1. A swine erysipelothrix rhusiopathiae GAPDH molecular antigen polypeptide for serum antibody detection, wherein the GAPDH molecular antigen polypeptide comprises at least one swine erysipelothrix rhusiopathiae GAPDH epitope ERepitope1 or ERepitope2, wherein the GAPDH epitope ERepitope1 corresponds to an ERG-1 polypeptide having an amino acid sequence of QLVSTDIIGMSY; the synthetic polypeptide corresponding to EREpitope2 is ERG-2, and the amino acid sequence thereof is as follows: ERG-2A: HAYTNDQNTLDGPHAKGDLRRGRAA, respectively; or ERG-2B: DQNTLDGPHAKGDLRRGRAAAQSII, respectively; or ERG-2C: DGPHAKGDLRRGRAAAQSIIPNSTG, respectively; or ERG-2D:

KGDLRRGRAAAQSIIPNSTGA。

2. the use of the antigenic polypeptide of the GAPDH molecule of Erysipelothrix rhusiopathiae for the detection of serum antibodies of claim 1 for the specific detection of Erysipelothrix rhusiopathiae.

3. The use of the polypeptide of the GAPDH molecule antigen of Erysipelothrix rhusiopathiae for detecting antibodies in serum according to claim 1 for evaluating the immunological effect of the GAPDH antigen.

4. The process of claim 1 for preparing GAPDH molecular antigen polypeptide for detecting erysipelothrix rhusiopathiae with serum antibody, which comprises the following steps: synthesizing ERG-1 polypeptide or ERG-2 by solid phase peptide synthesis method, namely adding amino acids to resin in sequence to construct peptide chain; after the synthesis is finished, firstly, protecting the Fmoc group at the N-terminal, then, protecting a side chain protecting group, and finally, cutting the polypeptide from the resin; after dissolving the crude peptide liquid sample, injecting the dissolved crude peptide liquid sample into a high performance liquid chromatograph, and testing the molecular weight and the purity of each part appearing in the chromatogram so as to confirm the content of the target polypeptide.

Background

Erysipelothrix rhusiopathiae (II)Erysipelothrix rhusiopathiaeAlso known as erysipelothrix rhusiopathiae and erysipelothrix rhusiopathiae) is a pathogen causing erysipelothrix rhusiopathiae. The swine erysipelas is one of the main bacterial diseases which endanger the pig industry in the world, and is called three major diseases of a pig farm in China together with swine erysipelas, swine plague and swine plague, and is still listed as a second class of animal diseases by the government of China at present. In recent years, the harm of swine erysipelas is reduced, but the clinical separation or detection rate of swine erysipelas is still in the front and still is the main bacterial epidemic disease in a pig farm. Swine erysipelas also causes the onset of human infections (erysipelas-like) and is a zoonotic pathogen.

The Erysipelothrix rhusiopathiae glyceraldehyde-3-phosphate dehydrogenase (GAPDH) molecule is an excellent protective antigen and has been used in the development of subunit vaccines. The evaluation of vaccine immunization requires the establishment of specific serum antibody detection methods. Since GAPDH is a glycolytic enzyme and has molecules with similar structures in other bacteria, antibodies which can cross-react with the GAPDH molecules of the erysipelothrix rhusiopathiae are easy to appear in the serum of the non-immunized pigs, so that it is difficult to establish a highly specific serum detection method for detecting the immune effect of the GAPDH antigens of the erysipelothrix rhusiopathiae. Therefore, the search for the specific sequence of the erysipelothrix rhusiopathiae GAPDH and the establishment of a highly specific serum antibody detection method become necessary requirements for effectively utilizing the erysipelothrix rhusiopathiae GAPDH antigen.

The invention aims to obtain an antigen amino acid sequence required by a method for establishing a specific detection method of serum antibody level after GAPDH antigen immunization by identifying the epitope of a swine erysipelothrix rhusiopathiae GAPDH molecule.

