CN114350636B

CN114350636B - Recombinant Mh-PGK protein and application thereof in detection of swine haemophilus mycoplasma

Info

Publication number: CN114350636B
Application number: CN202111479647.3A
Authority: CN
Inventors: 付媛; 石团员; 袁秀芳; 孙洪超; 王德前; 徐丽华; 李军星; 苏菲; 郝力力; 薛红叶; 刘娱霏; 李天恩; 余斌
Original assignee: Zhejiang Academy of Agricultural Sciences
Current assignee: Zhejiang Academy of Agricultural Sciences
Priority date: 2021-12-06
Filing date: 2021-12-06
Publication date: 2023-06-20
Anticipated expiration: 2041-12-06
Also published as: CN114350636A

Abstract

The invention discloses a recombinant Mh-PGK protein and application thereof in detection of swine haemophilus mycoplasma. The invention firstly discloses a recombinant Mh-PGK protein with an amino acid sequence shown as SEQ ID NO. 1. The invention further discloses an indirect ELISA detection kit and a detection method for the swine haemophilus antibody by taking the recombinant Mh-PGK protein as an ELISA plate of a coating antigen. The invention establishes an indirect ELISA detection method for the Mycoplasma hyopneumoniae antibody based on the recombinant Mh-PGK protein, which is used for detecting the level of the Mycoplasma hyopneumoniae antibody, has no cross reaction with part of other pig disease serum, has good specificity and repeatability, and provides an effective means for clinical diagnosis, epidemiological research and immunodetection of Mycoplasma suis, mycoplasma parvum and Mycoplasma haemosuis.

Description

Recombinant Mh-PGK protein and application thereof in detection of swine haemophilus mycoplasma

Technical Field

The present invention relates to the field of biotechnology. More particularly, relates to a recombinant Mh-PGK protein and application thereof in detection of swine haemophilus mycoplasma.

Background

Haemophilus mycoplasma (Hemotropic mycoplasma, HM also known as eperythrozoon) is a species of mycoplasma that infects many mammals, parasitizes on the surface of erythrocytes and in bone marrow (Liu Chengping, ed. Veterinary microbiology [ M ]. Fourth edition. Beijing: chinese agriculture press 2010:240-241). The destruction of erythrocytes by HM causes hemolysis, infectious anemia in the host, and can cause abortion and stillbirth in sows, anemia in piglets, jaundice and wasting (Hoelzle L E, felder K M, hoelzle K. Portland erythrozoosis: from Eperythrozoon suis to Mycoplasma suis [ J ]. Tierarztl Prax Ausg G Grosstiere Nutztier.2011, 39 (4): 215-220.; messack, J.B., hemotrophic mycoplasmas (thermoplastas): a review and new insights into pathogenic potential. Vet Clin Pathol,2004.33 (1): p.2-13.). Three HMs were performed in the chinese herd, including early identified Mycoplasma hyopneumoniae (m.suis, old-fashioned pig eperythrozoon) (Zhang Haoji, xie Mingquan, zhang Jian, etc.) swine eperythrozoon 16S rRNA gene sequencing and phylogenetic analysis [ J ]. Animal husbandry journal 2005 (06): 596-601.), 2011 molecular biology level identified Mycoplasma minor (Mycoplasma parvum, m.parvum) (Watanabe Y, fujihara M, obara H, et al.two genetic clusters in swine hemoplasmas revealed by analyses of the 16S rRNA and RNase P RNA genes[J ]. J Vet Med sci.2011,73 (12): 1657-1661.) and new-fashioned Mycoplasma hyophelophilum reported in the company of Zhejiang 2017 (Mycoplasma haemosuis, m.haos, old-fashioned as Candidatus m.haemeis) (fur Y, xu H, et al of a novel Hemoplasma species from pigs in Zhejiang province, chiend j.3579, j.80 j.ve 2017).

PCR method pathogenic epidemiological investigation found that M.haemauis appeared to be popular in Zhejiang, jiangsu, henan, etc. China, and that mixed infection of HM of other two swine sources occurred more frequently, sow infection rate was higher, and M.haemauis (Seo M G, kwon O D, kwak D.2019.prevvalance and phylogenetic analysis of hemoplasma species in domestic pigs in Korea. Parasites vector, 12 (1): 378; stadler J, ade J, ritzmann M, et al 2020.detection of a novel haemoplasma species in fattening pigs with skin alterations, fe and anaemia. Ve Record,187 (2): 66) were also detected in swine herd. Serological and molecular biological epidemiological studies have reported M.suis and M.parvum serum antibody detection methods (Amit Kadam, wang Jinxiu, xie Gaojin, et al) reveal that the micromanipulation/eperythrozoon is Hainan island major Mycoplasma hyophelophilum [ J ]. Proc. M. Of medical insects, 2018,25 (03): 140-147;. Hsu F S, liu M C, chou S M, et al. Evaluation of an enzyme-linked immunosorbent assay for detection of Eperythrozoon suis antibodies in swine [ J ]. Am J Vet Res.1992,53 (3): 352-354;. Hoelzle L E, hoelzle K, helbling M, et al MSG1, a surface-localised protein of Mycoplasma suis is involved in the adhesion to erythrocytes [ J ]. Microbe Infect.2007,9 (4): 466-474), and the lack of M.haemas antibody detection methods.

ELISA method has the advantages of sensitivity, rapidness, simplicity, easy standardization and the like, hoelzle and the like prove that the first adhesion protein MSG1 of M.suis has glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, the ELISA method established by the recombinant protein has better specificity, is more sensitive and effective than the bacterial antigen ELISA (Hoelzle K, doser S, ritzmann M, et al, vaccine with the Mycoplasma suis recombinant adhesion protein MSG1 elicits a strong immune response but fails to induce protection in pigs [ J ]. Vaccine.2009,27 (39): 5376-5382), the ELISA detection method of M.parvum truncated protein mp-tGAPDH is also established by the auxiliary chromatography and the like, and the M.parvum serum antibody positive rate is 36.69 percent (91/248) (auxiliary chromatography, dan Tuanyuan, sun Hongchao. The micro mycoplasma antibody indirect ELISA detection method based on recombinant truncated protein mp-tGAPDH is established and applied [ J ]. Zhejiang agriculture, 2021,33 (05): 5376-5382).

Phosphoglycerate kinase (Phosphoglycerate kinase PGK) is an important enzyme for the energy metabolism of the glycolytic pathway and an essential enzyme for the survival of organisms. PGK is involved in the second stage of glycolysis and catalyzes the conversion of 1, 3-diphosphoglycerate to 3-phosphoglycerate, producing ATP. Haemophilus relies on the host blood to provide energy, glycolysis being the primary source of energy supply. Meanwhile, PGK is also involved in pathogenic effects caused by pathogenic bacteria such as cell homeostasis, candida albicans and the like adhering to host cells, interactions of nucleic acids, tumor development, cell death, viral replication and the like (FuQ, yu Z.phosphoglycate kinase 1 (PGK 1) in cancer: A promising target for diagnosis and therapy. Life Sci.2020Sep 1;256:117863.Pii: S0024-3205 (20) 30613-5.Doi:10.1016/j.lfs.2020.117863.PubMed PMID: 32479953; rojas-Pirela M, andrade-Alvi rez D, rojas V, kemmerling U, C.ceresAJ, michels PA, concepci n JL,

W. phosphoglyceride kinase structural aspects and functions, with special emphasis on the enzyme from Kinetoplastea. Open biol.2020Nov;10 (11) 200302.Doi:10.1098/rsob.200302.PubMed PMID: 33234025.). PGK crystal structure of pig, horse and yeast has been analyzed, it is a monomer enzyme, it is composed of two spherical structures, C-terminal peptide fragment is very important in maintaining stability of structure (Mas MT, result ZE. Structure-function relationships in 3-phosphoglycerate kinase: role of the carboxy-terminal peptide protein.1988; 4 (1): 56-62.doi:10.1002/prot.340040108.PubMed PMID:3054872.; shang Yuanbing, qi Jingjing, wang Yu, etc. clone expression of chicken synovial mycoplasma phosphoglycerate kinase and enzymatic Activity determination [ J ]]Chinese animal infectious diseases school report 2021,29 (03): 71-79.). PGK of haemophilus mycoplasmaRelated studies have only prokaryotic expression of the canine haemophilus PGK protein and immunogenicity (Song Qiqi, qian Limin, su Miao, in dawn, qin Shunyi, yue. Prokaryotic expression, purification of canine eperythrozoon PGK and preparation of polyclonal antibodies [ J)]Proc. Tianjin agricultural college, 2018,25 (03): 42-46.). However, the study on the prokaryotic expression of the swine mycoplasma hyopneumoniae PGK protein is not reported.

