Two cynoglossus semilaevis anti-vibrio disease genes and application method

Abstract

The present application relates to the technical field of genetic engineering, in particular to two Cynoglossus semilaevis anti-vibrio disease genes and application methods. The present application mainly studies the role of CD79a and CD79b in Cynoglossus semilaevis in resisting vibrio disease, and verifies from two directions of in vivo and in vitro, further promotes the progress of Cynoglossus semilaevis disease resistance breeding, and shows that CD79a and CD79b have the role of inhibiting Vibrio harveyi.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to two genes for resistance to vibrio in the half-smooth tongue sole and their application methods. Background Technology

[0002] Like mammals, fish possess both innate and adaptive immune systems. Fish were the first species to develop an adaptive immune system, making them highly valuable for research. The spleen and prokidneys are the main immune organs in fish, followed by the mucosal immune system, including intergillary lymphoid tissue, first discovered in salmon. [1] Gut-associated lymphoid tissue and skin-associated lymphoid tissue also play important immune roles. [2-3] Sex hormones also affect the immune response of fish. [4] In bony fish, there are three types of immunoglobulins: IgM, IgT, and IgD, with IgM being a key effector molecule in fish immunity. [5] IgT is considered to be unique to bony fish. [6] lgD exhibits genetic diversity across different species. [8] Similar to mammals, the diversity of immunoglobulins in bony fish is also formed through gene recombination in heavy and light chain regions, as well as somatic cell mutations. [9] .

[0003] CD molecules are primarily expressed on the surface of immune cells, specific endothelial and epithelial cells.

[10] Because CD molecules are expressed relatively specifically, they are usually regarded as cell surface markers. By recognizing CD molecules, white blood cell types and cell differentiation and development stages can be distinguished. Currently, a total of 371 members of the CD family have been discovered and named (CD1-CD371).

[11] These molecularly specialized functions are expressed only on specific cells.

[12] Monoclonal antibodies using CD molecules have been studied and proven to treat a variety of malignant tumors and autoimmune diseases.

[11] For example, studies in mammals have shown that antibody-drug conjugates targeting CD79b can treat relapsed and refractory diffuse large B-cell lymphoma.

[12] Targeting CD79 monoclonal antibodies to induce a "numeric" state in B cells, thus treating autoimmune diseases.

[13] Among fish, studies have found that large yellow croaker...

[14] and Nile tilapia

[15] CD59 in fish exhibits antibacterial activity; CD22 in fish can inhibit the activation of peripheral blood leukocytes.

[16] .

[0004] CD79 is a dimer formed by CD79a and CD79b linked by disulfide bonds. It is located on the B cell membrane and forms the B cell antigen receptor (BCR) with membrane immunoglobulins (mlg). In mammals, CD79 has been shown to participate in the intracellular transduction of BCR signaling. [17-18] When an extracellular antigen binds to a receptor, intracellular CD79b may be activated first, followed by activation of the ITAM domain of CD79a.

[19] This further phosphorylates various downstream adaptor proteins, activating downstream signaling pathways such as PI3K-AKT and NF-κB. These signaling pathways can interact with Toll-like receptors, CD40, and other signaling pathways.

[20] It is closely related to cell proliferation, differentiation, development, individual aging, and immunity. [20-21] Among bony fish, CD79 is found in the red-lipped barb.

[22] Nile tilapia

[23] , head sea bream

[24] While CD79 has been reported in studies, research on its function in the BCR signaling pathway in bony fish remains limited. The tongue sole is an important economically important fish species, and infection with Vibrio harveyi can result in a mortality rate as high as 80%. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides the application of CD79a and CD79b as molecular markers in the screening of disease-resistant varieties, and the application of reagents for overexpressing CD79a and CD79b genes in the preparation of drugs for treating vibrio infection in half-smooth tongue sole. This invention primarily studies the role of CD79a and CD79b in resistance to vibrio infection in half-smooth tongue sole, and verifies this from both in vivo and in vitro perspectives, further advancing the process of disease-resistant breeding of half-smooth tongue sole.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of two molecular markers (CD79a and CD79b) for resistance to vibrio in half-smooth tongue sole in breeding.

[0008] This invention also provides the application of reagents that overexpress the CD79a gene in the preparation of drugs for treating vibrio semismooth tongue infection.