Disclosure of Invention

The technical problem solved by the invention is as follows: a highly specific amino acid sequence is obtained by identifying the epitope of the pig erysipelothrix rhusiopathiae GAPDH, so that the detection method capable of specifically detecting the serum antibody level of the pig erysipelothrix rhusiopathiae GAPDH after the immunization of the antigen is established.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: the polypeptide formed by the polypeptide has the characteristic of high specific specificity, and can only react with the erysipelothrix rhusiopathiae GAPDH hyperimmune serum or the erysipelothrix rhusiopathiae hyperimmune serum antibody, but can not react with the hyperimmune serum of common bacteria in other farms.

Preferably, the GAPDH molecular antigen polypeptide comprises at least one GAPDH epitope ERepitope1 or ERepitope2 of erysipelothrix suis, wherein GAPDH epitope ERepitope1 corresponds to ERG-1 polypeptide having the amino acid sequence QLVSTDIIGMSY; the synthetic polypeptide corresponding to EREpitope2 is ERG-2, and the amino acid sequence thereof is as follows: ERG-2A: HAYTNDQNTLDGPHAKGDLRRGRAA, respectively; or ERG-2B: DQNTLDGPHAKGDLRRGRAAAQSII, respectively; or ERG-2C: DGPHAKGDLRRGRAAAQSIIPNSTG, respectively; or ERG-2D:

KGDLRRGRAAAQSIIPNSTGA。

the GAPDH molecular antigen polypeptide for detecting the swine erysipelas by the serum antibody can be applied to the aspect of specific detection of the swine erysipelas.

The GAPDH molecular antigen polypeptide for detecting the swine erysipelothrix through the serum antibody can be applied to the evaluation of the immune effect of the GAPDH antigen.

The preparation method of the GAPDH molecular antigen polypeptide for detecting the erysipelothrix rhusiopathiae by the serum antibody comprises the following preparation steps: synthesizing ERG-1 polypeptide or ERG-2 by solid phase peptide synthesis method, namely adding amino acids to resin in sequence to construct peptide chain; after the synthesis is finished, firstly, protecting the Fmoc group at the N-terminal, then, protecting a side chain protecting group, and finally, cutting the polypeptide from the resin; after dissolving the crude peptide liquid sample, injecting the dissolved crude peptide liquid sample into a high performance liquid chromatograph, and testing the molecular weight and the purity of each part appearing in the chromatogram so as to confirm the content of the target polypeptide.

The invention has the beneficial effects that: compared with the prior art, although the prior art can detect the generation of antibodies after immunization of the GAPDH molecules by using the recombinant protein of the GAPDH molecules of the erysipelothrix rhusiopathiae as an antigen, the GAPDH molecules exist in different bacteria, and the antibodies of the GAPDH molecules of different bacteria can have cross reactivity, so that the specificity of the detection method established by the technical means adopted by the prior art is poor, and the immune effect of the GAPDH molecules of the erysipelothrix rhusiopathiae is difficult to effectively detect. The invention finds a polypeptide sequence which can specifically react with the swine erysipelas GAPDH antibody by analyzing the GAPDH epitope, the polypeptide does not react with the common bacterial rehabilitation serum in a pig farm, has high specificity, and can be used for establishing a detection method for evaluating the serum antibody level of the swine erysipelas GAPDH after immunization.

Drawings

FIG. 1 shows the epitope capability of the pig erysipelothrix rhusiopathiae GAPDH and the linear B cell epitope prediction result; using online software Bepipred-2.0: (http://www.detaibio.com/tools/epitope-prediction- vr.html) The sequence analyzed was the GAPDH sequence of strain SE38

FIG. 2 is the predicted position of the epitope on the GDS GAPDH molecule; the amino acid sequence of the diagram is the primary structure of the Pasteurella C51-17 strain GAPDH, and the epitope predicted by this study is underlined

FIG. 3 shows the results of indirect ELISA to detect the reaction between the polypeptide and the hyperimmune serum;

FIG. 4 shows the indirect ELISA method for detecting the reaction between the polypeptide and the common bacterial immune serum in the pig farm. A, the coating antigen is recombinant protein of a swine erysipelothrix rhusiopathiae GAPDH full-length molecule; and B, the coating antigen is ERG-1 polypeptide.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.

