Anti-channa argus IgM antigen epitope polypeptide, polyclonal antibody as well as preparation method and application of anti-channa argus IgM antigen epitope polypeptide and polyclonal antibody

Abstract

The invention belongs to the technical field of fish immunodetection, and particularly relates to antigenic epitope polypeptide of anti-channa argus IgM, a polyclonal antibody as well as a preparation method and application of the polyclonal antibody. Specifically, the invention successfully constructs a preparation technology system based on the snakehead IgM specific antigen epitope polypeptide and the corresponding polyclonal antibody. Meanwhile, through combination of bioinformatics epitope prediction and immunogenicity optimization, the linear epitope polypeptide with high reactogenicity is successfully screened and obtained. And finally constructing to obtain the anti-channa argus IgM polyclonal antibody, and the verification shows that the polyclonal antibody has high specific recognition capability on channa argus mucus IgM protein. The successful preparation of the antibody fills the blank of a reagent for detecting the mucosal immune globulin of the freshwater economic fish, and provides a core reagent basis for establishing standardized detection platforms such as fish vaccine titer evaluation and immune response dynamic monitoring, so that the antibody has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of fish immune detection, and specifically relates to an anti-Channa argus IgM antigen epitope polypeptide, a polyclonal antibody, and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] In recent years, researchers have made significant progress in the field of aquatic vaccine research and development. The development of new preparations such as inactivated vaccines, subunit vaccines and genetically engineered vaccines has entered the experimental research stage. However, the process of vaccine industrialization and application still faces key technical bottlenecks, among which the standardization of vaccine efficacy evaluation system has become a prominent problem restricting the development of the industry. As the main evaluation method, the traditional virus attack experiment method has the biosafety risk of causing the spread of the disease. Its test results are easily affected by multiple factors such as the host's physiological state, environmental parameters and the incubation period of the pathogen, resulting in significant deviations in the determination of the protection rate.

[0004] In view of the above technical defects, the establishment of a quantitative evaluation system based on dynamic monitoring of immune response has become the current research focus. This system needs to systematically analyze the laws of vaccine-induced cellular immunity and humoral immune response, and establish core biomarkers that are significantly correlated with immune protection titers. At the level of humoral immunity assessment, the quantitative detection of specific immunoglobulin IgM has important diagnostic value.

[0005] The IgM antibody-based immune detection platform (including ELISA, immunochromatographic test strips and immunofiltration technology, etc.) can realize the rapid quantitative detection of IgM antibodies against pathogenic antigens (Nocardia, Aeromonas hydrophila, etc.) in fish mucus and serum. Combined with cellular immune detection methods such as flow cytometry and immunofluorescence analysis, the number of immune cell subpopulations and changes in functional status can be simultaneously evaluated. This dual-track evaluation model can not only avoid the biosafety risks of traditional virus attack experiments, but also realize early dynamic monitoring of vaccine immune responses, providing a scientific basis for disease early warning and optimization of immune programs.

[0006] However, the inventors found that there is still a gap in the current research on immunoglobulin antibodies for freshwater economic fish. In particular, there are few reports on the research of mucus IgM antibodies of important aquaculture species such as Channa argus, and a systematic database has not yet been established for the correlation between their immune response characteristics and vaccine protective titers. Therefore, in-depth application research on anti-Channa argus IgM polyclonal antibodies will not only help to improve the vaccine evaluation technology system for this species, but also provide theoretical references for the establishment of standardized immune assessment programs across fish species, and promote the substantial transformation of aquatic vaccines from laboratory research to industrial applications. Summary of the invention

[0007] Based on the above-mentioned deficiencies of the prior art, the present invention provides an antigen epitope polypeptide, polyclonal antibody and preparation method and application thereof against Channa argus IgM. The present invention obtains the antigen epitope polypeptide of Channa argus IgM by conducting in-depth research on Channa argus mucus IgM, and successfully prepares the anti-Channa argus mucus IgM polyclonal antibody using it as an antigen. Based on the above-mentioned research results, the present invention is completed.

[0008] Specifically, the technical solution of the present invention is as follows:

[0009] The first aspect of the present invention provides an antigenic epitope polypeptide of an anti-Channa argus IgM antibody, wherein the antigenic epitope polypeptide is selected from:

[0010] (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0011] (b) A polypeptide derived from (a) having an antigenic epitope function, wherein the amino acid sequence in (a) is substituted, deleted or added with one or more amino acids.

[0012] The second aspect of the present invention provides a nucleic acid molecule encoding the above-mentioned antigen epitope polypeptide.

[0013] The third aspect of the present invention provides the use of the above antigen epitope polypeptide in the preparation of anti-Channa argus IgM antibodies.

