japonicus interferon IFNd and receptor thereof and application

Technical Field

The invention relates to interferon and application of an interferon receptor, in particular to an antiviral composition containing lateolabrax japonicus interferon IFNd and an interferon receptor and application thereof.

Technical Field

Lateorax maculirus is an important seawater economic fish in China, the culture period is short, the growth speed is high, the meat quality is delicious, the nutrition is rich, the annual output exceeds 16 million tons, and the Lateorax maculirus is the second place of seawater culture fish. With the popularization of intensive and high-density culture modes, the development of the lateolabrax japonicus culture industry is seriously influenced by fish viral diseases.

Interferons (IFNs) are a family of class II alpha-helical cytokines with antiviral activity that play multiple roles in a variety of cellular response processes by inducing a number of IFN-stimulating factors (ISGs), of which antiviral responses in the body are particularly important. After the body is infected by virus, type I interferon is produced and the response cells are altered by inducing a group of ISGs with regulatory or antiviral functions, thereby inducing the cells to enter an antiviral state. Based on the number of cysteines and phylogenetic relationships, type I IFNs are clearly divided into groups I and II, where group I has 2 cysteines and group II has 4 cysteines, and further divided into subgroups a, d, e, h and b, c, f, etc., respectively. Most fish type I IFNs contain three subgroups a, c and d, a recently discovered IFNh, found only in the order perciformes. Interferons need to be able to signal down by binding to interferon receptors on cell membranes. In mammals, the initiation of type I IFN-mediated signaling pathways is dependent on the interaction between type I IFN and its heterodimeric receptor complex consisting of IFNAR1 and IFNAR 2. IFNAR1 is considered to be a low affinity receptor chain comprising four FNIII extracellular domains and a shorter cytoplasmic domain, while the high affinity receptor chain IFNAR2 comprises two FNIII extracellular domains and a longer intracellular domain. Both IFNAR1 and IFNAR2 belong to the class II cytokine receptor family, known by comparative immunologists in fish as fish cytokine receptor family b (crfb). Whole genome sequence analysis showed that at least 17 CRFB members were present in zebrafish and puffer fish. Phylogenetically, CRFB1 and CRFB2 may be homologous genes of mammalian IFNAR2, and CRFB5 may be homologous genes of mammalian IFNAR 1. Two group I IFNs in zebrafish, IFNa and IFNd respectively, signal through the receptor complex consisting of CRFB1 and CRFB5, while group II IFNs, classified as IFNc, signal through the unique receptor complex consisting of CRFB2 and CRFB 5. In lateolabrax japonicus, the mode and function of interaction between type I IFNs and their receptors is unknown. The research on the I-type interferon immune system of the lateolabrax japonicus is beneficial to improving the prevention and control of viral diseases of the lateolabrax japonicus.

Disclosure of Invention

It is an object of the present invention to provide an antiviral composition comprising lateolabrax japonicus interferon IFNd and its receptor.

Specifically, the antiviral composition containing the lateolabrax japonicus interferon IFNd and the receptor thereof comprises the lateolabrax japonicus interferon IFNd and a lateolabrax japonicus type I interferon receptor, wherein the lateolabrax japonicus type I interferon receptor is one or more than two of CRFB1, CRFB2 and CRFB 5.

Preferably, the antiviral composition comprising lateolabrax japonicus interferon IFNd and its receptor comprises lateolabrax japonicus interferon IFNd, CRFB2 and CRFB 5.

More preferably, the antiviral composition comprising lateolabrax japonicus interferon IFNd and its receptor comprises lateolabrax japonicus interferon IFNd, CRFB1 and CRFB 5.

The second purpose of the invention is to provide the application of the antiviral composition containing the lateolabrax japonicus interferon IFNd and the receptor thereof, in particular to the application of the antiviral composition containing the lateolabrax japonicus interferon IFNd and the receptor thereof in the preparation of aquatic animal antiviral drugs; the antiviral composition containing the lateolabrax japonicus interferon IFNd and the receptor thereof is applied to the preparation of the medicine for enhancing the antiviral immunity of aquatic animals.

The Virus is Infectious Spleen and Kidney Necrosis Virus (ISKNV), nervous Necrosis Virus (VNN), frog Virus (RGV).

The invention has the advantages that:

1. the antiviral composition containing the lateolabrax japonicus interferon IFNd and the receptor thereof can protect the activity of brain cells (LMB) and reduce the copy number of ISKNV (infectious spleen and kidney necrosis virus) (ISKNV) in the lateolabrax japonicus head kidney cells after the Infectious Spleen and Kidney Necrosis Virus (ISKNV) infects the lateolabrax japonicus. The compound has good effect on the induction of downstream antiviral genes (Mx, viperin, ISG15 and PKR), wherein the combination consisting of IFNd + CRFB1+ CRFB5 has the best effect on the induction of the downstream antiviral genes (Mx, viperin, ISG15 and PKR), and has the highest protective effect on the cell activity of virus-infected phoxinus scaphigera muscle cells (FHM).

2. The antiviral composition containing the lateolabrax japonicus interferon IFNd and the receptor thereof provided by the invention not only can enhance the virus antagonistic capacity of the lateolabrax japonicus, but also can improve the virus antagonistic capacity of other aquatic animals.

Drawings

Figure 1 is an expression analysis of LmCRFBs in different tissues. The different letters indicate significant differences between groups (P < 0.05).

FIG. 2 is the spatiotemporal expression of CRFCs under ISKNV stimulation.

FIG. 3-1 is an SDS-PAGE analysis of LmIFNd cell disruption;

m, marker; 1-2, precipitating; 3: and (6) supernatant fluid.