Example 1 Erysipelothrix rhusiopathiae GAPDH epitope prediction

(1) Gene sequence sources the present invention first analyzes the amino acid sequence of Erysipelothrix rhusiopathiae GAPDH in genome data of Erysipelothrix rhusiopathiae in which genome sequencing has been completed: SE38 (NZ _ CP011861.1), G4T10 (NZ _ CP011860.1), SY1027 (NC _021354.1), ZJ (NZ _ CP041995.1), ML 101 (NZ _ CP029804.1), WH13013 (NZ _ CP017116.1), GXBY-1 (NZ _ CP014861.1), Fujisawa (NC _015601.1), NCTC8163 (NZ _ LR134439.1), KC-Sb-R1 (NZ _ CP 033601.1). All sequences were obtained from the public database of the national center for Biotechnology information database NCBI-GenBank.

(2) Biological materials and major reagents all hyperimmune serum has been prepared and stored in previous experiments. ELISA plates were purchased from Costar, USA, HRP mouse secondary antibody from Biosharp, and TMB single component chromogenic solution from England Biotech, Inc., Huzhou.

(3) Comparison of the amino acid sequences of GAPDH of the different strains multiple sequence alignments were performed using the online software Clustal Omega (http:// www.clustal.org/Omega /), comparing the similarity between the GAPDH amino acid sequences of the different strains of Erysipelothrix rhusiopathiae in (1). The GAPDH amino acid sequences of the swine erysipelothrix rhusiopathiae SE38, G4T10, SY1027, ZJ, ML 101, WH13013, GXBY-1, Fujisawa, NCTC8163 and KC-Sb-R1 are completely identical through multiple sequence comparison (Identity =100%, so that multiple sequence alignment results are not shown), and the swine erysipelothrix rhusiopathiae GAPDH is shown to have a high conservation characteristic.

(4) Erysipelothrix rhusiopathiae GAPDH epitope prediction the ability of each amino acid residue on the Erysipelothrix rhusiopathiae GAPDH molecule (strain SE 38) to constitute an epitope was analyzed using the online software Bepipred-2.0 (http:// www.detaibio.com/tools/epitope-prediction-vr. html). A threshold of 0.5 by default for the software is set above which a polypeptide with a longer continuous sequence is predicted to be a linear B cell epitope of the GAPDH molecule. The predicted distribution of the linear B-cell epitopes of Erysipelothrix rhusiopathiae GAPDH is shown in FIG. 1. There were multiple contiguous sequences above the software default threshold of 0.5, and the longest three fragments (designated as ERepitope1, ERepitope2, ERepitope3, table 1, fig. 2, respectively) were selected for subsequent studies as predicted linear B-cell epitopes.

FIG. 1 epitope prediction results and corresponding polypeptide synthesis

Example 2 identification of epitopes of Erysipelothrix rhusiopathiae GAPDH

(1) Chemical synthesis of epitope polypeptide aiming at the predicted linear B cell epitope of Bepipred-2.0, a linear polypeptide of a corresponding sequence is chemically synthesized by Nanjing Kingsler Biotech GmbH. The synthesis procedure is briefly described as follows, and a polypeptide is synthesized using a solid phase peptide synthesis method (SPPS), in which amino acids are sequentially added to a resin to construct a peptide chain. When the synthesis is complete, the N-terminal Fmoc group is first deprotected, then the side chain protecting group is deprotected, and finally the polypeptide is cleaved from the resin. After dissolution of the crude peptide liquid sample, the sample is injected into a High Performance Liquid Chromatography (HPLC) instrument to perform molecular weight and purity tests on each fraction appearing in the chromatogram to confirm the content of the target polypeptide. The polypeptides corresponding to the predicted epitope (designated ERG-1, ERG-2 (A-D), and ERG-3, respectively) were successfully obtained by artificial chemical synthesis (Table 1). Since the length of the epitope EREpitope2 exceeds 25 amino acid residues, the conventional synthesis is difficult, so that overlapping polypeptides capable of covering the corresponding epitope are synthesized to complete subsequent experiments.

