Two cynoglossus semilaevis vibriosis-resistant genes and application method thereof

Abstract

The invention relates to the technical field of gene engineering, in particular to two cynoglossus semilaevis vibriosis-resistant genes and an application method thereof. According to the invention, the effects of the CD79a and the CD79b in the cynoglossus semilaevis in resisting vibriosis are mainly studied, verification is respectively carried out in vivo and in vitro, the process of disease-resistant breeding of the cynoglossus semilaevis is further promoted, and it is indicated that the CD79a and the CD79b have the effect of inhibiting vibrio harveyi.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to two half-smooth tongue sole anti-vibriosis genes and application methods. Background Art

[0002] Fish immunity, like mammals, has both natural and adaptive immune systems. Fish are the first species to have an adaptive immune system, so they are very valuable for research. The spleen, pronephros, and spleen are the main immune organs of fish, followed by the mucosal immune system, including the interbranchial lymphoid tissue first discovered in salmon. [1] , gut-associated lymphoid tissue, and skin-associated lymphoid tissue also play an important role in immunity [2-3] , sex hormones also affect the immune response of fish [4] In teleost fish, there are three types of immunoglobulins: IgM, IgT, and IgD, with LgM being the key effector molecule in fish immunity. [5] , IgT is considered to be unique to bony fish [6] lgD has genetic diversity among different species [8] Similar to mammals, the diversity of teleost immunoglobulins is also formed by gene recombination and somatic mutation of the heavy and light chain regions. [9] .

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

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

[11] These molecules have specialized functions and are only expressed in certain cells.

[12] , monoclonal antibodies against CD molecules have been studied and proven to be able to treat a variety of malignant tumors and autoimmune diseases

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

[12] ; Targeting CD79 monoclonal antibodies to induce B cells into an “anergic” state to treat autoimmune diseases

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

[14] and Nile tilapia

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

[16] .

[0004] CD79 is a dimer formed by the disulfide bond between CD79a and CD79b. It is located on the B cell membrane and forms a B cell antigen receptor (BCR) with membrane immunoglobulin (mlg). In mammals, it has been shown that CD79 is involved in the intracellular transduction of BCR signals. [17-18] When the extracellular antigen binds to the receptor, the intracellular CD79b may be activated first, and then the ITAM domain of CD79a is activated.

[19] , further phosphorylating various downstream adaptor proteins, activating downstream PI3K-AKT, NF-KB and other signaling pathways, which can interact with Toll-like receptors, CD40 and other signaling pathways

[20] , which is closely related to cell proliferation, differentiation, development, individual aging and immunity [20-21] In teleosts, CD79 is expressed in mullet

[22] , Nile tilapia

[23] , sea bream

[24] However, the functional studies of CD79 in the BCR signaling pathway in teleost fish are still limited. Cynoglossus semilaevis is an important economic fish, and the mortality rate after infection with Vibrio harveyi is as high as 80%. Summary of the invention

[0005] In order to solve the above problems, the present invention provides the use of CD79a and CD79b as molecular markers in the screening of disease-resistant varieties, and the use of reagents overexpressing CD79a and CD79b genes in the preparation of drugs for treating Vibrio disease in half-smooth tongue sole. The present invention mainly studies the role of CD79a and CD79b in half-smooth tongue sole in resistance to Vibrio disease, and verifies them from both in vivo and in vitro directions, further promoting the process of disease-resistant breeding of half-smooth tongue sole.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The invention provides application of two molecular markers (CD79a and CD79b) of half-smooth tongue sole resistance to Vibrio disease in improved variety breeding.

[0008] The present invention also provides the use of a reagent for overexpressing the CD79a gene in the preparation of a drug for treating Vibrio semilaevis disease.

[0009] The present invention also provides the use of a reagent for overexpressing the CD79b gene in the preparation of a drug for treating Vibrio semilaevis disease.