Figure 3-2 is an SDS-PAGE analysis and western blot of LmIFNd protein purification.

FIG. 3-3 is an SDS-PAGE analysis of the proteins of CRFBs after induction of large amounts of bacteria;

m: marker, 1: CRFB2 supernatant, 2: CRFB2 precipitate, 3: CRFB5 supernatant, 4: precipitation, 5: CRFB1 supernatant, 6: CRFB1 precipitated.

FIG. 4-1 shows the ISGs gene expression changes after in vivo injection of LmIFNd-His recombinant protein. FIGS. (a) and (b) show the head kidney and spleen, respectively.

FIG. 4-2 shows ISGs gene expression after LMB cells are incubated with recombinant protein LmIFNd-His.

FIGS. 4-3 are ISKNV (1X 10) after incubation of LMB cells for 12h with different concentrations of LmIFNd-His protein^-3TCID50/ml) activity of cells 12h after infection.

Figures 4-4 are the effect of LmIFNd-His recombinant protein on ISKNV proliferation.

Figure 5-1 is the in vitro interaction of LmIFNd with LmCRFBs protein. Pull-down experiments;

a: LmIFNd-His recombinant protein is combined with GST on magnetic beads and LmCRFBS-GST protein, after denaturation and elution, a protein complex is dissociated out, and Western Blot is carried out by using a His antibody to verify whether LmIFNd-His protein exists in an elution mixture;

b: anti-GST antibody validation of GST or CRFBs-GST protein bound to magnetic beads;

lane 1: CRFB 1; lane 2: CRFB 5; lane 3: CRFB 2.

FIG. 5-2 shows the expression level of ISGs gene when IFNd expression plasmid is co-transfected into FHM cells with different receptor combinations of CRFB1, CRFB2 and CRFB 5. The IFNd expression plasmid was cotransfected with pcDNA3.1 empty vector as a control.

FIG. 6 shows that the IFNd expression plasmid, CRFB1, CRFB2 and CRFB5 are co-transfected into FHM cell for ISKNV challenge and the activity of FHM cell is detected. a. b, c represent differential significance (p < 0.05).

Detailed Description

The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.

The cDNA of lateolabrax japonicus type I interferon receptor genes CRFB1, CRFB2 and CRFB5 can be prepared by the following method:

according to the EST sequence of the cytokine family B (CRFBs) in the constructed Lateolabrax japonicus cDNA library, after the EST sequence of the target gene is verified, PCR primers are designed, and the full coding sequences of the CRFB1, CRFB2 and CRFB5 genes of Lateolabrax japonicus are obtained by cloning by combining the RACE technology. The total length of the lateolabrax japonicus CRFB1 gene cDNA is 2730bp, the total length of the lateolabrax japonicus CRFB1 gene cDNA comprises 172bp 5 'UTR (untranslated region), 871bp 3' UTR and 1587bp open reading frame, 529 amino acids are coded, and the molecular weight of the coded protein of the lateolabrax japonicus CRFB1 is predicted to be 58.14 KD. The length of ORF of the weever CRFB2 gene is 1572bp, 524 amino acids are coded, and the molecular weight of the encoded protein of the weever CRFB2 is predicted to be 56.86 KD. The total length of the lateolabrax japonicus CRFB5 gene cDNA is 1542bp, comprises 99bp 5 'UTR (untranslated region), 171bp 3' UTR and 1272bp open reading frame, encodes 424 amino acids, and predicts the molecular weight of the protein encoded by the lateolabrax japonicus CRFB1 to be 47.21 KD. The lateolabrax japonicus type I interferon receptors CRFB1, CRFB2 and CRFB5 can be induced to express in immune organs after being stimulated by viruses.

The cDNA and amino acid sequence of LmCRFB1 are as follows. The start and stop codons are boxed, the predicted signal peptide is in bold, the IFN binding region is in single-dashed lines, and the transmembrane region is in double-dashed lines.

The cDNA and amino acid sequence of LmCRFB2 are as follows. The start and stop codons are boxed, the predicted signal peptide is in bold, the IFN binding region is in single-dashed lines, and the transmembrane region is in double-dashed lines.

The cDNA and amino acid sequence of LmCRFB5 are as follows. The start and stop codons are boxed, the predicted signal peptide is in bold, the IFN binding region is in single-dashed lines, and the transmembrane region is in double-dashed lines.

1. Extraction of tissue total RNA and preparation of full-length cDNA template

1.1 extraction of Total RNA

Healthy and fresh and alive Lateolabrax japonicus with the body mass of 20 +/-5 g and the body length of 15 +/-5 cm is temporarily cultured in a 500L plastic bucket for one week (7d) in a Zhuhai base of the Nanhai aquatic research institute of the Chinese aquatic science institute. 2/3 of culture water was changed every day. Selecting 5 healthy lateolabrax japonicus, anesthetizing for 2min by using 50mg/ml MS222, respectively taking blood cells, spleen, head and kidney, liver, gill, heart, muscle, intestine, skin, stomach and other tissues, immediately putting the tissues into liquid nitrogen for temporary storage, and then extracting the total RNA of each tissue by using Trizol reagent according to the instruction. And (3) detecting the concentration, purity and integrity of the extracted total RNA, and freezing and storing the total RNA in a refrigerator at the temperature of-80 ℃ for later use after the total RNA is detected to be qualified.