Therefore, the establishment of the indirect ELISA detection method for the mycoplasma hyopneumoniae antibody based on the new mycoplasma hyopneumoniae PGK protein has important significance for clinical diagnosis, epidemiological research and immunodetection of mycoplasma hyopneumoniae.

Disclosure of Invention

The first aim of the invention is to provide a new recombinant PGK protein of haemophilus parasuis (called recombinant Mh-PGK protein for short) for detecting the antibody level and epidemiological monitoring of haemophilus parasuis of swine, and evaluating the role of the disease in the epidemic of haemophilus parasuis of swine.

The second object of the invention is to provide the application of the recombinant Mh-PGK protein in detecting mycoplasma hyopneumoniae and/or in detecting mycoplasma hyopneumoniae antibodies.

The third aim of the invention is to provide an indirect ELISA detection kit for the mycoplasma hyopneumoniae antibody.

The fourth object of the invention is to provide an indirect ELISA detection method for the mycoplasma hyopneumoniae antibody.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a novel recombinant PGK protein of haemophilus parasuis, which is simply called recombinant Mh-PGK protein, and the amino acid sequence of the recombinant Mh-PGK protein is shown as SEQ ID NO. 1.

The recombinant Mh-PGK protein is prepared by the following method:

constructing a recombinant plasmid by using the optimized Mh-PGK gene shown in the 7 th-1239 th position of SEQ ID NO.2, and expressing the recombinant Mh-PGK protein by an expression system; wherein, the optimized Mh-PGK gene carries out codon optimization on the Mh-PGK gene shown in SEQ ID NO.3 (the Mh-PGK protein with the coding amino acid sequence shown in 22 th to 431 th positions of SEQ ID NO. 1), and 3 TGAs are optimized to be obtained by the codon TGG which can code tryptophan in escherichia coli and partial codons are optimized to be escherichia coli preferential codons.

In a specific embodiment of the invention, the recombinant plasmid is pET28-mh-pgk. The pET28-Mh-PGK is a recombinant plasmid obtained by replacing a DNA fragment between NdeI enzyme and XhoI enzyme recognition sequences of pET28 a (+) plasmid with optimized Mh-PGK genes shown in SEQ ID NO.2 positions 7-1239 in a sequence table, and the recombinant plasmid can express recombinant Mh-PGK proteins shown in SEQ ID NO.1 in the sequence table; wherein, the 1 st to 21 st positions of SEQ ID NO.1 in the sequence table are the amino acid sequences encoded by DNA sequences on pET-28a (+) plasmid, and the 22 nd to 431 st positions of SEQ ID NO.1 are the Mh-PGK protein sequences.

Further, the expression system is an E.coli expression system.

In a specific embodiment of the invention, the expression is induced by transforming pET28-Mh-PGK into E.coli BL21 (DE 3) to obtain recombinant Mh-PGK protein.

In a second aspect, the invention provides the use of a recombinant Mh-PGK protein as described above in any one of the following:

1) Detecting whether serum of an animal to be detected contains a swine haemophilus antibody;

2) The application of the kit in preparing a mycoplasma hyopneumoniae antibody product for detecting whether the serum of an animal to be detected contains swine blood;

3) Detecting whether an animal to be detected is infected or infected with mycoplasma hyopneumoniae;

4) The application of the kit in preparing the product for detecting whether the animal to be detected is infected or not or is infected by the mycoplasma hyopneumoniae.

The Mycoplasma hyopneumoniae is Mycoplasma suis, mycoplasma parvum and/or Mycoplasma haemosuis.

In a third aspect, the invention provides an indirect ELISA detection kit for a mycoplasma hyopneumoniae antibody, which comprises an ELISA plate taking the recombinant Mh-PGK protein as a coating antigen.

Further, the coating concentration of the recombinant Mh-PGK protein was 0.31 μg/mL.

Further, the kit also comprises one or more of coating liquid, sealing liquid, washing liquid, M.haemas positive serum, M.haemas negative serum, enzyme-labeled secondary antibody, substrate chromogenic liquid and stop solution.

Further, the coating liquid was a carbonate buffer (1.59 g Na ₂ CO ₃ And 2.93g NaHCO ₃ Deionized water to 1000 mL), PBS (pH 7.4.01M (8.0 g NaCl, 0.2g KCl, 2.86g Na) ₂ HPO ₄ ·12H ₂ O and 0.27g KH ₂ PO ₄ Adding deionized water to 1000 mL) or Tris-HCl (1M Tris-HCl is obtained by adding about 80mL deionized water from 12.11g Tris base, adding concentrated HCl to adjust the pH value to 8.0 after dissolution, and fixing the volume to 100 mL) with pH of 8.0.01M; 1mL of 1M Tris-HCl is taken and 99mL of deionized water is added to obtain Tris-HCl with pH of 8.0.01M; tris-HCl, pH8.00.01M, is preferred.

Further, the blocking solution was 2.5% nonfat milk powder (100 mLPBST added with 2.5g nonfat milk powder), 5% nonfat milk powder (100 mLPBST added with 5g nonfat milk powder), 7.5% nonfat milk powder (100 mLPBST added with 7.5g nonfat milk powder), 1% BSA (100 mLPBST added with 1g BSA), 2% BSA (100 mLPBST added with 2g BSA), or 3% BSA (100 mLPBST added with 3g BSA); preferably 5% skimmed milk powder.

Further, the washing solution was PBST (8.0 g NaCl, 0.2g KCl, 2.86g Na) ₂ HPO ₄ ·12H ₂ O、0.27g KH ₂ PO ₄ And 0.5mL Tween-20 to 1000 mL).

Further, the second enzyme-labeled antibody was horseradish peroxidase-labeled goat anti-pig IgG (IgG-HRP) (available from KPL).

Further, the dilution of the second enzyme-labeled antibody was 1:12500.

Further, the substrate color developing solution was TMB substrate color developing solution (available from Shanghai Bioengineering Co., ltd., product number E661007).

Further, the termination liquid was 0.5. 0.5M H ₂ SO ₄ (16.4 mL of concentrated sulfuric acid was slowly added dropwise to 400mL of deionized water, and the volume was made up to 500 mL).

The application of the indirect ELISA detection kit for the mycoplasma hyopneumoniae antibody in the detection of the mycoplasma hyopneumoniae antibody is also within the protection scope of the invention.