[0009] This invention also provides the application of reagents that overexpress the CD79b gene in the preparation of drugs for treating vibrio semismooth tongue infection.

[0010] Preferably, the vibriosis is caused by Vibrio harveyi.

[0011] The beneficial effects of this invention are:

[0012] This invention mainly studies the role of CD79a and CD79b in the resistance to vibrio disease in half-smooth tongue sole. The results were verified both in vivo and in vitro, further advancing the process of disease-resistant breeding of half-smooth tongue sole. The results show that CD79a and CD79b have the effect of inhibiting Vibrio harveyi. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0014] Figure 1-1 The amino acid sequence analysis diagram of Cs-CD79a;

[0015] Figure 1-2 The amino acid sequence analysis diagram of Cs-CD79b is shown.

[0016] Figure 2 Phylogenetic analysis of Cs-CD79a, Cs-CD79b, Cs-CD22, and Cs-CD59;

[0017] Figure 3 The expression levels of Cs-CD79a and Cs-CD79b mRNA in various tissues of healthy tongue sole: Liver, Skin, Blood, Spleen, Gill, Foregut, Midgut, Hindgut, Kidney, and Muscle.

[0018] Figure 4 The expression levels of Cs-CD79a and Cs-CD79b mRNA in Vibrio harveyi-infected half-smooth tongue sole tissues; Skin (skin), Spleen (spleen), Kidney (kidney), Intestines (intestine);

[0019] Figure 5 Expression of Cs-CD79a and Cs-CD79b in susceptible and resistant pedigrees of *S. semismooth tongue sole*; susceptible pedigree (S), resistant pedigree (R), Spleen (spleen), Kidney (kidney); AB: 2017 pedigree; CD: 2018 pedigree; EF: 2019 pedigree;

[0020] Figure 6 Histological localization of CD79a and CD79b in *Cocculus semismoothienosus*. AC: gills, kidneys, and spleen, CD79a antisense probe; DF: gills, kidneys, and spleen, CD79a positive sense probe; GI: gills, kidneys, and spleen, CD79b antisense probe; JL: gills, kidneys, and spleen, CD79b positive sense probe.

[0021] Figure 7 CD79a-overexpressing cells were stimulated with different concentrations of bacteria for 3 hours.

[0022] Figure 8 CD79b-overexpressing cells were stimulated with different concentrations of bacteria for 3 hours.

[0023] Figure 9 CD79a-overexpressing cells were stimulated with different concentrations of bacteria for 6 hours;

[0024] Figure 10 CD79b-overexpressing cells were stimulated with different concentrations of bacteria for 6 hours.

[0025] Figure 11 CD79a-overexpressing cells were stimulated with different concentrations of bacteria for 9 hours.

[0026] Figure 12 CD79b-overexpressing cells were stimulated with different concentrations of bacteria for 9 hours.

[0027] Figure 13 CD79a-overexpressing cells were stimulated with different concentrations of bacteria for 12 hours.

[0028] Figure 14 CD79b-overexpressing cells were stimulated with different concentrations of bacteria for 12 hours. Detailed Implementation

[0029] This invention provides a method for detecting two genes, CD79a and CD79b, in the fish *Cyprinus semismooth tongue sole*, which are responsible for vibrio infection. In this invention, the vibrio infection is caused by *Vibrio harveyi*.

[0030] This invention also provides the use of reagents overexpressing the CD79a gene in the preparation of drugs for treating vibrio harveyi infection. In this invention, the vibrio infection is preferably caused by Vibrio harveyi.

[0031] This invention also provides the use of reagents overexpressing the CD79b gene in the preparation of drugs for treating vibrio harveyi infection. In this invention, the vibrio infection is preferably caused by Vibrio harveyi.

[0032] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] 1. Materials and Methods

[0035] 1.1 Experimental Fish

[0036] Clinically healthy tongue sole were purchased from Huanghai Aquatic Products Co., Ltd. in Shandong Province, China, and kept at 20°C in aerated seawater. Prior to the experiment, the fish were acclimatized in the laboratory for two weeks, confirming the absence of bacterial pathogens.