1.2cDNA template preparation

Using SMART 5 'RACE and 3' RACE kits (TaKaRa, Japan), 1. mu.g of total RNA was taken according to the instruction requirements and reacted at 42 ℃ for 1h under the action of reverse transcriptase to synthesize the first strand of cDNA, and linkers were added to both ends of the template under the action of DNA polymerase and DNA ligase using the first strand as a template, finally obtaining a template for cloning full-length cDNA.

2. Cloning of cDNA complete sequence of lateolabrax japonicus type I interferon receptor genes CRFB1, CRFB2 and CRFB5

2.1 validation of core fragments

Splicing sequences of CRFB1, CRFB2 and CRFB5 are obtained in a weever transcriptome database according to gene annotation information, specific primers are designed according to the splicing sequences, the primer sequences are shown in the table below, and the amplification sizes are 1587bp, 1572bp and 1272bp respectively.

The synthesized cDNA is used as a template, and PCR amplification is carried out by using a specific primer, wherein the reaction system is as follows: mu.L of cDNA template, 1. mu.L of each of the 10nmol/L upstream and downstream primers, 2. mu.L of 10mmol/L dNTP, 2.5. mu.L of 10 XExtaq buffer, and 17. mu.L of ultrapure water, totaling to 25. mu.L. The PCR reaction conditions are as follows: denaturation at 94 deg.C for 3 min; high temperature denaturation at 94 ℃ for 30s, low temperature annealing at 58 ℃ for 30s, and extension at 72 ℃ for 1min for 30s, for 33 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃. And after the amplified PCR product is detected to be qualified by 1.2% agarose gel electrophoresis, purifying and recovering the PCR product of the target fragment from the gel. Then connecting the purified PCR product to a pMD18-T vector, then transforming the PCR product into an escherichia coli competent cell DH-5 alpha, selecting a positive clone single strain according to the blue-white spot screening, shaking for 4h in a cell culture box with the temperature of 37 ℃ and the rpm for amplification culture, upgrading plasmid DNA, and sequencing by using a universal primer M13.

2.2 obtaining full-length cDNA of CRFB1, CRFB2 and CRFB5

Specific primers were designed based on the sequence of the cDNA fragments obtained. PCR Amplification is performed on the 3 'end and the 5' end of the target gene using Rapid Amplification of CDNA Ends (RACE) technology. The 3 '/5' RACE primers were synthesized according to the sequence as follows:

adopting the synthesized full-length cDNA as a template, adopting a falling-down PCR and nested PCR method according to

RACE 5'/3' kit (TaKaRa, Japan) was used for 3' RACE PCR amplification. The first reaction system is 20 mu L, and the first amplification conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 72 ℃ (1 ℃) for 30s, 10 cycles, and annealing temperature reduction of 1 ℃ in each cycle; extending for 1.5h at 72 ℃; denaturation at 95 ℃ for 30s, annealing at 65 ℃ for 30s, and extension at 72 ℃ for 1.5min for 25 cycles; extending for 10min at 72 ℃; storing at 4 ℃. The second amplification system is 50 μ l, and the second amplification conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 1.5min for 30 cycles; extension at 72 ℃ of 10 miin; storing at 4 ℃. The obtained PCR products were separated by 1.2% agarose gel electrophoresis, the objective product was purified and recovered from the gel, and then cloned into pMD18-T vector, and M13 was subjected to sequencing reaction to obtain 3 'and 5' RACE fragments. The length of CRFB13 'UTR is 871bp, and the length of 5' UTR is 172bp, and the full-length cDNA obtained after splicing is 2730 bp. The length of CRFB 53 'UTR is 171bp, and the length of 5' UTR is 99bp, and the full-length cDNA obtained after splicing is 1542 bp.

2.3 bioinformatic analysis of Micropterus fasciatus CRFB1, CRFB2, and CRFB5

Homology analysis was carried out using Blast (http:// www.ncbi.nim.nih.gov /), and the results showed that the CRFB1 gene has high homology with CRFB1 genes such as mandarin fish, swordfish, largemouth bass, large yellow croaker and sebastes dorsalis, and revealed that the gene is the CRFB1 gene. The CRFB2 gene has high homology with CRFB2 of mandarin fish, grouper pompholyx, flag fish, yellowtail and turbot, etc., and the gene is revealed to be CRFB2 gene. The CRFB5 gene has high homology with CRFB5 genes of mandarin fish, epinephelus punctatus, epinephelus lanceolatus, largemouth bass, fusceva sylvestris, eelweiss and the like, and the CRFB5 gene is revealed. The first 19 amino acids (MNCLPLVVYLMLLLDAVHS) of the N-terminal of the LmCRFB1 protein are predicted to be signal peptides by SignalIP. The extracellular domain sequence comprises a 211 amino acid, 33 amino acid transmembrane region sequence, and 284 amino acid intracellular domain sequence. The functional domain predicts that the first 113-212 amino acids of the protein are interferon binding region, the 24-99 region amino acids and the 136-200 region amino acids are FN3 protein superfamily domain. The first 19 amino acids (MTALIWMLTWLPQVLPAVS) of the N-terminal of the LmCRFB2 protein are predicted to be signal peptides by SignalIP. The first 223 amino acids are the ectodomain sequence, the middle 23 amino acids are the transmembrane region sequence, and the endodomain sequence has 277 amino acids. Functional domains predict that the first 119-216 amino acids of the protein are the interferon binding domain, and the amino acids in the 23-214 region are the FN3 protein superfamily domain. The Signal predicts that the first 32 amino acids (MGSTWKPVPQKIFAMSAAFLACFLSACLQSNA) of the N end of the LmCRFB5 protein are Signal peptides. 1-249 amino acids are the ectodomain sequence, the middle 32 amino acids of the transmembrane region sequence, and the last 151 amino acids are the endodomain sequence. The functional domain predicts that 140-244 amino acids of the protein are interferon binding regions. Amino acids in the 37-127 and amino acids in the 142-232 regions are the superfamily domain of the FN3 protein.