In a fourth aspect, the invention provides an indirect ELISA detection method for a mycoplasma hyopneumoniae antibody, which comprises the following steps:

1) Coating antigen: coating the ELISA plate with the coating solution by taking the recombinant Mh-PGK protein as a coating antigen, and washing with a detergent;

2) Closing: adding a sealing liquid, sealing and washing with a detergent;

3) Adding serum: adding serum of an animal to be tested, reacting, and washing with a detergent;

4) Adding enzyme-labeled secondary antibodies: adding enzyme-labeled secondary antibody, reacting, and washing with a detergent;

5) TMB color development: adding a substrate color development solution to react;

6) Reaction termination: adding a stopping solution to stop the reaction;

7) Detecting OD ₄₅₀ Value: measurement of the OD of the serum of each test animal Using an enzyme-labeled Instrument ₄₅₀ A value;

8) Interpretation of the results: the criterion is OD ₄₅₀ Value of>0.505 person is judged to be positive (namely, the serum of the animal to be tested is positive serum of the mycoplasma hyopneumoniae, that is, the serum of the animal to be tested contains the mycoplasma hyopneumoniae antibody or the animal to be tested is infected or has been infected with the mycoplasma hyopneumoniae), OD ₄₅₀ Value of<0.397 person judges as negative (namely, the serum of the animal to be tested is the serum negative for mycoplasma hyopneumoniae, that is, the serum of the animal to be tested does not contain mycoplasma hyopneumoniae antibodies or the level of mycoplasma hyopneumoniae antibodies is lower than the detection lower limit), and OD is not less than 0.397 ₄₅₀ The patient with the value less than or equal to 0.505 is judged to be suspicious (namely, the serum of the animal to be tested is suspected to be the positive serum of the mycoplasma hyopneumoniae, that is, the serum of the animal to be tested is suspected to contain the mycoplasma hyopneumoniae antibody or the animal to be tested is suspected to be infected or infected by the mycoplasma hyopneumoniae).

In the invention, through optimizing coating liquid, coating concentration, sealing liquid, sealing time, serum dilution, serum reaction time, enzyme-labeled secondary antibody dilution and enzyme-labeled secondary antibody action time, tris-HCl with the optimal coating liquid of pH 8.0.01M is obtained, the optimal coating concentration is 0.31 mu g/mL, the optimal sealing liquid is 5% skim milk powder, the optimal sealing time is 1h, and the optimal serum dilution is 1:8, the optimal reaction time of the serum is 1h, and the optimal dilution of the enzyme-labeled secondary antibody is 1:12500 and the optimal reaction time of the enzyme-labeled secondary antibody is 1h.

In a preferred embodiment of the invention, the method for indirectly detecting the mycoplasma hyopneumoniae antibody by ELISA comprises the following steps:

1) Coating antigen: the recombinant Mh-PGK protein is taken as a coating antigen, and the recombinant Mh-PGK protein is incubated at 37 ℃ for 1h and then placed at 4 ℃ for overnight to coat an ELISA plate with 100 uL/hole at a coating concentration of 0.31 mu g/mL by using Tris-HCl with pH of 8.0.01M, and the ELISA plate is washed 3 times by using PBST for 3min each time;

2) Closing: 250 μl of 5% skimmed milk powder was added, sealed at 37deg.C for 1h, and washed 1 time with PBST;

3) Adding serum: adding serum of an animal to be tested with the dilution of 1:8, reacting for 1h at 37 ℃ at 100 mu L/hole, and washing with PBST for 3 times;

4) Adding enzyme-labeled secondary antibodies: adding horseradish peroxidase-labeled goat anti-pig IgG with a dilution of 1:12500, reacting at 37 ℃ for 1h, and washing with PBST for 3 times;

5) TMB color development: adding TMB substrate, 100 mu L/hole, and reacting for 8min;

6) Reaction termination: adding 0.5. 0.5M H ₂ SO ₄ 50. Mu.L/well, terminate the reaction;

7) Detection OD450 value: measuring the OD450 value of the serum of each animal to be tested by using an enzyme-labeled instrument;

In the present invention, the Mycoplasma hyopneumoniae is an early identified Mycoplasma hyopneumoniae (m.suis), mycoplasma minutissimum (Mycoplasma parvum, m.parvum) and/or Mycoplasma hyopneumoniae (Mycoplasma haemosuis, m.haemauis). The animal to be tested is a pig.

The beneficial effects of the invention are as follows:

the invention establishes an indirect ELISA detection method for the Mycoplasma hyopneumoniae antibody based on the recombinant Mh-PGK protein, which is used for detecting the level of the Mycoplasma hyopneumoniae antibody, has no cross reaction with part of other pig disease serum, has good specificity and repeatability, and provides an effective means for clinical diagnosis, epidemiological research and immunodetection of Mycoplasma suis, mycoplasma parvum and Mycoplasma haemosuis.

Drawings

The following describes the embodiments of the present invention in further detail with reference to the drawings.

FIG. 1 shows the expression of recombinant Mh-PGK protein in E.coli BL21 by SDS-PAGE analysis; wherein M1 and M2 represent Protein markers; 1 represents the expression of recombinant proteins in recombinant E.coli pET28/BL21 (DE 3); 2 represents the expression of the recombinant protein in the uninduced recombinant E.coli pET28-mh-pgk/BL21 (DE 3); 3 represents the expression of recombinant proteins in IPTG-induced recombinant E.coli pET28-mh-pgk/BL21 (DE 3); 4 represents SDS-PAGE detection result of the purified recombinant Mh-PGK protein.

FIG. 2 shows the immunoreactivity of recombinant Mh-PGK protein by Western-blot analysis; wherein M represents a pre-dyed protein Marker;1 represents Western Blot reaction of mouse anti-recombinant Mh-PGK protein serum and recombinant Mh-PGK protein, and the arrow indicates reaction band; 2 represents the Western Blot reaction of mouse negative serum and recombinant Mh-PGK protein.

Detailed Description

In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments and the accompanying drawings. Like parts in the drawings are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.

Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified; wherein the main materials, reagents and instrument sources are as follows:

coli Transetta (DE 3) competent cells were purchased from Beijing all gold biotechnology Co., ltd;

pET-28a (+) plasmid was purchased from Shanghai bioengineering Co., ltd and was assigned the designation B540183;

the M.haemauis negative serum is imported pig serum, is provided by the inspection and quarantine technical center of the Zhejiang province, and is negative through PCR detection, and the total weight is 135 parts.

The M.haemauis positive serum is serum collected from sows identified as positive by a qPCR method, and 25 parts of serum are taken in total;

three mycoplasma hyopneumoniae (M.suis, M.parvum, M.haemasuis) are all identified as positive pathogens by a PCR method, and then the positive serum of the antibodies is identified by a whole-bacterial antigen ELISA and stored;

after the positive serum of the toxoplasma gondii is identified as positive by the laboratory through a PCR method, the toxoplasma gondii IHA diagnostic kit of the animal research institute of Lanzhou is used for identifying positive antibodies and then is stored; the foot-and-mouth disease positive serum is stored after the foot-and-mouth disease indirect hemagglutination antibody kit of the Lanzhou veterinary research institute identifies the positive of the antibody;

positive serum of porcine circovirus disease, swine fever, porcine pseudorabies, porcine reproductive and respiratory syndrome is obtained from a pig disease research laboratory of the national institute of livestock and poultry in Zhejiang province, and is detected, identified and stored by using an ELISA detection kit corresponding to IDEXX company;

Peroxidase-labeled goat anti-porcine IgG-HRP was purchased from KPL company;

affinity chromatography nickel beads Ni-argros were purchased from Invitrogen company;

TMB substrate color development was purchased from Shanghai Biotechnology engineering services Co., ltd, cat# E661007;

the enzyme-labeled instrument is Spectra Max M5, which is a product of Molecular Devices company in the United states;

the protein electrophoresis apparatus is a product of Bio-Rad company.