[0037] 1.2 Bacterial strain culture and bacterial infection

[0038] The Gram-negative bacterium *Vibrio harveyi* used in this invention was obtained from the Yellow Sea Fisheries Research Institute. Competent cells were purchased from TransGen Biotech Ltd. The bacteria were cultured on tryptic soy peptone broth (TSB) in a shaker at 28°C and 180 rpm / min. Five experimental fish were intraperitoneally injected with 500 μL of 1.0 × 10⁻⁶ bacteria. 7 Five fish were anesthetized at 0, 6, 12, 24, 48, 72 h and 96 hpi after injection, and their kidneys, spleen, liver, intestines, gills and skin were collected.

[0039] 1.3 Sequence Analysis

[0040] CsCD79a (GenBank accession number XM_008323971.3) and CsCD79b (GenBank accession number XM_008314006.3) amino acid sequences were analyzed using the BLAST program from the National Center for Biotechnology Information (NCBI). Domain searches were performed using NCBI's conserved domain search program. The theoretical molecular mass and theoretical isoelectric point were predicted using EditSeq in the DNASTAR (Madison, WI) software package. Multiple sequence alignment was performed using Smart.

[0041] 1.4 Real-time quantitative PCR

[0042] Real-time quantitative PCR (qRT-PCR) was performed using an FQD-96C instrument (BIOER) to analyze the expression of CD79a and CD79b gene transcripts in *Coccus semilaevis* under normal physiological conditions. Tissues (blood, intestine, kidney, foregut, midgut, hindgut, gills, spleen, muscle, and liver) were extracted from five healthy *Coccus semilaevis*. The expression of the CD79 gene in immune tissues of *Coccus semilaevis* infected with *Vibrio harveyi* was analyzed, with tissues (kidney, spleen, liver, intestine, gills, and skin) extracted from five infected fish. The expression levels of CD79 transcripts in immune tissues of *Coccus semilaevis* from resistant and susceptible families from different years were analyzed. All tissues were analyzed using the TIANGEN Animal Tissue Total RNA Extraction Kit and the TaKaRa Reverse Transcription Kit. All qRT-PCR experiments were performed using ArtiCan. ATMThe SYBR qPCR Mix kit (TsingKe) was used, with a total volume of 20 μL. The reaction mixture was incubated at 95 °C for 2 min, then cycled at 95 °C for 10 s, at 60 °C for 20 s, and at 72 °C for 30 s.

[0043] 1.5 Probe Synthesis and In Situ Hybridization

[0044] To detect the localization of CD79a and CD79b in the gills, spleen, and kidneys of tongue sole, dig-labeled primers and ribosomal probes were synthesized at Qingdao Ruiboxingke Biotechnology Co., Ltd. Transcription was performed for 3 hours, followed by purification using LiCl. Hybridization was carried out at 65℃, followed by staining at 30℃ for 6 hours using a 25xNBT / 25xBCIP kit (Solarblo), and mounting with glycerol-gelatin. Three biological replicates of each gene were performed in each tissue. Scanning was performed using a 3DHIETCH Pannormic MIDI digital slide scanning workstation, and images were taken with SlideViewer.

[0045] 1.6 Inactivation of Vibrio harveyi

[0046] Vibrio harveyi was inoculated into LB liquid medium; cultured at 28°C and 180 rpm / min; centrifuged at 4°C and 6000 g / min for 15 minutes, the supernatant was discarded, and the culture was washed three times with PBS; resuspended in PBS containing 0.5% formaldehyde and incubated at 4°C for 48 hours; plate culture was performed to detect the presence of viable bacteria; the culture was washed three times with PBS; the OD value was measured using a UV spectrophotometer, and the OD value was adjusted accordingly. 600 =1.

[0047] 1.7 293 Cell Transfection and Bacterial Infection Experiment

[0048] The CsCD79a gene sequence was ligated into a GFP vector, and the CsCD79b gene sequence was ligated into an RFP vector to construct luminescent plasmids. CsCD79a and CsCD79b were transfected into 293 cells for overexpression. The luminescence was observed under a microscope 48 hours after transfection into 24-well plates (500 μL of medium per well).

[0049] Five gradients of Vibrio harveyi culture were set up for cell infection, with each well containing 50 μL / 500 μL, 75 μL / 500 μL, 100 μL / 500 μL, and 125 μL / 500 μL of bacterial culture. Three replicates were set up for each gradient, and cell status was observed at 3 h, 6 h, 9 h, and 12 h of stimulation.