3, detecting the distribution of CRFB1, CRFB2 and CRFB5 genes in different tissues of the lateolabrax japonicus by PCR

Separately, 1. mu.g of RNA from blood cells, spleen, head kidney, liver, gill, heart, muscle, intestine, skin, stomach, etc. of lateolabrax japonicus was mixed with 0.5. mu.l of reverse transcription Oligo- (dT) (50. mu. mol/L) and 0.5. mu.L of Random 6 mers (100. mu. mol/L), 2. mu.L of 5 XPrimeScript buffer and 0.5. mu.L of reverse transcriptase mixture PrimeScript RT Enzyme Mix I were added, and the mixture was supplemented with water to 10. mu.L. The reaction process is 15min at 37 ℃ and 5s at 85 ℃, and the diluted solution is used as a template after being diluted by 10 times.

Amplifying CRFB1 gene by using primers CRFB1-qF and CRFB1-qR in real-time fluorescent quantitative PCR; amplifying a CRFB2 gene by using primers CRFB2-qF and CRFB 2-qR; CRFB5 gene was amplified using primers CRFB5-qF and CRFB 5-qR. Amplification of reference Gene 18s RNAFor internal control, the above synthesized cDNA was used as a template. The reaction was 12.5. mu.L containing 6.25. mu.L of TB Green Premix Ex Taq II (TaKaRa, Dalian, China), 2. mu.L of cDNA template, 0.5. mu.L of 10. mu. mol/L forward and reverse primers, and 3.25. mu.L deionized water. The PCR reaction condition is pre-denaturation at 95 ℃ for 30 s; denaturation at 94 ℃ for 5s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for a total of 40 cycles. The dissolution curve analysis was 65-95 ℃ to ensure amplification of each single product. Through 2^-ΔΔCtThe method analyzes the relative expression levels of weever CRFB1, CRFB2 and CRFB 5. The results are shown in FIG. 1, and it can be seen that there is expression in all tissues tested. Wherein, CRFB1 has the highest expression in blood cells, and secondly, gill and intestine; CRFB2 was most highly expressed in the head kidney, followed by spleen and gill; CRFB5 was most highly expressed in the spleen, followed by the head kidney and gill.

Primer:

CRFB1-qF：5’-CAGCGCACTGGTTGTATGTG-3’；

CRFB1-qR：5’-TGTGTGGCTCAAAGTCCGTT-3’。

CRFB2-qF：5’-ACATTAACCTTCGGCAGGCA-3’；

CRFB2-qR：5’-ACATCTGTGCTTAGGTGCCC-3’。

CRFB5-qF：5’-GAGTGTCCAGTCACGCTACG-3’；

CRFB5-qR：5’-GATCTGCCACCACGGAGTAGCGGTATC-3’。

18s-F：5’-GGGTCCGAAGCGTTTACT-3’；

18s-R：5’-TCACCTCTAGCGGCACAA-3’。

PCR detection of CRFB1, CRFB2 and CRFB5mRNA expression analysis under virus infection

Injecting 200 μ L ISKNV (2.9 × 10) into abdominal cavity of healthy Lateolabrax japonicus with a body mass of 20 + -5 g²copies/. mu.L) virus, the control group was injected with an equal amount of PBS, and after injection, the head and kidney tissues were taken at time points of 0h, 6h, 12h, 24h, 48h, 72h, and 96h, respectively, and snap-frozen in liquid nitrogen. The total RNA extraction method and the reverse transcription method were as described above. Meanwhile, 18s RNA is selected as an internal reference gene, and the internal reference gene primer is shown in the specification. The quantitative system and the reaction procedure were as described above. The results are shown in FIG. 2. LmCRFB1 and LmCRFB5 were significantly up-regulated at 72h post viral infection,the expression level of LmCRFB2 and LmCRFB5 still varied significantly at 96h LmCRFB 1.

5. Lateolabrax japonicus IFNd and preparation of recombinant proteins of receptors CRFB1, CRFB2 and CRFB5 thereof

Using cDNA as a template, amplifying all open reading frames of IFNd and receptors CRFB1, CRFB2 and CRFB5 thereof by PCR, adding enzyme cutting sites in front of primers, wherein the PCR primers are respectively as follows:

rIFNd-F：5’-CCGGAATTCCTGAGCTGCAGATGGATGGATC-3’；

rIFNd-R：5’-CCCAAGCTTGTTGGTGGTGAGTAGAGATGAAACC-3’。

rCRFB1-F：5’-CGCGGATCCGTGCTCGGCACCCTTCCT-3’；

rCRFB1-R：5’-CCGCTCGAGAGTGAAGACACAATTCCAAGTGCTT-3’。

rCRFB2-F：5’-CGCGGATCCAAACTCCTTCAGCCTGTCAACC-3’；

rCRFB2-R：5’-CCGCTCGAGAGCGTCTGCCGTAAAGATGC-3’。

rCRFB5-F：5’-CGCGGATCCGTGGAAGCGGAGCTGCCT-3’；

rCRFB5-R：5’-CCGCTCGAGCCACGGAGTAGCACCTTCGG-3’。

the PCR reaction conditions were 94 ℃ for 3min, 94 ℃ for 30s, 60 ℃ for 30s, 72 ℃ for 50s, for 30 cycles. The LmIFNdPCR product is cloned into pET21a vector plasmid to form recombinant plasmid pET21a-LmIFNd (wherein Lm refers to lateolabrax), and the LmCRFB1, LmCRFB2 and LmCRFB5 PCR products are cloned into pGEX-4T vector plasmid to form recombinant plasmid. The recombinant plasmids pET21a-LmIFNd, pGEX-LmCRFB1, pGEX-LmCRFB2 and pGEX-LmCRFB5 were transformed into E.coli BL21(DE3) strain. Four E.coli BL21 cells were expanded in LB medium containing 100. mu.g/mL ampicillin. When the OD600 of the E.coli BL21 cell solution reached between 0.6 and 0.8, isopropyl-. beta. -D-thiogalactopyranoside (IPTG) was added to LB medium at a final concentration of 0.6mol/L, and induced for expression at 37 ℃ and 220rpm for 12 hours. The recombinant LmIFNd-His protein was purified by Ni-NTA affinity chromatography according to the His-binding Purification Kit instructions (Beijing kang was a century). The purified protein was confirmed by SDS-PAGE and Western blot to have a distinct band around 22kDa (FIGS. 3-1,3-2), using the modifiedThe BCA protein assay kit (TaKaRa) detects the concentration of LmIFNd-His recombinant protein. And then storing the purified LmIFNd-His recombinant protein in an ultra-low temperature refrigerator at-80 ℃ for later use. Using a Beyogold^TMGST-tag Purification Resin purified LmCRFB1-GST, LmCRFB2-GST and LmCRFB5-GST recombinant proteins, and a thicker band appeared at about 49KD, 49.9KD and 52KD, respectively. The sizes of the LmCRFB1-GST, LmCRFB2-GST and LmCRFB5-GST are close to the theoretical value of the recombinant protein (figure 3-3).

Immune function study of LmIFNd-His protein

5.1LmIFNd-His protein induces expression of ISGs

By injecting LmIFNd recombinant protein into the abdominal cavity of the lateolabrax japonicus and incubating LMB cells with the recombinant protein in vitro, the expression changes of ISGs after in vivo and in vitro induction are respectively analyzed. In vitro experiments: LmIFNd-His recombinant protein (2 mu g/g) is injected into experimental lateolabrax japonicus, and the head kidney and spleen of the lateolabrax japonicus are respectively taken at 0h, 12h, 24h, 48h, 72h and 96 h. Five fish were taken per time group to reduce individual variation. In vivo experiments: to 6-well plates, LmIFNd was added at a final concentration of 4. mu.g/ml for induction treatment, and to control groups, an equal volume of DPBS was added as a control, and 3 replicates of all experiments were set up and cultured at 28 ℃. And (4) sampling after incubation for 0h, 3h, 6h, 12h and 24h, extracting total RNA of tissues and cells, and detecting ISGs gene expression by RT-PCR. The results show that: both in vivo and in vitro, LmIFNd-His recombinant protein activated the expression of downstream ISGs genes (FIGS. 4-1, 4-2).

5.2 analysis of ISKNV-resistant immune protection effect of LmIFNd-His recombinant protein

Perch brain cells were plated in 96-well plates, and 100. mu.L of an 10-fold dilution gradient of LmIFNd protein (10) was added to each well^-1-10^-8) Each concentration was 3 replicates. Negative controls (equal amounts of PBS only, no IFNd protein added to each well) and a blank zero-adjustment control (equal amounts of media added without cells) were set. After incubation at 28 ℃ for 12h, 100. mu.l of ISKNV virus dilution (1X 10) was added to each well^-3TCID₅₀And/ml), continuously culturing at 28 ℃ for 12h, and detecting the cell activity by using an MTT reduction method. The results show that: the cell activity gradually increased along with the increase of the concentration of the recombinant protein LmIFNd-His, which indicates that the recombinant protein LmIFNd-His pairISKNV infected cells were protective (FIG. 5-1). To further determine the immunoprotection effect of LmIFNd recombinant protein on Lateolabrax japonicus, one group was intraperitoneally injected with LmIFNd recombinant protein (2. mu.g/g) after one week of temporary rearing of Lateolabrax japonicus, the other group was intraperitoneally injected with PBS, and after 6h, both groups were intraperitoneally injected with 100. mu.L of ISKNV (2.9X 102 copies/. mu.L) virus, 30 fish per group. Taking the head and kidney tissues of two groups of lateolabrax japonicus at 12h, 24h, 48h, 96h and 128h after the challenge respectively, and using the tissues to detect the copy number of ISKNV. The results show that: LmIFNd-His recombinant protein was found to significantly reduce the copy number of ISKNV virus in head and kidney tissues (FIGS. 4-3, 4-4).

Interaction and transcriptional Regulation assay of LmIFNd with receptors

6.1 interaction of LmIFNd with Acceptor

To verify whether LmIFNd could bind to the extracellular domains of LmCRFB1, LmCRFB2, and LmCRFB5 in vitro, the present experiment performed pull down experiments on LmIFNd-His protein with three receptors, LmCRFB1-GST, LmCRFB2-GST, and LmCRFB5-GST, respectively. 100 μ L of CRFB1-GST, LmCRFB2-GST and LmCRFB5-GST proteins were added to 100 μ L of equilibrated GST beads, respectively, and GST-tag proteins were used as controls. Slowly shaking the mixture on a side shaking table for 1-2h at 4 ℃. 13000g, 1min, centrifuge and discard the supernatant. PBS was washed three times. To a 2ml EP tube, 100. mu.L LmIFNd protein was added and resuspended by pipetting. The mixture was slowly shaken overnight at 4 ℃ on a side-shaking table. 13000g, 1min, discard the supernatant. PBS was washed three times. Protein loading buffer was added directly to the Beads, boiled and run on SDS-PAGE gels. The results showed that the target protein LmIFNd-His could be detected in the experimental groups of LmCRFB1-GST, LmCRFB2-GST, and LmCRFB5-GST fusion proteins, but no target protein band could be detected in the GST group (FIG. 5-1A), indicating that the target proteins LmIFNd-His and LmCRFB1-GST, LmCRFB2-GST, and CRFB5-GST could be directly bound in vitro in the three receptor ectodomains (FIG. 5-1).