Example 1 preparation of an Indirect ELISA antigen against Mycoplasma hyopneumoniae antibody and detection of antigenicity

1. Preparation of antigens

1) Optimization of mh-pgk genes

According to the preference of escherichia coli codons, the Mh-PGK gene (the nucleotide sequence of which is shown as SEQ ID NO.3 and consists of 1233 nucleotides, the new mycoplasma hyopheles PGK protein (simply referred to as Mh-PGK protein) of which the amino acid sequence is shown as SEQ ID NO.1 and 22-431 positions of which is amplified to obtain a new mycoplasma hyopheles (M.haemauis) ZJ1102 strain is subjected to codon optimization, 3 TGAs (due to the specificity of a gene coding system of mycoplasma, TGA codes for tryptophan non-stop codons) are optimized to obtain a codon TGG capable of coding tryptophan in escherichia coli and a part of codons are optimized to the escherichia coli preference codons, and the optimized Mh-PGK gene (the nucleotide sequence of which is shown as SEQ ID NO.2 and consists of 1233 nucleotides and the amino acid sequence of the coded Mh-PGK protein is unchanged) is synthesized by the biological technology of Suzhou Jin Weizhi.

2) Inducible expression of recombinant proteins

NdeI cleavage site (5 'catatg3') and XhoI cleavage site (5 'ctcgag 3') were added to the 5 'end and the 3' end of the optimized mh-pgk gene (the sequence was synthesized by Souzhou Jin Weizhi Biotechnology Co., ltd.) as shown in SEQ ID NO.2, and double cleavage was performed with NdeI and XhoI, and simultaneously double cleavage of pET-28a (+) plasmid with NdeI and XhoI was performed. Connecting the optimized mh-pgk gene after enzyme digestion and the pET-28a (+) plasmid by using DNA ligase to construct a recombinant plasmid pET28-mh-pgk, converting the recombinant plasmid pET28-mh-pgk into escherichia coli Top10, extracting the plasmid, and determining that the insertion sequence is correct by sequencing and identification of Shanghai bioengineering company. The pET28-Mh-PGK is a recombinant plasmid obtained by replacing a DNA fragment between NdeI enzyme and XhoI enzyme recognition sequences of pET-28a (+) plasmid with optimized Mh-PGK genes shown in SEQ ID NO.2 at positions 7-1239 in a sequence table, and the recombinant plasmid can express fusion proteins shown in SEQ ID NO.1 in the sequence table, namely new mycoplasma hyopneumoniae recombinant PGK protein, which is simply called recombinant Mh-PGK protein; wherein, the 1 st to 21 st positions of SEQ ID NO.1 in the sequence table are the amino acid sequences encoded by DNA sequences on pET-28a (+) plasmid, and the 22 nd to 431 st positions of SEQ ID NO.1 are the Mh-PGK protein sequences.

The recombinant plasmid pET28-mh-pgk with the correct sequence is transformed into escherichia coli BL21 (DE 3) to obtain recombinant escherichia coli pET28-mh-pgk/BL21 (DE 3). Recombinant E.coli pET28-mh-pgk/BL21 (DE 3) was expressed by induction with 0.8mM IPTG (37 ℃ C., 4 h), and bacterial solutions were collected before induction and after induction for 4h, respectively. And replacing pET28-mh-pgk with pET28a, repeating the test to obtain recombinant escherichia coli pET28/BL21 (DE 3), and collecting bacterial liquid after 4h induction. Three parts of bacterial liquid are respectively taken for 100 mu L, 5min at the speed of 5000 rpm at 4 ℃ and are centrifuged to precipitate bacterial bodies, the bacterial bodies are resuspended in 40 mu L of double distilled water, 10 mu L of 5 multiplied by SDS loading buffer are added for boiling, and the bacterial liquid is respectively used for SDS-PAGE to detect the expression of recombinant proteins in the recombinant escherichia coli pET28-mh-pgk/BL21 (DE 3) which are not induced and are induced by IPTG and the expression of recombinant proteins in the recombinant escherichia coli pET28/BL21 (DE 3). As a result, as shown in FIG. 1, recombinant E.coli pET28-Mh-PGK/BL21 (DE 3) after IPTG induction showed an expression protein band at about 45kD, the size of which was consistent with the expectation, indicating that recombinant Mh-PGK protein was expressed; the recombinant E.coli pET28/BL21 (DE 3) and the uninduced recombinant E.coli pET28-mh-pgk/BL21 (DE 3) have no expression of the corresponding proteins.

3) Purification of recombinant proteins

The bacterial solution of recombinant E.coli pET28-mh-pgk/BL21 (DE 3) induced by IPTG was centrifuged, and the pellet was resuspended in PBS and sonicated. Centrifuging again for 10min, washing the precipitate with PBS (pH 7.4) for 2 times, adding 19.7mL of buffer A (Tris-HCl 6.06g,EDTA 0.186g,NaCl 2.92g, glycerol 50mL, sterile deionized water to 1L) and 0.3mL of 20% Sarcosyl (SKL) stock solution, suspending the precipitate for standing overnight, centrifuging for 10min, discarding the precipitate, taking the supernatant, adding 20% PEG4000 (2 g PEG4000 and sterile deionized water to 10 mL) to a final concentration of 0.2%, adding 50mmol/L of oxidized glutathione (0.153 g oxidized glutathione deionized water to 5 mL) to a final concentration of 1mmol/L, adding 100mmol/L of reduced glutathione (0.153 g reduced glutathione deionized water to 5 mL), standing for 30min-2h, and dialyzing with 0.01MPBS (pH 7.4) for 2 days to obtain an extracted inclusion body;

about 20mL of inclusion bodies were taken, and 5mL of Ni-Agarose equilibrated with pH7.4 Binding buffer (purchased from Shanghai Biotechnology, cat. No. C600303) was added thereto and bound at room temperature for 30 minutes. During this period, the mixture was shaken 3-5 times at 4000rpm for 5min, the supernatant was discarded, and the pellet was washed 4 times with 20mM, 30mM, 40mM, and 50mM imidazole Binding buffer. 250mM Elution buffer 5mL/time, eluting for 3 times, collecting and combining the eluted Elutation proteins, dialyzing for 24 hours by PBS (phosphate buffer solution) pH7.4, concentrating to half the volume, centrifuging at 10000rpm for 10min, and collecting the supernatant to obtain the purified recombinant Mh-PGK protein. Measuring the concentration (the concentration of the purified recombinant Mh-PGK protein is 730 mu g/mL) according to a specification by using a Shanghai worker BAC protein quantitative detection kit, and then sub-packaging for freezing at-20 ℃ for later use; SDS-PAGE detection is carried out on the purified recombinant Mh-PGK protein, the result is shown in figure 1, the result shows that a single band only appears at about 45kD, the purification recovery result is good, and the obtained recombinant Mh-PGK protein has an amino acid sequence shown as SEQ ID NO. 1.

2. Antigenicity detection

1) Preparation of mouse anti-recombinant Mh-PGK protein serum:

BALB/c mice were selected and female 5 were prepared for antibodies according to the following procedure:

dissolving purified recombinant Mh-PGK protein (i.e. antigen) in PBS, uniformly mixing with equal volume Freund's complete adjuvant by double pushing method, and performing multi-point injection on BALB/C mice via back subcutaneous and leg muscles at a dose of 40 μg/mouse; and (2) avoiding: 14d, uniformly mixing the antigen with the incomplete Freund's adjuvant in equal amount, and immunizing at a dose of 50 mug/dose; after 28d, the equivalent antigen and the incomplete Freund's adjuvant are uniformly mixed and immunized, the dosage is 50 mug/dose, and the titer is measured by ELISA; after the last immunization, serum titer can be measured by tail-breaking blood sampling, and if the expected purpose is achieved, antisera can be collected by bleeding. And (5) taking blood from the mice with high titers, and separating serum to obtain the anti-recombinant Mh-PGK protein serum of the mice.