[0050] 2. Results

[0051] 2.1 Sequence characteristics of CsCD79a and CsCD79b

[0052] CsCD79a consists of 224 amino acid residues, with a calculated molecular weight of 25.0 kDa and a theoretical isoelectric point (pI) of 5.649; CsCD79b also consists of 224 amino acids, with a calculated molecular weight of 25.31 kDa and a theoretical isoelectric point (pI) of 6.297. Similar to other CD79s, CsCD79s possess an extracellular hydrophobic signal peptide (residue 1e21) and an lg-like domain, a transmembrane domain (residue 22e91), and an intracellular ITAM domain (residue 92e107). The basic structure of the ITAM domain in bony fishes is: D / E(x)7D / ExxYxxLx(6-8)xxxI / L( Figure 1-1 and Figure 1-2 Phylogenetic analysis showed that CsCD79s is closely related to bony fish such as flounder and Nile tilapia; CsCD59 is closely homologous to bluethorn, zebrafish, and spotted fish; and CsCD22 is closely related to zebrafish and golden-eyed bass. Figure 2 It has been concluded that the two may form dimers extracellularly via non-covalent bonds, and that the transmembrane dimer is stabilized by hydrogen and ionic bonds between glutamate and glutamine.

[25] .

[0053] CD79a nucleotide sequence (SEQ ID No. 1):

[0054] Atgggagctgtacacagtgagcttacttggaggcctgacaacccctacatgagttttgccctttttgaacctgctgagctgatgtgctgcttcaccgtcacatctgggaacacaggttccgtccactgggtcagaagtttcaacggcgctggcacaatatctgtgaacctgaaggatggcgtgacctcgaacacccaaagttaccgaggcgaaaactgtagtgtcctgatctttaagtcagtcaagctgaacgacagtggactgtaccagtgcttgcttaacaccaaaggccacttctctcatggcacctacatgcaagtcttccagcccctgaagaagaggataaatctcaaggaaaccaccaagaacagcatcctcaccgcggagggaatcctgctgctgctctgtgtactcttgccttcagccacccttttatgcaagtcaaagaaactggttcaactagaaaagaagaaggcgggaaaagaagaggagaacatttatcaggggctgaatctggatgagtgttctgccacatatgatgagattgtacgcacacaggggcagtacctgtacgaggatgtgggtaacatgaaagaagaactggaagaggaggaaatccagctggagaaaccctga。

[0055] CD79a amino acid sequence (SEQ ID No.2):

[0056] MGAVHSELTWRPDNPYMSFALFEPAELMCCFTVTSGNTGSVHWVRSFNGAGTISVNLKDGVTSNTQSYRGENCSVLIFKSVKLNDSGLYQCLLNTKGHFSHGTYMQVFQPLKKRINLKETTKNSILTAEGILLLLCVLLPSATLLCKSKKLVQLEKKKAGKEEENIYQGLNLDECSATYDEIVRTQGQYLYEDVGNMKEELEEEEIQLEKP。

[0057] CD79b nucleotide sequence (SEQ ID No.3):

[0058] atgactgcagtgactgtgagacacacgtttttattgccttttaaaattatttctttcttgcatgttatcatgcgctggttactggttggatgcttcattttgggtctgaaccacatctcagcacataggattactcagaagccccggttcattggagtcaaaatgggctcctccgtgttgattcaatgttccatgactgaacgtgtggactcactgaacatgcagtggtacagagctgacgcgtacgaccagaagaaagtagtgctacagctggaaagggacgtgtctggtaacaggagtttgaccaaagacaaccttctcaccttacacagagttcaagtagaggacagtggagtgtacttctgccaagctggcaacacaatggggcctggaactcaactgatagtcgtcaaggccacagacttgactgcagcccagcacaggaccaccatcaaggacgggctcattattgtccaggctctgttgttggccttgtttatcgctgctctgctgatgcagaaacgaaacctgtctgaaaaggaggatagcgaatatgagatgcctgaaaatgaccacatctatcagggcttggcaattgagacgtgtaatggaggaatgtatgaggagctgacagtttatgctcagccagatggatctgaggccccgtgggaatga。