[0014] A fourth aspect of the present invention provides an anti-Channa argus IgM antibody, which specifically binds to Channa argus mucus IgM protein and specifically recognizes the above-mentioned antigen epitope polypeptide.

[0015] A fifth aspect of the present invention provides a method for preparing the above-mentioned anti-Channa argus IgM antibody, the method comprising: immunizing a non-human animal based on an antigen consisting of the above-mentioned antigen epitope polypeptide to obtain the anti-Channa argus IgM antibody.

[0016] A sixth aspect of the present invention provides a detection kit, which comprises the above-mentioned anti-Channa argus IgM antibody.

[0017] A seventh aspect of the present invention provides a method for detecting Channa argus IgM protein, the method comprising: using the above-mentioned anti-Channa argus IgM antibody and / or the above-mentioned detection kit to detect a sample to be tested.

[0018] Beneficial technical effects of one or more of the above technical solutions:

[0019] The above technical scheme successfully constructed a technical system for the preparation of specific antigenic epitope polypeptides and corresponding polyclonal antibodies based on the IgM of Channa argus. At the same time, through bioinformatics epitope prediction combined with immunogenicity optimization, linear epitope polypeptides with high reactivity were successfully screened. Finally, a polyclonal antibody against Channa argus IgM was constructed, and it was verified that the polyclonal antibody had a highly specific recognition ability for the IgM protein of Channa argus mucus. The successful preparation of this antibody fills the gap in the detection reagents of mucosal immunoglobulins of freshwater economic fish, and provides a core reagent basis for the establishment of standardized detection platforms such as fish vaccine potency evaluation and dynamic monitoring of immune responses, so it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings in the specification, which constitute a part of the present application, are used to provide further understanding of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute improper limitations on the present application.

[0021] Figure 1 This is a rare codon analysis diagram of the IgM protein in the mucus of Channa argus in the embodiment of the present invention.

[0022] Figure 2 This is a hydrophilicity analysis diagram of the IgM protein in the mucus of Channa argus in the embodiment of the present invention.

[0023] Figure 3 This is a diagram for analyzing the transmembrane domain of the IgM protein in the mucus of Channa argus in an embodiment of the present invention.

[0024] Figure 4 This is a predicted map of the signal peptide of the IgM protein in the mucus of Channa argus in the embodiment of the present invention.

[0025] Figure 5 This is a graph showing the antigenicity analysis of the IgM protein in the mucus of Channa argus in an embodiment of the present invention.

[0026] Figure 6 HPLC analysis chart of the polypeptide in the example of the present invention.

[0027] Figure 7 This is the MS analysis diagram of the polypeptide in the example of the present invention.

[0028] Figure 8 The figure is the SDS-PAGE analysis diagram of the purified IgM polyclonal antibody of Channa argus mucus in the embodiment of the present invention; M: protein molecular weight standard; 1: antibody purification analysis result.

[0029] Fig. 9The polyclonal antibody ELISA method is used to detect the changes in the IgM protein level in the mucus and bile of Channa argus in the embodiment of the present invention. A, B, C, and D are the ELISA results of IgM in the skin, gill, intestinal mucus and bile of Channa argus, respectively. * indicates the significant difference level of protein expression level in each group at different times after inactivated Aeromonas hydrophila immune stimulation (P<0.05).

[0030] Fig.10 The immunohistochemical results of Channa argus intestine detected by antibodies in the examples of the present invention are shown in Figure 1. A and a are the immunohistochemical results of Channa argus intestine with rabbit anti-Channa argus IgM polyclonal antibody and non-immunized rabbit serum, respectively. Scale bar: 100 μm. DETAILED DESCRIPTION

[0031] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0032] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0033] As mentioned above, there is still a gap in the current research on immunoglobulin antibodies for freshwater economic fish. In particular, there are few reports on the research on IgM antibodies of important aquaculture species such as Channa argus.

[0034] In view of this, in a typical embodiment of the present invention, an antigenic epitope polypeptide of an anti-Channa argus IgM antibody is provided, wherein the antigenic epitope polypeptide is selected from:

[0035] (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0036] (b) A polypeptide derived from (a) having an antigenic epitope function, wherein the amino acid sequence in (a) is substituted, deleted or added with one or more amino acids.

[0037] In another specific embodiment of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the above-mentioned antigen epitope polypeptide.

[0038] In another specific embodiment of the present invention, there is provided use of the above antigen epitope polypeptide in the preparation of anti-Channa argus IgM antibodies.

[0039] Wherein, the anti-Channa argus IgM antibody may be a monoclonal antibody or a polyclonal antibody. In a specific embodiment of the present invention, the anti-Channa argus IgM antibody is a polyclonal antibody.