2) Western-blot: the purified recombinant Mh-PGK protein is adjusted to have the concentration of 365 mu g/mL, 10 mu L/hole is loaded, SDS-PAGE electrophoresis is carried out, the adhesive tape is cut to a proper size after finishing, electrotransfer solution is balanced, 5min multiplied by 3 times, nitrocellulose (NC) membrane and filter paper are cut in advance to be the same as the adhesive tape in size, and the adhesive tape is immersed into the electrotransfer solution for balancing for 10min.

Electric conversion: placing the filter paper according to the flat positive electrode, placing the filter paper, placing the NC film, the gel, the filter paper and the negative electrode in sequence, aligning the filter paper, the NC film and the gel, and switching on current according to the gel area of 0.65mA-1.0mA/cm < 2 >, and electrically transferring for 2h.

Dyeing: and (3) turning off the power supply, lifting off all layers one by one, transferring the gel into ponceau staining solution, staining until protein bands appear on the membrane, and taking a picture. The nitrocellulose filter was rinsed with deionized water to a protein-free band.

Closing: the transferred nitrocellulose filters were blocked with 1% BSA in PBST for 2h at room temperature, and the blocking solution was discarded.

Binding of antibody to target protein: PBST diluted primary antibody, mouse anti-recombinant Mh-PGK protein serum as the primary antibody was diluted 1:500, and nitrocellulose membrane transformed with Mh-PGK protein was laid flat on a shaker for 4℃overnight, and nitrocellulose filter was washed with PBST for 5min X3 times.

Secondary antibody binding: PBST was diluted 1:3000 horseradish peroxidase (HRP) -labeled rabbit anti-mouse secondary antibodies, and incubated with nitrocellulose membrane on a shaker at room temperature for 2h. The nitrocellulose filters were washed with PBST 5min X3 times. Adding a chemiluminescent reagent for color development: the nitrocellulose filter was placed in 1mL of ECL luminophore (Shanghai Biotechnology, cat. C510043), and photographed by exposure.

The Western-blot was repeated by replacing the mouse anti-recombinant Mh-PGK protein serum with mouse negative serum.

The immune reactivity of the mouse anti-recombinant Mh-PGK protein serum and the recombinant Mh-PGK protein is analyzed, the result is shown in figure 2, and the result shows that the recombinant Mh-PGK protein can be combined with the mouse anti-recombinant Mh-PGK protein serum, and has no combination reaction with the mouse negative serum, so that the protein has good antigenicity.

Example 2 establishment of indirect ELISA detection method for swine haemophilus parasuis antibody and condition optimization

1. Determination of optimal coating concentration and optimal serum dilution

1) Coating antigen: the recombinant Mh-PGK protein was coated with a coating solution (pH 9.60.01M carbonate buffer, 1.59g Na by square titration ₂ CO ₃ And 2.93g NaHCO ₃ Adding deionized water to 1000mL, performing multiple dilution (antigen concentrations of 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078 μg/mL, respectively), coating 1 row of each concentration, coating 7 concentrations, blank control of the last row, incubating at 37deg.C for 1 hr, coating ELISA plate at 4deg.C overnight, taking out washing solution (PBST, 8.0g NaCl, 0.2g KCl, 2.86g Na) ₂ HPO ₄ ·12H ₂ O、0.27g KH ₂ PO ₄ And 0.5mL Tween-20 to 1000 mL), 3 times each for 3min;

2) Closing: adding 250 μl of blocking solution (obtained by adding 5g of skimmed milk powder into 100mL of PBST), blocking at 37deg.C for 1h, and washing with PBST for 1 time;

3) Adding serum: performing transverse multiple dilution (dilution ratios are 1:4,1:8,1:16,1:32,1:64,1:128 respectively) on the first 6 columns and the last 6 columns of the ELISA plate respectively by using M.haemauis positive serum (one part is randomly selected from serum collected from sows identified as positive by the qPCR method) and M.haemauis negative serum (one part is randomly selected from the 135 parts of M.haemauis negative serum subjected to PCR detection), and performing 7-hole repetition longitudinally for each dilution, wherein 100 mu L/hole, and PBST is washed 3 times after reaction for 1h at 37 ℃;

4) Adding enzyme-labeled secondary antibodies: adding horseradish peroxidase labeled goat anti-pig IgG (IgG-HRP) (diluted with blocking solution) at a dilution of 1:10000, reacting at 37deg.C for 1h, and washing with PBST for 3 times;

5) TMB color development: adding a substrate color development liquid (TMB substrate color development liquid), and reacting for 8min at room temperature with 100 mu L/hole;

6) Reaction termination: adding stop solution (0.5M H) ₂ SO ₄ Slowly dripping 16.4mL of concentrated sulfuric acid into 400mL of deionized water, adding the deionized water to 500mL, and stopping the reaction at 50 mu L/hole;

7) Detection OD450 value: measuring the OD values of the M.haemasuis positive serum and the M.haemasuis negative serum respectively (namely positive serum OD) at the wavelength of 450nm by using an enzyme-labeled instrument ₄₅₀ Value and negative serum OD ₄₅₀ Value), and calculates the P/N value. By positive serum OD ₄₅₀ The value (P) starts to vary greatly, while the corresponding negative serum OD ₄₅₀ The value (N) is substantially unchanged for the corresponding antigen concentration and serum dilution as the antigen optimal coating concentration and serum optimal dilution.

The recombinant Mh-PGK protein matrix titration test result shows that the positive serum OD when the antigen concentration is between 0.500 mug/hole and 0.031 mug/hole ₄₅₀ The value change is not obvious, and the OD of positive serum is between 0.031 mug/well and 0.015 mug/well dilution ₄₅₀ The value change is greatly reduced. Thus, positive serum OD was selected ₄₅₀ The recombinant Mh-PGK protein was coated at an antigen concentration at which a significant decrease in the value began to occur (0.031 μg/well, i.e., 0.31 μg/mL) as the optimal coating concentration for the antigen.

Serum positive serum OD between 1:8-1:128 dilutions ₄₅₀ The value is greatly reduced, and the positive serum OD is obtained when the antigen is diluted at 1:8 ₄₅₀ Value and negative serum OD ₄₅₀ The ratio of the values (P/N) is the largest, negative serum OD ₄₅₀ The value is substantially unchanged. Thus, 1:8 was chosen as the optimal dilution of serum.

2. Optimal coating liquid selection

According to steps 1) to 7), the coating solution pH 9.6.0.01M carbonate buffer was replaced with PBS pH 7.4.0.01M (8.0 g NaCl, 0.2g KCl, 2.86g Na) ₂ HPO ₄ ·12H ₂ O and 0.27g KH ₂ PO ₄ Deionized water was added to 1000mL to obtain), tris-HCl (pH 8.0.01M Tris-HCl (1M Tris-HCl is prepared by adding about 80mL deionized water from 12.11g Tris base), adding concentrated HCl to adjust pH to 8.0 after dissolution, and constant volume to 100mL. 1mL of 1M Tris-HCl is taken and added with 99mL of deionized water to obtain Tris-HCl with pH of 8.0.01M and carbonate buffer with pH of 9.6.0.1M to coat the ELISA plate, the influence of each coating liquid on the P/N value is detected under the optimal condition, and other steps are unchanged. 5 parts of M.haemauis positive serum (5 parts of the serum collected from the sow identified as positive by the qPCR method) and 5 parts of each coating liquid are preparedM.haemauis negative serum (5 randomly selected from the 135 parts of M.haemauis negative serum negative by PCR) test, 2 wells each were repeated, and OD was measured ₄₅₀ Value, i.e. positive serum OD ₄₅₀ Value (P) and negative serum OD ₄₅₀ And (3) calculating P average value, N average value and P/N value of different coating liquids, and selecting the coating liquid with the maximum P/N value as the optimal coating liquid.