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

[0011] Beneficial effects of the present invention:

[0012] The present invention mainly studies the role of CD79a and CD79b in half-smooth tongue sole in resistance to Vibrio disease, and verifies them in vivo and in vitro, further advancing the process of disease-resistant breeding of half-smooth tongue sole, indicating that CD79a and CD79b have the effect of inhibiting Vibrio harveyi. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

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

[0015] Figure 1-2 is the amino acid sequence analysis diagram of Cs-CD79b;

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

[0017] Figure 3 is the expression level of Cs-CD79a and Cs-CD79b mRNA in various tissues of healthy tongue sole; Liver, Skin, Blood, Spleen, Gill, Foregut, Midgut, Hindgut, Kidney, Muscle;

[0018] Figure 4 is the expression level of Cs-CD79a and Cs-CD79b mRNA in the tissues of Semilaevis semilaevis infected with Vibrio harveyi; Skin, Spleen, Kidney, Intestines;

[0019] Figure 5 The expression of Cs-CD79a and Cs-CD79b in the tissues of susceptible and resistant families of Cynoglossus semilaevis; susceptible family (S), resistant family (R), Spleen (spleen), Kidney (kidney); AB: 2017 family; CD: 2018 family; EF: 2019 family;

[0020] Figure 6 Tissue localization of CD79a and CD79b in Cynoglossus semilaevis. AC: gills, kidneys and spleen, CD79a antisense probe; DF: gills, kidneys and spleen, CD79a sense probe; GI: gills, kidneys and spleen, CD79b antisense probe; JL: gills, kidneys and spleen, CD79b sense probe;

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

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

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

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

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

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

[0027] Fig.13 CD79a-overexpressing cells were stimulated with different concentrations of bacteria for 12 h;

[0028] Fig.14 CD79b-overexpressing cells were stimulated with different concentrations of bacteria for 12 hours. DETAILED DESCRIPTION

[0029] The present invention provides a method for detecting two anti-vibriosis genes CD79a and CD79b of Cynoglossus semilaevis. In the present invention, the vibriosis is caused by Vibrio harveyi.

[0030] The present invention also provides the use of a reagent for overexpressing the CD79a gene in the preparation of a drug for treating Vibrio semilaevis. In the present invention, the Vibrio disease is preferably caused by Vibrio harveyi.

[0031] The present invention also provides the use of a reagent for overexpressing the CD79b gene in the preparation of a drug for treating Vibrio semilaevis. In the present invention, the Vibrio disease is preferably caused by Vibrio harveyi.

[0032] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they 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 soles were purchased from Yellow Sea Aquatic Products Co., Ltd., Shandong Province, China, and maintained in aerated seawater at 20 °C. Before the experiment, the fish were acclimated in the laboratory for two weeks and confirmed to be free of bacterial pathogens.

[0037] 1.2 Strain culture and bacterial infection

[0038] The Gram-negative bacteria Vibrio harveyi used in the present invention were obtained from the Yellow Sea Fisheries Research Institute, and the competent cells were purchased from TransGen Biotech Co., Ltd. The bacteria were cultured in tryptic soy broth (TSB) at 28°C and 180 rpm / min in a shaker. Five experimental fish were intraperitoneally injected with 500 μL of 1.0×10 7 CFU / mL500 μL, 0, 6, 12, 24, 48, 72 h, and 96 hpi, 5 fish were anesthetized and the kidney, spleen, liver, intestine, gill, and skin were collected.

[0039] 1.3 Sequence analysis

[0040] The amino acid sequences of CsCD79a (GenBank Accession No. XM_008323971.3) and CsCD79b (GenBank Accession No. XM_008314006.3) were analyzed using the BLAST program of the National Center for Biotechnology Information (NCBI). Domain searches were performed using the conserved domain search program of NCBI. Theoretical molecular masses and theoretical isoelectric points were predicted using EditSeq in the DNASTAR (Madison, WI) software package. Multiple sequence alignments were created using Smart.