[0059] CD79b amino acid sequence (SEQ ID No.4):

[0060] MTAVTVRHTFLLPFKIISFLHVIMRWLLVGCFILGLNHISAHRITQKPRFIGVKMGSSVLIQCSMTERVDSLNMQWYRADAYDQKKVVLQLERDVSGNRSLTKDNLLTLHRVQVEDSGVYFCQAGNTMGPGTQLIVVKATDLTAAQHRTTIKDGLIIVQALLLALFIAALLMQKRNLSEKEDSEYEMPENDHIYQGLAIETCNGGMYEELTVYAQPDGSEAPWE。

[0061] 2.2 Expression patterns of CsCD79 in various tissues of tongue sole

[0062] qRT-PCR analysis of the transcriptional expression of CsCD79s in the immune tissues of tongue sole under normal physiological conditions revealed that CsCD79 expression was highest in the spleen and kidneys, followed by the gills, while low expression was observed in other tissues, and even absent in the intestines. This expression was directly related to the blood content of the tissues. Figure 3 To determine the effect of pathogen infection on CsCD79s expression, the expression of CsCD79s in immune tissues at 0h, 6h, 12h, 24h, 48h, 72h, and 96h after Vibrio harveyi infection was detected by qPCR. Figure 4 The results showed that the highest expression levels were observed in the skin, spleen, and gills at 24 hours after infection, while the high expression in the gills at 6 hours was likely unrelated to bacterial stimulation. CD79b showed high expression in the kidneys, but almost no expression in the intestines and liver, thus ruling out the possibility of irritative expression. To advance disease-resistant breeding, Academician Chen Songlin's team established multi-generational selected disease-resistant and susceptible families of *C. semi-smooth tongue sole*. Immunohistochemical qPCR results from 2017-2019 showed that the expression levels of CsCD79a and CsCD79b in the disease-resistant families were significantly higher than those in the susceptible families. Figure 5 ).

[0063] 2.3 Location of CsCD79s in the slice

[0064] In situ hybridization results of paraffin sections from healthy tongue sole tissues showed that CD79a and CD79b were distributed in similar locations in these three tissues. Figure 6 In the kidneys, it is mainly distributed in various renal tubules, abundant in dense macula, and extensively distributed in the renal cortical blood vessels; in the gills, it is distributed in the capillaries of the gill filaments and gill arches; in the spleen, it is abundant in the arteries and surrounding capillaries.

[0065] 2.4 Cell transfection and antibacterial effect

[0066] After bacterial stimulation, CD79a and CD79b proteins showed a certain inhibitory effect on Vibrio harveyi. Three hours after stimulation with inactivated Vibrio harveyi, a small number of cells in the experimental group died, and a few cells floated. The cell morphology did not show significant differences compared to the control group. Figure 7 and Figure 8 After 6 hours of stimulation, cell mortality increased, while the morphology of living cells remained unchanged. In contrast, the control group cells showed obvious cracks, a greater number of deaths, and altered cell morphology. Figure 9 and Figure 10 After 9 hours of stimulation, the cell death area became more pronounced, and cells appeared as clumps floating; the control group showed a significant decrease in the number of adherent cells, larger intercellular spaces, and obvious changes in cell morphology. Figure 11 and Figure 12 After 12 hours of stimulation, very few cells remained in the control group, and the culture medium became severely turbid. The experimental group still had some adherent cells. Figure 13 and Figure 14 In summary, CD79a and CD79b proteins exhibit significant antibacterial effects. While there was no significant difference in antibacterial effects across different concentration gradients over a short period as the concentration of bacterial stimulation increased, the differences became more pronounced with prolonged stimulation time. The optimal antibacterial effects were observed at bacterial stimulation concentrations of 70 μl / 500 μl and 100 μl / 500 μl.

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[0093] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of reagents that overexpress the CD79a gene in the preparation of drugs and feed additives for treating vibrio infection in half-smooth tongue sole, wherein the vibrio infection is caused by Vibrio harveyi.

2. Application of reagents overexpressing the CD79b gene in the preparation of drugs and feed additives for treating vibrio infection in half-smooth tongue sole, wherein the vibrio infection is caused by Vibrio harveyi.

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