The P-means, N-means and P/N values of the different coating liquids are shown in Table 1, and it can be seen from the P/N values that the P/N value is highest when the coating liquid is Tris-HCl of pH 8.0.01M, i.e., the coating effect is the best, and therefore, the best coating liquid is Tris-HCl of pH 8.0.01M.

TABLE 1 optimization results of optimal coating solution for indirect ELISA detection method

3. Selection of optimal blocking liquid and blocking time

According to steps 1) to 7), the blocking solution was blocked by replacing 5% skimmed milk powder with 2.5% skimmed milk powder (100 mLPBST to which 2.5g skimmed milk powder was added), 5% skimmed milk powder (100 mLPBST to which 5g skimmed milk powder was added), 7.5% skimmed milk powder (100 mLPBST to which 7.5g skimmed milk powder was added), 1% BSA (100 mLPBST to which 1g BSA was added), 2% BSA (100 mLPBST to which 2g BSA was added), or 3% BSA (100 mLPBST to which 3g BSA was added), respectively, and the effect of each blocking solution on the P/N value was examined under the above-mentioned optimal conditions, and the other steps were unchanged. 5 parts of M.haemasuis positive serum (5 parts randomly selected from the serum collected from sow identified as positive by the qPCR method) and 5 parts of M.haemasuis negative serum (5 parts randomly selected from the 135 parts of M.haemasuis negative serum detected as negative by PCR) were tested for each blocking solution, 2 wells were repeated, and OD was measured ₄₅₀ Value, i.e. positive serum OD ₄₅₀ Value (P) and negative serum OD ₄₅₀ And (3) calculating P average value, N average value and P/N value of different sealing solutions, and selecting the sealing solution with the maximum P/N value as the optimal sealing solution.

The P-means, N-means and P/N values of the different blocking solutions are shown in Table 2, from which it can be seen that the P/N value is highest when the blocking solution is 5% nonfat dry milk, and therefore, the optimal blocking solution is 5% nonfat dry milk.

TABLE 2 optimization results of optimal blocking solution for indirect ELISA detection method

According to steps 1) -7), the closing time is replaced by 0.5h, 1h and 1.5h from 1h, the influence of different closing times on the P/N value is detected under the optimal condition, and other steps are unchanged. Each blocking time was tested for 5 parts of M.haemauis positive serum (5 parts randomly selected from the sera collected from sows identified as positive by the qPCR method) and 5 parts of M.haemauis negative serum (5 parts randomly selected from the 135 parts of M.haemauis negative serum that were negative by PCR), 2 wells were repeated to determine OD ₄₅₀ Value, i.e. positive serum OD ₄₅₀ Value (P) and negative serum OD ₄₅₀ And (3) calculating P mean value, N mean value and P/N value of different closing time, and selecting the closing time with the maximum P/N value as the optimal closing time.

The P-means, N-means and P/N values for the different blocking times are shown in Table 3, from which it can be seen that the P/N value is highest when 5% of the skimmed milk powder is blocked for 1h, and thus the optimal blocking time is 1h.

TABLE 3 optimization of blocking time of Indirect ELISA detection method

4. Serum optimal reaction time

According to steps 1) -7), the serum reaction time is replaced by 0.5h, 1h and 1.5h from 1h, the influence of each serum reaction time on the P/N value is detected under the optimal condition, and other steps are unchanged. Each serum reaction time was tested for 5 parts of M.haemauis positive serum (5 parts randomly selected from the sera collected from sows identified as positive by the qPCR method) and 5 parts of M.haemauis negative serum (5 parts randomly selected from the 135 parts of M.haemauis negative serum that were negative by PCR), 2 wells were repeated to determine OD ₄₅₀ Value, i.e. positive serum OD ₄₅₀ Value (P) and negative serum OD ₄₅₀ And (3) calculating P average value, N average value and P/N value of different serum reaction time, and selecting the serum reaction time with the maximum P/N value as the serum optimal reaction time.

The P-means, N-means and P/N values of the different serum reaction times are shown in Table 4, and it can be seen from the P/N values that the P/N value is maximum when the serum reaction time is 1h, and thus, the optimal serum reaction time is determined to be 1h.

TABLE 4 optimization of serum reaction time results for indirect ELISA detection methods

5. Determination of optimal dilution of second enzyme-labeled antibody and optimal reaction time of second enzyme-labeled antibody

According to steps 1) -7), the dilutions of horseradish peroxidase-labeled goat anti-pig IgG (IgG-HRP) were replaced by 1:7500, 1:10000, 1:12500 and 100. Mu.L/well from 1:10000, and the effect of each enzyme-labeled secondary antibody dilution on the P/N value was examined under the optimal conditions, and the other steps were unchanged. 5 parts of M.haemasuis positive serum (5 parts randomly selected from serum collected from sows identified as positive by the qPCR method) and 5 parts of M.haemasuis were tested for each dilution of the second enzyme-labeled antibody, 2 wells were repeated, and OD was measured ₄₅₀ Value, i.e. positive serum OD ₄₅₀ Value (P) and negative serum OD ₄₅₀ And (3) calculating P average value, N average value and P/N value of different enzyme-labeled secondary antibody dilutions, and selecting the enzyme-labeled secondary antibody dilution with the maximum P/N value as the optimal enzyme-labeled dilution.

The P-means, N-means and P/N values of the different secondary antibody dilutions are shown in Table 5, from which it can be seen that the P/N value is highest when the secondary antibody dilution is 1:12500, and therefore, the optimal secondary antibody dilution is determined to be 1:12500.

TABLE 5 optimization results of enzyme-labeled secondary antibody dilution for indirect ELISA detection method

According to steps 1) -7), the action time of horseradish peroxidase-labeled goat anti-pig IgG (IgG-HRP) is replaced by 30min, 60min and 90min from 1h, the influence of the reaction time of each enzyme-labeled secondary antibody on the P/N value is detected under the optimal condition, and other steps are unchanged. 5 parts of M.haemauis positive serum (5 parts randomly selected from the serum collected from the sow identified as positive by the qPCR method) and 5 parts of M.haemauis negative serum (5 parts randomly selected from the 135 parts of M.haemauis negative serum detected as negative by PCR) were tested for each enzyme-labeled secondary antibody reaction time, 2 wells were repeated for each, and OD was measured ₄₅₀ Value, i.e. positive serum OD ₄₅₀ Value (P) and negative serum OD ₄₅₀ And (3) calculating P average value, N average value and P/N value of the reaction time of different enzyme-labeled secondary antibodies, and selecting the enzyme-labeled secondary antibody reaction time with the maximum P/N value as the enzyme-labeled secondary antibody optimal reaction time.

The P average value, N average value and P/N value of the reaction time of the different enzyme-labeled secondary antibodies are shown in Table 6, and the P/N value can be seen that the P/N value is highest when the reaction time of the enzyme-labeled secondary antibodies is 60min, so that the optimal reaction time of the optimal enzyme-labeled secondary antibodies is determined to be 60min.