[0040] Furthermore, the anti-Channa argus IgM antibody can be used to detect Channa argus IgM protein.

[0041] In another specific embodiment of the present invention, an anti-Channa argus IgM antibody is provided, wherein the anti-Channa argus IgM antibody specifically binds to the Channa argus mucus IgM protein and specifically recognizes the above-mentioned antigen epitope polypeptide.

[0042] In another specific embodiment of the present invention, a method for preparing the above-mentioned anti-Channa argus IgM antibody is provided, the method comprising: immunizing a non-human animal based on the antigen consisting of the above-mentioned antigen epitope polypeptide to obtain the anti-Channa argus IgM antibody.

[0043] Furthermore, the anti-Channa argus IgM antibody may be a polyclonal antibody; and the non-human animal is a rabbit. In this case, the prepared antibody is a rabbit polyclonal antibody to the antigenic epitope polypeptide of the anti-Channa argus IgM antibody.

[0044] In another specific embodiment of the present invention, a detection kit is provided, wherein the detection kit comprises the above-mentioned anti-Channa argus IgM antibody, and the detection kit can be used for qualitative or quantitative detection of Channa argus IgM protein.

[0045] The kit may be a kit based on immunohistochemical staining or ELISA, etc. In another embodiment of the present invention, the kit may also include a buffer solution, reagents, etc. commonly used for antibody detection, which are not specifically limited here.

[0046] In another specific embodiment of the present invention, a method for detecting Channa argus IgM protein is provided, the method comprising: using the above anti-Channa argus IgM antibody and / or the above detection kit to detect the sample to be tested. The detection can be qualitative and / or quantitative detection.

[0047] The sample to be tested may be a sample of skin, gill, intestinal mucus and / or bile of Channa argus, which is not specifically limited herein.

[0048] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0049] Example

[0050] 1. Screening of IgM antigen epitopes and preparation of polyclonal antibodies

[0051] 1. Antigenic epitope design

[0052] Based on Channa argus IgM protein (GenBank: EU822510.1, Channa argus immunoglobulin M heavy chain mRNA, complete cds), specific antigenic epitopes were screened out through bioinformatics analysis (including antigenicity, hydrophilicity and transmembrane domain prediction), and the antigenic epitope polypeptide sequence was determined: KSYTRIPGRPTQ-C (SEQ ID NO: 1), which is the 462-473 amino acid sequence of Channa argus mucus IgM protein. The polypeptide was chemically synthesized and coupled with a carrier protein (KLH / BSA / OVA) as a complete antigen, and the purity of the synthetic polypeptide was >90% (verified by HPLC and mass spectrometry).

[0053] 2. Animal Immunization

[0054] After the complete antigen (dose 0.5 mg / time) was emulsified with Freund's adjuvant, two New Zealand rabbits were immunized. The immunization scheme was as follows:

[0055] Immunization cycle: After the first immunization, booster immunization is performed every 14 days, for a total of 5 immunizations;

[0056] Blood collection test: titer monitoring begins after the third immunization, and the last blood collection time is the 56th day.

[0057] 3. Antibody Purification and Validation

[0058] The antiserum was purified by Protein A / G affinity chromatography, and the purity of the purified antibody was >85% (SDS-PAGE verification). Western Blot results showed that the antibody could specifically recognize the target protein in the recombinant protein of Channa argus IgM and tissue lysate.

[0059] 2. Antibody titer and specificity testing

[0060] 1.ELISA titer analysis

[0061] The antibody titer was determined by indirect ELISA method. The specific conditions were as follows: antigen coating concentration: 2 μg / mL; serum dilution gradient: 1:500 to 1:29,524,500; titer determination standard: OD 450nm (Sample) / OD 450nm (Blank) ≥ the highest dilution of 2.1.

[0062] Results: The titer of the purified antibody was >9,841,500 (Table 1), indicating that the antibody had high affinity.

[0063] Table 1 Detection results of purified antibodies

[0064]

[0065] 2. Western Blot Verification

[0066] After purification, the antibody was subjected to SDS-PAGE electrophoresis and stained with Coomassie Brilliant Blue, which was consistent with the expected size of the IgM heavy chain. It can be seen that the purity of the purified antibody was above 85%.

[0067] 3. Dynamic detection of IgM protein levels in Channa argus mucosa

[0068] 1. Construction of immune model

[0069] Healthy Channa argus were divided into two groups: the injection group: intraperitoneal injection of 1×10 8 CFU / mL Aeromonas hydrophila inactivated vaccine (100 μL / tail); immersion group: immersed in the same concentration of bacterial solution for 30 minutes.