(2) Reaction of epitopes with GAPDH hyperimmune serum the reactivity of polypeptides with serum was identified using indirect ELISA and briefly described below. ELISA plates (10. mu.g/well, control not coated with any antigen) were coated with each of the above polypeptides and incubated at 37 ℃ for 2 h. After 5 washes with PBST, 5% skim milk was used for 1 h at 37 ℃. Next 1/200 diluted Erysipelothrix suis SE38 strain GAPDH hyperimmune serum was incubated at 37 ℃ for 1 h. After 5 washes with PBST, HRP-conjugated goat anti-mouse secondary antibodies were incubated for 30 min at 37 ℃. After 5 times of washing, 100. mu.l of TMB single-component color developing solution was added, and after 10 min of reaction, 50. mu.l of stop solution (2M sulfuric acid) was added. Finally, reading the absorbance of 450nm on a microplate reader.

The synthetic polypeptides (ERG-1, ERG-2 (A-D)) corresponding to two epitopes of Erepitope1 and Erepitope2 of Erephorum erysipelothrix GAPDH can obviously react with the swine erysiphe GAPDH hyperimmune serum (P <0.05), but the synthetic polypeptide (ERG-3) corresponding to Erepitope3 cannot react with the swine erysiphe GAPDH hyperimmune serum (P > 0.05). This result confirmed that the predicted EREpitope1 and EREpitope2 are epitopes of GAPDH of Erpitocin, particularly EREpitope1 (ERG-1 polypeptide, QLVSTDIIGMSY) reacts most strongly and are suitable for subsequent experiments.

Example 3 reaction of epitope Polypeptides with common bacterial immune sera in pig farms

ELISA plates (10. mu.g/well, control group not coated with any antigen) were coated with recombinant protein of the whole length molecule of Erwinia suis GAPDH or polypeptide ERG-1 and incubated at 37 ℃ for 2 h. After 5 washes with PBST, 5% skim milk was used for 1 h at 37 ℃. Then 1/500 diluted swine-origin bacterial hyperimmune serum (immunized strains are respectively the swine erysipelothrix rhusiopathiae SE38 strain, the actinobacillus pleuropneumoniae JL03 strain, the pasteurella multocida E0630 strain, the escherichia coli PCN033 strain, the haemophilus parasuis SH0165 strain, the salmonella choleraesuis C500 strain, the bacillus subtilis BS168 strain, the lactococcus lactis MG1363 strain, the streptococcus equi subsp zooepidemicus ST171 strain and the streptococcus suis R61 strain) is incubated, and the swine-origin bacterial hyperimmune serum is incubated at 37 ℃ for 1 h. After 5 washes with PBST, HRP-conjugated goat anti-mouse secondary antibodies were incubated for 30 min at 37 ℃. After 5 times of washing, 100. mu.l of TMB single-component color developing solution was added, and after 10 min of reaction, 50. mu.l of stop solution (2M sulfuric acid) was added. Finally, reading the absorbance of 450nm on a microplate reader. The results are shown in FIG. 4, where antisera from other bacteria showed varying degrees of cross-reactivity when the coating antigen was the recombinant protein of Erwinia suis GAPDH in ELISA assay (FIG. 4A), but did not show significant cross-reactivity when the coating antigen was the ERG1 polypeptide (FIG. 4B). The result shows that ERG1 has high specificity, and can be used for the antibody level evaluation after the pig erysipelas GAPDH immunization, and even can be used for the identification of the pig erysipelas infection.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.