6.2 use of LmIFNd preferential acceptors

There has been experimental evidence that there is little cross-species reaction between type I IFN ligands and receptors in fish of different orders, e.g., salmonidae fish type I interferon ligands do not function on carp cell lines. However, the major downstream signaling pathway of type I IFN receptors, the JAK-STAT pathway, is relatively conserved among different fish species, and therefore we used fish FHM cell lines to test receptor usage of type I IFNd in lateolabrax japonicus. We cloned the ORFs for IFNd, CRFB1, CRFB2, and CRFB5, respectively, onto the PCDNA3.1-myc-his (-) vector, and then co-transfected IFNd ligands with different receptor combinations, respectively, into FHM cells. After 48h, the cells were harvested and the change in the expression level of ISGs was detected. The results showed that both the combination of LmIFNd + CRFB1+ CRFB5 and the combination of LmIFNd + CRFB2+ CRFB5 induced the transcription of ISGs genes in FHM cells, and that the combination of LmIFNd + CRFB1+ CRFB5 was found to have a higher induction effect, indicating that LmIFNd can preferentially select the combination of CRFB1+ CRFB5 receptor complex for signaling (fig. 5-2). Therefore, the antiviral composition provided by the invention can be suitable for various fishes to enhance the antiviral capability.

Use of different combinations of LmIFNd and receptor in antiviral applications

Based on the above research results, we co-transfected IFNd ligand and different receptor combinations into FHM cells, and after transfection for 24h, aspirated cell culture solution was discarded, and ISKNV virus (2.6X 10) was added to the cells^-5TCID50/ml)), the cells were challenged, the virus was removed after 1h incubation, the cells were washed 2 times with DPBS, fresh cell culture medium was added, and the cell activity was measured 24h later by MTT reduction. The results showed that the combination of LmIFNd + CRFB1+ CRFB5 and the group of LmIFNd + CRFB2+ CRFB5 had significantly higher activity on cells than the other combinations (fig. 6).

Sequence listing

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

<120> antiviral composition containing lateolabrax japonicus interferon IFNd and receptor thereof and application

<160> 33

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2730

<212> DNA

<213> Lateorax maculotus)