TABLE 6 optimization results of enzyme-labeled secondary antibody reaction time for indirect ELISA detection method

6. An indirect ELISA detection method for swine haemophilus influenzae antibody comprises the following steps:

according to the optimization test in the step 1-5, the method for determining the indirect ELISA detection method of the optimized mycoplasma hyopneumoniae antibody comprises the following steps:

1) Coating antigen: recombinant Mh-PGK protein is taken as a coating antigen, and the coated ELISA plate is coated with PBST for 3 times and 3min each time at a coating concentration of 0.31 mug/mL and 100 uL/hole by using a coating solution (Tris-HCl with the pH of 8.0.01M), after incubation for 1h at 37 ℃;

2) Closing: adding 250 μl of blocking solution (5% skimmed milk powder), blocking at 37deg.C for 1h, and PBST washing for 1 time;

4) Adding enzyme-labeled secondary antibodies: adding horseradish peroxidase-labeled sheep anti-pig IgG (IgG-HRP) (diluted with blocking solution) at a dilution of 1:12500, reacting at 37deg.C for 1h, and washing with PBST for 3 times;

6) Reaction termination: adding stop solution (0.5M H) ₂ SO ₄ ) 50. Mu.L/well, terminate the reaction;

7) Detection OD450 value: the OD value of the serum of each animal to be tested was measured at a wavelength of 450nm using an enzyme-labeled instrument.

7. Determination of positive critical value of swine haemophilus influenzae antibody indirect ELISA detection method

135 parts of M.haemasuis negative serum sample are screened by PCR detection, indirect ELISA detection is carried out according to the swine haemophilus parasuis antibody indirect ELISA detection method optimized in the step 6, and the OD of the M.haemasuis negative serum is measured ₄₅₀ Value, calculate average

And Standard Deviation (SD) of less than +.>

The value is negative, between->

And->

The value between them is judged as suspicious, greater than +.>

And judging positive. Calculated mean +.>

Since the standard deviation SD value was 0.181 and 0.107, the judgment standard was OD ₄₅₀ Value of>0.505 person is judged to be positive (namely, the serum of the animal to be tested is positive serum of the mycoplasma hyopneumoniae, that is, the serum of the animal to be tested contains the mycoplasma hyopneumoniae antibody or the animal to be tested is infected or has been infected with the mycoplasma hyopneumoniae), OD ₄₅₀ Value of<0.397 person judges as negative (namely, the serum of the animal to be tested is the serum negative for mycoplasma hyopneumoniae, that is, the serum of the animal to be tested does not contain mycoplasma hyopneumoniae antibodies or the level of mycoplasma hyopneumoniae antibodies is lower than the detection lower limit), and OD is not less than 0.397 ₄₅₀ The patient with the value less than or equal to 0.505 is judged to be suspicious (namely, the serum of the animal to be tested is suspected to be the positive serum of the mycoplasma hyopneumoniae, that is, the serum of the animal to be tested is suspected to contain the mycoplasma hyopneumoniae antibody or the animal to be tested is suspected to be infected or infected by the mycoplasma hyopneumoniae).

8. Indirect ELISA detection kit for mycoplasma hyopneumoniae antibody

The indirect ELISA detection kit for the mycoplasma hyopneumoniae antibody comprises an ELISA plate taking recombinant Mh-PGK protein shown as SEQ ID NO.1 in a sequence table as a coating antigen, a coating liquid, a sealing liquid, a washing liquid, M.haemasuis positive serum, M.haemasuis negative serum, an ELISA secondary antibody, a substrate color development liquid and a termination liquid;

wherein the coating liquid is Tris-HCl with the pH of 8.0.01M; the sealing liquid is 5% skimmed milk powder; the washing liquid is PBST; the enzyme-labeled secondary antibody is horseradish peroxidase labeled goat anti-pig IgG (IgG-HRP); the substrate color development liquid is TMB substrate color development liquid; the termination liquid is 0.5M H ₂ SO ₄ 。

Example 3 specificity and sensitivity test of an indirect ELISA method for detecting Mycoplasma hyopneumoniae antibody of porcine origin

1. Specificity test

The detection method of indirect ELISA of swine haemophilus parasuis antibody established in example 2 was used to detect 8 common swine positive sera of M.suis, M.parvum, M.haemauis, porcine circovirus disease (PCV), swine fever (CSFV), porcine Reproductive and Respiratory Syndrome (PRRS), toxoplasma gondii (PT) and foot-and-mouth disease (FMD), respectively, and to determine OD ₄₅₀ And (3) determining the specificity of serum by an indirect ELISA detection method of the swine haemophilus influenzae antibody.

The specific detection results are shown in Table 7, and the results show that the three pig-derived mycoplasma hyopneumoniae (M.suis, M.parvum, M.haemauis) positive serum can be detected simultaneously without cross reaction with the serum of common swine diseases (porcine circovirus disease, swine fever, porcine reproductive and respiratory syndrome, toxoplasma gondii and foot-and-mouth disease).

TABLE 7 ELISA specificity detection results

2. Repeatability test

Batch-to-batch repeatability test: from 135 parts of M.haemaggles negative serum sample screened by PCR detection, 7 parts of M.haemaggles negative serum (3 replicates are arranged for each part of serum) are randomly selected, 3 batches (0802, 0804 and 0808) of recombinant Mh-PGK protein are used for coating ELISA plates according to OD of each part of serum respectively by using the indirect ELISA detection method of the swine haemophilus parasuis antibody established in example 2 ₄₅₀ Respectively calculating average values

Standard Deviation (SD) and Coefficient of Variation (CV).

The results are shown in Table 8, and the ELISA detection OD of different batches of antigen recombinant Mh-PGK protein ₄₅₀ The coefficient of variation is between 2.3% and 8.9%, which indicates good reproducibility between antigen batches.

TABLE 8 coefficient of variation between different batches of recombinant Mh-PGK protein coated batches by indirect ELISA

Example 4 application of indirect ELISA detection method for mycoplasma hyopneumoniae antibody

Collecting 795 parts of pig blood from 18 pig farms such as Zhejiang, henan, anhui, jiangsu and the like, separating serum from a laboratory, and preserving at-20 ℃ for later use;

the results of the detection of 795 parts of serum by using the indirect ELISA detection method for the mycoplasma hyopneumoniae antibody established in example 2 are shown in Table 9, and the result shows that the serum antibody positive rate of the mycoplasma hyopneumoniae is 37.9% (301/795).

25 parts of positive serum are randomly selected from 202 parts of positive serum, and all positive serum are detected by whole-cell antigen ELISA, so that the positive coincidence rate of the invention is 100%.

TABLE 9 indirect ELISA detection of serum antibodies to Mycoplasma hyopneumoniae

It should be understood that the foregoing examples of the present invention are provided merely for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention, and that various other changes and modifications may be made therein by one skilled in the art without departing from the spirit and scope of the present invention as defined by the appended claims.

SEQUENCE LISTING

<110> academy of agricultural sciences in Zhejiang province

<120> recombinant Mh-PGK protein and application thereof in detection of swine haemophilus mycoplasma

<130> JLP21I1528

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 431

<212> PRT

<213> amino acid sequence of recombinant protein

<400> 1

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Val Val Phe Asn Lys Ser Thr Leu Ser Asp Leu