[0041] 1.4 Real-time quantitative PCR

[0042] The expression of CD79a and CD79b transcripts in tongue sole under normal physiological conditions was analyzed by real-time quantitative PCR (qRT-PCR) using FQD-96C instrument (BIOER). Tissues (blood, intestine, kidney, foregut, midgut, hindgut, gill, spleen, muscle and liver) were extracted from 5 healthy tongue soles. The expression of CD79 gene in tongue sole immune tissues after Vibrio harveyi infection was analyzed. Tissues (kidney, spleen, liver, intestine, gill and skin) were extracted from 5 infected fish. The expression of CD79 transcripts in tongue sole immune tissues in disease-resistant and susceptible families from different years were analyzed. All tissues were extracted using TIANGEN animal tissue total RNA extraction kit and TaKaRa reverse transcription kit, and all qRT-PCR experiments were performed using ArtiCan ATMSYBR qPCR Mix Kit (TsingKe) was used in a total volume of 20 μL. The reaction mixture was incubated at 95°C for 2 min, followed by cycles of 95°C for 10 s, 60°C for 20 s, and 72°C for 30 s.

[0043] 1.5 Probe synthesis and section in situ hybridization

[0044] To detect the localization of CD79a and CD79b in the gills, spleen, and kidney of tongue sole. Dig-labeled primers were synthesized at Qingdao Ruiboxingke Biotechnology Co., Ltd., and ribosomal probes were synthesized. The transcription time was 3 hours and purified by Licl. The hybridization temperature was 65°C, and the 25xNBT / 25xBCIP kit (Solarblo) was used for staining at 30°C for 6 hours, and glycerol-gelatin was used for sealing. Three biological replicates were performed for each gene in each tissue. The digital slide scanning workstation 3DHIETCH Pannormic MIDI was used to scan and take pictures with SlideViewer.

[0045] 1.6 Inactivation of Vibrio harveyi

[0046] Inoculate Vibrio harveyi into LB liquid culture medium; culture at 28°C, 180rpm / min; centrifuge at 4°C, 6000g / min for 15 minutes, remove the supernatant and wash with PBS three times; resuspend in PBS containing 0.5% formaldehyde and place at 4°C for 48 hours; culture on plates to detect the presence of live bacteria; wash with PBS three times by centrifugation; detect OD value with UV spectrophotometer, and adjust OD 600 =1.

[0047] 1.7 293 cell transfection and bacterial infection experiments

[0048] The CsCD79a gene sequence was connected to the GFP vector; the CsCD79b gene sequence was connected to the RFP vector to construct a luminescent plasmid. CsCD79a and CsCD79b were transfected into 293 cells for overexpression, and the luminescence was observed under a microscope 48 hours after transfection in a 24-well plate (500ul culture medium per well).

[0049] The volume of the Vibrio harveyi bacterial solution in each well was set to 5 gradients of 50μL / 500μL, 75μL / 500μL, 100μL / 500μL, and 125μL / 500μL to infect the cells. Three repeated experiments were set for each gradient, and the cell status was observed at 3h, 6h, 9h, and 12h of stimulation.