<400> 1

acatgggggt tgaacacaac ttggtggaac gtctctaact gtgggcaact tcaccattat 60

ttcgcttttc cacctcggac gttgaaacga gccacaagtg gtgtgtcgac agcactgatg 120

cagtgtatgg aggctgagga agaccctcct gctgcctcat gagtctgaaa ggatgaactg 180

tctgcctctt gttgtgtatc tcatgttact gcttgatgcc gttcactcag tgctcggcac 240

ccttcctgtt ccggtcaatg tttcagtcca ctccgtcaac ttcaaccatg tcctgctttg 300

gaaccccggg catggcaccc caccaggaac acagtacatg atcaggagga actgcgggga 360

aaaaaagaaa tatctgaaaa agaccacgat gacatcattt aagctgaagc taaacaccta 420

caagaaatac tgcctgactg tccaggcgtc ctacaaccaa accctgtctc cagtgtccag 480

cagtgtcatt tttacccctt tcacagacac caaaataggt cctccaaaac tctcactggc 540

tggatgtggc aactgcatcc aaatcaacat tacactgcct aaggcaaata aaacctcaag 600

agactttgat gtccacaatt tctacaaacc tcaatataga gtcttttgga ggaaacctaa 660

agacgcagtg gagcactctg aaacagggaa cactagcttt actattgaga acttggagaa 720

ttggacggag tactgtgtac aggtggagac aaagattcgt gtcaacaaaa acactgtgcc 780

aagcacttgg aattgtgtct tcactagcat tgcaccgaac agagcccctg ttcttttagg 840

tacaattgct gctgtccaga tttttgtatt cggtggactg atgatcttcg tgtattgcct 900

ctacagcact ggattccttt gtaaactaaa ggccctaccc agacaactaa ttgcggacct 960

gagccaaggc cacaccctga caccagagaa gacgatctgt gaccgcatct ccattagctc 1020

acagacggag agacagagga cccgcaacaa tcccacaacc ccacaagctg ccaccagggg 1080

tgataacttg gaggaggatg aagaggatga tgaagaggag ggagaggaag tctacatgga 1140

cagaggtgca gagctctcct caggtgacag ttcctgccag gactgtagtg aggtgtcggg 1200

gaacagaact ctggctgctt caggggactc tgggtgtttg actgcggagg cagaggcact 1260

ggctgcagag tttgaggtcg cagatggggg gcttgataaa gacgaggcag aagcagcaga 1320

ggtttctttc atccctgagg ggggtcaaac tggagtccag ggacatgtca cggacgaaga 1380

ggaggacgcg ttggtgtgtg acagttcagg caatgtcgat ctgttctcag tcaccctcag 1440

cgcactggtt gtatgtgagg aagaggaaca aaatacaaga gactctctca cagacttttt 1500

gaaactgtcc gacacggagc ttctgctgcc cacaaactca aaacggactt tgagccacac 1560

agattcacaa actgagtcag gtgacgggac taccgtggca ttcatgttgc ctacacaaga 1620

agacttgact gcgactgtgt gtgaaggcag acgtgcagac actttgtcag gctgccgcaa 1680

aagctgtgat ggtgagacgc agcatgaaga gacacaagag gaggaggagg aattctctgg 1740

atatatgagc cacaaataat atgttggtct gatgtgtgtt actgtatgtg tacaaattcc 1800

aatggggtac attacctttt tgttaaagga aaaatccagc gaaaacattt aaatgtagtt 1860

aaggaggttg attttgttgt tttgtttgtg tatttttctt taggacacaa cacattgata 1920

cattgcccct gcagctacaa tgccattata gccacctgca ttttgacagt ttttgaaggc 1980

agcagagaat tgtgtttgag tgaccaaata tgccagtaca ttggcagaac atttaagtaa 2040

agataaatgt tttatctgca tcaacatgct tttggtaaac atttcatgac agtctgaaag 2100

atggaataaa atgtgtttgg tttgttatgg gaaaggaaat caagtgtcag caaacaccat 2160

tttgtaaatg tgtaagtctt gactggaggt tcttcttgag ctcacggctc actgttttgg 2220

ctgacagcat gctatatttg catgtccatt tacctaatgt atagtcaaac cagcaagatc 2280

atgccagcat catgtaaaga gtgttcatgt aacatccaag tcataatgtt atgactgtga 2340

atctattctg agagctcgga ttgggaagtg cctgaaaagc caaacaaata aaaataactc 2400

acgttagcca aaagttcagt gtaattatac ccaaatttaa tggatataac gttatacagt 2460

tgtttttcac agtattaatt ttgacaggtg ttattaataa cattaaaggc tcttttccat 2520

ccaagtgtcc cagtaagcca tgccagtgtg acagttagac agcataaaca ataccaggac 2580

cctgaaactg aagcagctaa gtggaattca gccatcatta attttattgt ttacatctgt 2640

gctttgttct gttaaaaaag cctatgttaa atccctcatt aaactcatgt ctgcaattta 2700

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2730

<210> 2

<211> 1572

<212> DNA

<213> Lateorax maculotus)

<400> 2

atgactgctc tcatttggat gctcacatgg ctgcctcagg tcctgccagc tgtaagtaaa 60

ctccttcagc ctgtcaacct gaacttgacc tcaaaccatt taagacatat gttgaaatgg 120

gagcctggac caggtacacc cacaggagtc tgctaccgtg ttgacgtaaa gacagaaacg 180

ggatctacct gggtgcaagt ggctggctgc gagtgtgtcc agcacacact ggtttgcaac 240

ttgacagagg ccttctccga cccaaaacag gtgtacttta cccaggttac agcactgctg 300

ggtgaacagg cctcgcaatc agtcttcccc ttaggatttc aacccatcag agacactcac 360

ctggacctgc ctctgctgac tgtgacgccc tgtggcatgg atctgtgtgt ggaccttaag 420

cctcccatgg ggcaccttag ggagatctat ggttctctgc attacaaact caggatcacg 480

agcaacagtt ccaacagagc ccagttcttt aaggtaacca agtttctgag aagagaggtt 540

ttggaggatc tggcccctgg cagacagtac tgtgtctcag tctgcttctt agacagtcag 600

gtaccaaagg agtccaacta cagcaaaccc gtatgtgcat tcaccccggg catctttacg 660

gcagacgctt tgatctcagc catgctgtgt ttgctggtga tgtctggtgt ggtcgctgta 720

gccctgctgg tctctgctgg tttcatctgc cttaaaaaga ggctacagcc atcggtcctg 780

acatccatcc atcatgtaga agaggttatg gtggttccca gccccacgtt cttgtcgtct 840

ctggtgagtc ttaaaccaac actgccctct tcaggtgaag agaggagcaa ccaaacatca 900

tcagatgaaa gtgatgagga gagtgtgaca gagagcactg atgggagcgg aggaggaggt 960

tatgaactga aggggggccc aaaccctctt tcttccttct ctacatcatc tttgtcccct 1020

cttttatctc ctgaacctga accctcccca agcttctcct caaaccaaac atcagatgtg 1080

ttcaaccccc agcctgaggc ccaaacacag acacattcca gttctggatg gatcggcgca 1140

ctgagctcac acaccgcctc acactctgac tctgtgccac agtcacccca gcgtgcctca 1200

gatccctgtc ttcctgcgga cacgcaaagt ttgactggag gaagaaccac gccaaatcag 1260

gaggagaagg aggcggcggg acaggggaac agcctggatg tgaacctcct cacattaacc 1320

ttcggcaggc aggaagagga gaacgaggag gatgaagaat cgcatcttga catgacagag 1380

gtggagctgg agccttcctc tgcctcagag gtgtacaacg ccactcaaat cctgctctca 1440

caaactcgga acacgaagga agttgccata gaaggggttt cctactctgc tgaggaagag 1500

gaggaagagc aagaagaata ctctggatat atggggcacc taagcacaga tgtcctaaaa 1560

aacttattat aa 1572

<210> 3

<211> 1542

<212> DNA

<213> Lateorax maculotus)