[0070] 2. Sample collection and testing

[0071] Five Channa argus were randomly selected from each group before immunization and 1d, 3d, 5d, 7d, 10d, 14d, 21d, 28d, 35d and 49d after immunization, and skin mucus, gill mucus, intestinal mucus and bile were collected respectively. The IgM level was detected by ELISA. The key parameters were as follows: coating antigen: inactivated Aeromonas hydrophila diluted to a protein concentration of 0.1 mg / mL; primary antibody: rabbit anti-Canna argus IgM polyclonal antibody (1:4000 dilution); secondary antibody: HRP-labeled goat anti-rabbit IgG (1:5000 dilution).

[0072] 3. Results Analysis

[0073] After immunization of Channa argus with two methods, ELISA results showed that the IgM protein levels in the exocrine fluids such as skin mucus, gill mucus, intestinal mucus and bile were first increased within 49 days, and then decreased to the level of the control group. In the injection group, the IgM levels in all exocrine fluids reached the peak 21 days after immunization; in the immersion group, the IgM peaks of skin and gill mucus appeared on the 7th and 14th days, respectively, and the peaks of intestinal mucus and bile were delayed to the 21st day; the results showed that the peaks of skin and gill mucus in the immersion group were significantly higher than those in the injection group (P<0.05), while the peaks of intestinal mucus and bile were lower.

[0074] 4. Localization of IgM in the mucosal tissue of Channa argus

[0075] The paraffin sections of the hindgut tissue of Channa argus were analyzed by immunohistochemical staining. Specifically, the above-mentioned anti-Channa argus mucus IgM polyclonal antibody was used to perform immunohistochemical staining on the hindgut of the Channa argus mucosal immune tissue, wherein the primary antibody was a rabbit anti-Channa argus mucus IgM recombinant protein polyclonal antibody (1:500); the secondary antibody was a goat anti-rabbit IgG (H+L) secondary antibody (1:100), and non-immune rabbit serum replaced the polyclonal antibody as a negative control; the results showed an obvious red positive signal, while no positive signal was generated in the non-immune serum group (i.e., the negative control).

[0076] The above results confirmed that the antibody can specifically locate IgM in mucosal tissues, providing a tool for studying mucosal immune responses.

[0077] In summary, the polyclonal antibodies prepared in the embodiments of the present invention can be used for:

[0078] 1. Detect the IgM protein level in the mucus of Channa argus to evaluate the immune effect of the vaccine;

[0079] 2. Develop early diagnosis kits for diseases of Channa argus;

[0080] 3. Analyze the mucosal immune mechanism and provide technical support for disease-resistant fish breeding.

[0081] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific implementation methods of the present invention, it is not intended to limit the protection scope of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or deformations that can be made by those skilled in the art without creative labor are still within the protection scope of the present invention.

Claims

1. An antigenic epitope polypeptide of an anti-Channa argus IgM antibody, characterized in that: The antigenic epitope polypeptide is selected from: (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) A polypeptide derived from (a) having an antigenic epitope function, wherein the amino acid sequence in (a) is substituted, deleted or added with one or more amino acids.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antigenic epitope polypeptide according to claim 1.

3. Use of the antigen epitope polypeptide according to claim 1 in the preparation of anti-Channa argus IgM antibodies.

4. The use according to claim 3, characterized in that: The anti-Channa argus IgM antibody is a monoclonal antibody or a polyclonal antibody; preferably, the anti-Channa argus IgM antibody is a polyclonal antibody.

5. An anti-Channa argus IgM antibody, characterized in that: The anti-Channa argus IgM antibody specifically binds to the Channa argus mucus IgM protein and specifically recognizes the antigen epitope polypeptide according to claim 1.

6. A method for preparing the anti-Channa argus IgM antibody according to claim 5, characterized in that: The method comprises: immunizing a non-human animal with an antigen consisting of the antigen epitope polypeptide according to claim 1 to obtain the anti-Channa argus IgM antibody.

7. The method according to claim 6, characterized in that The anti-Channa argus IgM antibody is a polyclonal antibody.

8. A detection kit, characterized in that: The detection kit comprises the anti-Channa argus IgM antibody according to claim 5 or the anti-Channa argus IgM antibody prepared by the preparation method according to any one of claims 6-7.

9. The detection kit according to claim 8, characterized in that The detection kit is used for qualitative or quantitative detection of Channa argus IgM protein; Furthermore, the detection kit also includes a buffer solution and reagents commonly used for antibody detection.

10. A method for detecting IgM protein of Channa argus, characterized in that: The method comprises: using the anti-Channa argus IgM antibody of claim 5 and / or the detection kit of any one of claims 8 to 9 to detect the sample to be tested; Furthermore, the samples to be tested are skin, gill, intestinal mucus and / or bile samples of Channa argus; and the detection is qualitative and / or quantitative detection.

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