[0050] 2. Results

[0051] 2.1 Sequence characteristics of CsCD79a and CsCD79b

[0052] CsCD79a is composed of 224 amino acid residues, with a calculated molecular weight of 25.0 kDa and a theoretical isoelectric point (pI) of 5.649; CsCD79b is also composed 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 has an extracellular hydrophobic signal peptide (residue 1e21) and an lg-like domain, a transmembrane domain (residue 22e91), and an intracellular ITAM domain region (residue 92e107). The basic structure of the ITAM domain in teleost fish is: D / E(x)7D / ExxYxxLx(6-8)xxxI / L( Figure 1-1 and Figure 1-2 Phylogenetic analysis showed that CsCD79s was closely related to bony fish such as flatfish and Nile tilapia; CsCD59 was closely homologous to blue stickleback, zebrafish, and spotted fish; and CsCD22 was closely related to zebrafish and golden eye seabass ( Figure 2 ). It has been concluded that the two may form dimers through non-covalent bonds outside the cell, and the dimers in the transmembrane region are stabilized by hydrogen bonds and ionic bonds formed 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 pattern 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 showed that CsCD79 was expressed most highly in the spleen and kidney, followed by gills, while it was expressed at low levels in other tissues and even absent in the intestine, which was directly related to the blood content of the tissues ( Figure 3 To determine the effect of pathogen infection on CsCD79s expression, qPCR was used to detect the expression of CsCD79s in various immune tissues at 0h, 6h, 12h, 24h, 48h, 72h, and 96h after infection with Vibrio harveyi ( Figure 4 ). The results showed that the expression levels were highest in the skin, spleen, and gills at 24 hours after infection, and the high expression in the gills at 6 hours should be unrelated to bacterial stimulation; CD79b showed high expression in the kidney, while it was basically not expressed in the intestine and liver, thus eliminating the possibility of stimulatory expression. In order to advance the process of disease-resistant breeding, the team of Academician Chen Songlin established multi-generational selection of disease-resistant and susceptible families of Semilaevis semilaevis. The qPCR results of immune tissues of disease-resistant and susceptible families from 2017 to 2019 showed that the expression levels of CsCD79a and CsCD79b in disease-resistant families were significantly higher than those in susceptible families ( Figure 5 ).

[0063] 2.3 Localization of CsCD79s in slices

[0064] The results of in situ hybridization of paraffin sections of healthy tissues of Cynoglossus semilaevis showed that the distribution of CD79a and CD79b in these three tissues was similar ( Figure 6 ). In the kidney, it is mainly distributed in various renal tubules, rich in macula densa, and distributed in large quantities in the blood vessels of the renal cortex; in the gills, it is distributed in the capillaries of the gill filaments and gill arches; in the spleen, it is rich in the arteries and surrounding capillaries.

[0065] 2.4 Cell transfection and antibacterial effect

[0066] After bacterial stimulation, it was found that CD79a and CD79b proteins had a certain antibacterial effect on Vibrio harveyi. After 3 hours of stimulation with inactivated Vibrio harveyi, a small number of cells in the experimental group died, a small number of cells floated, and there was no significant difference in cell morphology compared with the control group ( Figure 7 and Figure 8 ); After 6 hours of stimulation, the cell death rate increased, and the morphology of living cells did not change. However, the cells in the control group showed obvious cracks, the death rate was greater, and the cell morphology had changed ( Fig. 9 and Fig.10 ); After 9 hours of stimulation, the cell death area was more obvious, and the cells appeared to float in blocks; the amount of attached cells in the control group was greatly reduced, the intercellular gap was larger, and the cell morphology changed significantly ( Fig.11 and Fig.12 ); After 12 hours of stimulation, there were few cells left in the control group and the culture medium was seriously turbid, while some adherent cells still existed in the experimental group ( Fig.13 and Fig.14 ). In summary, CD79a and CD79b proteins have obvious antibacterial effects. As the concentration of bacterial stimulation increases, there is no obvious difference in the antibacterial effects of each concentration gradient in a short period of time. As the stimulation time increases, the difference becomes increasingly obvious. When the bacterial stimulation amount is 70μl / 500ul and 100μl / 500μl, the antibacterial effect is the best.

[0067]

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[0093] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of CD79a and CD79b as molecular markers for resistance to Vibrio infection in the breeding of semilaevis semilaevis.

2. Application of reagents that overexpress the CD79a gene in the preparation of drugs and feed additives for the treatment of Vibrio semilaevis disease in half-smooth tongue sole.

3. Application of reagents that overexpress the CD79b gene in the preparation of drugs and feed additives for the treatment of Vibrio semilaevis disease in half-smooth tongue sole.

4. The use according to any one of claims 1 to 4, characterized in that: The vibriosis is caused by the bacterium Vibrio harveyi.

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