<400> 3

acatggggac atcggttcag atcggccata gttgagacct tagactgtgt tcagagtgga 60

gagtcgagct gagttcgacc gagccgagcc tgggaggaga tgggaagcac atggaaacca 120

gtacctcaaa aaatattcgc catgtccgct gctttccttg cctgctttct ctctgcgtgc 180

ctccaaagca acgctgtgga agcggagctg cctgctccac agcacgtggc catgaagacc 240

atgaacacca attacaaagt gagctgggag tgggaccata gtgctgcaga ggaccacact 300

gtcaccttca ccacacaata tgttgcgaag tacaagctga atatcaagaa gaagagtcca 360

gaatggtgca caacatgtga caagacgtcg cacaggtcat gtgaccttac agcgttcaag 420

ctgcactacc tgggcatcta catgattcgt gtacgagcca acgcgaacgg gagtcactct 480

gcctgggtgc agaaggagtt ctgccctgat aaagatgcta atgtgggtcc tccagctcaa 540

gtgcatcttt ctcctgctga aagtaacctg gacgtttaca tcgttgaccc tctgaccagc 600

accaacagct ccatgaggga gcatcttccc gatctgtact accacatcct ctactgggaa 660

cgctctctag acacacaggt ctcaagaccc cagactttga acagaagtgc caacatggtg 720

actttgtcaa acatgaaggc ctggacttgg tactgtgtga gtgtccagtc acgctacgac 780

tactacaaca agagcagcag cttcactgca ccccagtgta tgcagaccga aggtgctact 840

ccgtggtggc agatctttct gtgcttcttg ctgtctctgg cgatctgctt tgtggtcgtg 900

gtgttcctac tctatggatc tttttggtgc tacaagtccg tcaaggcagt gctgtacccc 960

tccatccagc ttcctccaca cctgaaggag tatttctgcg actctcctgg atctgacatt 1020

cctcgcctcc tcatttcgga ttcagaatca gagctcttgt gtgataaggt gaccgtctgt 1080

cccgagccac cagtcctgga aatccacaac cctcccccag aggccctgga gccagacagc 1140

agtggcagac acagtcgtca ggacagcagg ggcagcggag actcaggggt ttactccaca 1200

gggggcagct ccagcctgcg gcagcccagc agcagtcagt cctctgcggg gggcgaagcc 1260

tcctggcagg gcccctttga ctctgagcag gtgaagttgg tggacattgc cccggggccc 1320

aagagtcaac ttatgatcgc agacgagggc attgtagaca tgtgtgtctg aaagcacgca 1380

aaaagacatt tgtggagaag aatgggaagt gaggatgacc gaaagacgga ggtttgtaga 1440

aaatgtgcag cgtgcctttt aaccagctgt cactgtgaga cgaggacaga aagtgtcagg 1500

tctgttatca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 1542

<210> 4

<211> 25

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgctcaaca ggatcttctt tgtct 25

<210> 5

<211> 26

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tgacagattt tagttggtgg tgagta 26

<210> 6

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgaactgtc tgcctcttgt tgtg 24

<210> 7

<211> 27

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ttatttgtgg ctcatatatc cagagaa 27

<210> 8

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgactgctc tcatttggat gct 23

<210> 9

<211> 30

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ttataataag ttttttagga catctgtgct 30

<210> 10

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgggaagca catggaaacc a 21

<210> 11

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tcagacacac atgtctacaa tgcc 24

<210> 12

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

caccagagaa gacgatctgt gacc 24

<210> 13

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cagaggaccc gcaacaatcc 20

<210> 14

<211> 22

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ggactgtagt gaggtgtcgg gg 22

<210> 15

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

caggaactgt cacctgagga gag 23

<210> 16

<211> 27

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cagaatcaga gctcttgtgt gataagg 27

<210> 17

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

agccagacag cagtggcaga c 21

<210> 18

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ctcgtctgcg atcataagtt gact 24

<210> 19

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cagagtcaaa ggggccctgc c 21

<210> 20

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cagcgcactg gttgtatgtg 20

<210> 21

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tgtgtggctc aaagtccgtt 20

<210> 22

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

acattaacct tcggcaggca 20

<210> 23

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

acatctgtgc ttaggtgccc 20

<210> 26

<211> 18

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

gggtccgaag cgtttact 18

<210> 27

<211> 18

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

tcacctctag cggcacaa 18

<210> 28

<211> 31

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ccggaattcc tgagctgcag atggatggat c 31

<210> 29

<211> 34

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

cccaagcttg ttggtggtga gtagagatga aacc 34

<210> 28

<211> 27

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

cgcggatccg tgctcggcac ccttcct 27

<210> 29

<211> 34

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ccgctcgaga gtgaagacac aattccaagt gctt 34

<210> 30

<211> 31

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

cgcggatcca aactccttca gcctgtcaac c 31

<210> 31

<211> 29

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ccgctcgaga gcgtctgccg taaagatgc 29

<210> 32

<211> 27

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

cgcggatccg tggaagcgga gctgcct 27

<210> 33

<211> 29

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

ccgctcgagc cacggagtag caccttcgg 29

Claims (3)

1. An antiviral composition containing weever interferon IFNd and a receptor thereof is characterized by comprising the weever interferon IFNd and a weever type I interferon receptor, wherein the weever type I interferon receptor is CRFB1 and CRFB 5.

2. The use of an antiviral composition comprising lateolabrax japonicus interferon IFNd and its receptor according to claim 1 in the preparation of an aquatic animal antiviral medicament, said virus being an infectious spleen and kidney necrosis virus, a nervous necrosis virus or a frog virus.

3. The use of the antiviral composition comprising lateolabrax japonicus interferon IFNd and its receptor according to claim 1 in the preparation of a medicament for enhancing the antiviral immunity of aquatic animals, wherein the virus is infectious spleen and kidney necrosis virus, nervous necrosis virus or frog virus.

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