20 25 30

Asp Leu Ser Ser His Lys Arg Val Leu Leu Arg Leu Asp Leu Asn Val

35 40 45

Pro Val Lys Asp Gly Val Ile Thr Asp Asn Asn Arg Ile Val Gln Thr

50 55 60

Leu Pro Thr Val Lys Lys Leu Leu Glu Asn Gly Leu Lys Ile Ile Ile

65 70 75 80

Leu Ser His Phe Ser Arg Ile Lys Asp Val Ser Glu Val Asn Glu Lys

85 90 95

Lys Lys Ser Leu Lys Val Val Phe Glu Glu Phe Lys Lys Leu Ile Pro

100 105 110

Asp Lys Glu Ile Lys Phe Ile Glu Ser Ile Lys Phe Glu Asp Val Arg

115 120 125

Lys Ser Val Asn Asp Asn Ser Ser Ala Asp Ile Ile Ile Leu Glu Asn

130 135 140

Thr Arg Tyr Tyr Asp Val Asp Glu Asn Asn Asn Pro Val Lys Trp Glu

145 150 155 160

Ser Lys Asn Ser Ser Glu Leu Ser Lys Phe Trp Ala Ser Leu Gly Asp

165 170 175

Leu Tyr Ile Asn Asp Ala Phe Gly Thr Cys His Arg Ala His Ala Ser

180 185 190

Asn Val Gly Leu Ala Ser Cys Leu Pro Ser Ala Ile Gly Phe Leu Val

195 200 205

Glu Lys Glu Leu Asn Phe Leu Ser Lys Ala Val Ile Ser Thr Asp Phe

210 215 220

Pro Lys Val Leu Ile Leu Gly Gly Ser Lys Val Ser Asp Lys Leu Lys

225 230 235 240

Leu Ile Asn Val Ile Ala Pro Lys Val Asp Lys Leu Leu Ile Gly Gly

245 250 255

Gly Met Ala Tyr Thr Phe Leu Lys Ala Gln Gly Lys Glu Val Gly Ala

260 265 270

Ser Ile Ile Glu Asn Glu Met Ile Glu Glu Cys Lys Gly Leu Leu Ser

275 280 285

Lys Tyr Gly Glu Lys Leu Leu Leu Pro Val Asp His Val Val Ala Pro

290 295 300

Glu Phe Lys Asp Val Val Gly Val Ile Lys Asp Ala Asp Asp Arg Asn

305 310 315 320

Trp Asn Gly Glu Met Ser Leu Asp Ile Gly Pro Lys Thr Ile Asp Leu

325 330 335

Phe Arg Arg Ala Leu Asp Asp Ala Arg Val Val Ile Trp Asn Gly Pro

340 345 350

Met Gly Val Phe Glu Phe Glu Asn Phe Gly Leu Gly Thr Lys Ser Val

355 360 365

Ala Glu Lys Leu Ala Glu Ile Thr Asn Lys Gly Ala Tyr Thr Val Ile

370 375 380

Gly Gly Gly Asp Ser Ala Ala Ala Ala Glu Lys Phe Asn Leu Ser Ser

385 390 395 400

Ser Met Ser Phe Ile Ser Thr Gly Gly Gly Ala Ser Leu Ser Phe Phe

405 410 415

Glu Gly Ser Asp Met Pro Gly Ile Ser Ser Ile Ser Asp Lys Lys

420 425 430

<210> 2

<211> 1245

<212> DNA

<213> artificially synthesized optimized sequences

<400> 2

catatggttg ttttcaacaa gagtaccctg agtgatctgg atctgagtag tcataaacgc 60

gttctgctgc gcctggatct gaatgttccg gtgaaagatg gtgttattac cgataataat 120

cgcattgtgc agaccctgcc gaccgttaaa aaactgctgg aaaatggtct gaaaattatt 180

attctgagcc attttagccg cattaaggat gttagcgaag tgaatgaaaa gaaaaaatct 240

ctgaaggtgg tttttgaaga gtttaaaaaa ctgatcccgg ataaagaaat caaattcatt 300

gaaagcatca agttcgaaga tgttcgcaaa agtgttaatg ataatagtag cgcagatatt 360

atcatcctgg aaaatacccg ttattatgat gtggatgaaa ataataaccc ggttaaatgg 420

gaaagcaaaa atagcagcga actgagtaaa ttttgggcca gtctgggtga cctgtatatt 480

aatgatgcct ttggtacctg ccatcgtgcc catgcaagca atgtgggcct ggcaagctgt 540

ctgccgagtg caattggctt tctggttgaa aaagaactga attttctgag caaagcagtt 600

attagtaccg attttccgaa agtgctgatt ctgggtggca gtaaagttag cgataaactg 660

aaactgatta acgttattgc tcctaaagtt gataagttat taataggggg aggtatggct 720

tatacttttc tgaaagcaca gggtaaagaa gtgggcgcaa gcattattga aaatgaaatg 780

attgaggagt gcaaaggcct gctgagtaaa tatggtgaaa aactgctgct gccggttgat 840

catgtggtgg caccggagtt taaagatgtg gttggcgtta ttaaggatgc cgatgatcgt 900

aattggaatg gtgaaatgag tctggatatt ggtccgaaaa ccattgatct gtttcgtcgt 960

gccctggatg atgcccgtgt tgttatttgg aatggtccga tgggtgtttt tgaatttgaa 1020

aattttggcc tgggtaccaa aagcgttgca gaaaaactgg cagaaattac caataagggt 1080

gcatataccg ttattggtgg cggcgatagc gccgccgccg cagagaaatt caatctgagc 1140

agtagcatga gctttattag taccggtggt ggcgcaagcc tgagcttttt cgaaggtagc 1200

gatatgccgg gtattagcag cattagcgat aaaaaataac tcgag 1245

<210> 3

<211> 1233

<212> DNA

<213> Artificial synthesis optimized Pre-sequence

<400> 3

gtggtattca ataaatctac cctttctgat ttagatttaa gttctcacaa aagagtttta 60

ttgagacttg atcttaatgt tcctgtaaag gatggagtta taactgataa taatcgtatt 120

gttcaaactc ttcctactgt aaaaaaactt cttgaaaatg gacttaagat cattattctt 180

agccattttt caaggattaa agatgtatct gaagttaatg aaaaaaagaa atctcttaaa 240

gtagtttttg aggagtttaa aaaattgatt cctgataaag aaattaaatt tattgaatct 300

atcaagtttg aagatgtaag aaaatctgtg aatgataatt cttctgcaga tattatcatt 360

cttgaaaaca ctcgttatta tgatgtggat gaaaacaata atcctgttaa atgagaatct 420

aagaattcct ctgagttatc taaattttga gcttctcttg gggatttgta tataaatgat 480

gcttttggta cttgtcatag agcccatgct tcgaatgtag gtttagcttc atgtcttcct 540

tctgctattg gctttttggt ggaaaaggaa ttaaattttc tttcaaaagc tgttatttca 600

acagattttc ctaaagtttt aatcttggga ggaagtaagg tttctgataa gttgaagctt 660

ataaatgtta ttgctcctaa agttgataag ttattaatag ggggaggtat ggcttatact 720

ttcttgaaag ctcaaggaaa agaagtgggc gcttcaatta ttgagaacga gatgattgaa 780

gaatgcaaag gtcttctttc taaatatgga gagaaattat tattgcctgt tgatcatgtt 840

gttgctcctg agtttaaaga tgttgttggg gttattaaag atgcggatga tagaaattga 900

aatggagaaa tgtctttgga tatcggacct aagactattg atttatttag aagagcttta 960

gatgatgcta gagttgttat ttggaatgga cctatgggtg tgtttgaatt tgaaaatttt 1020

ggcctaggaa ctaaatcggt agctgagaaa cttgctgaaa ttacaaataa aggagcttat 1080

acagtcattg gaggaggaga ttctgcagca gcagctgaaa agttcaattt atcttctagt 1140

atgagtttta tttctacagg aggaggagct tccttatctt tctttgaagg ttctgatatg 1200

ccaggcatta gttctatttc tgataaaaaa taa 1233

Claims

1. The recombinant Mh-PGK protein is characterized in that the amino acid sequence of the recombinant Mh-PGK protein is shown as SEQ ID NO. 1.

2. The recombinant Mh-PGK protein of claim 1, wherein the recombinant Mh-PGK protein is prepared by the following method:

constructing recombinant plasmid by using optimized Mh-PGK gene shown in SEQ ID NO.2 at 7-1239, and expressing by an expression system to obtain the recombinant Mh-PGK protein.

3. The recombinant Mh-PGK protein of claim 2, wherein the recombinant plasmid is pET28-Mh-PGK; the pET28-mh-pgk is a recombinant plasmid obtained by replacing a DNA fragment between NdeI enzyme and XhoI enzyme recognition sequences of pET28 a (+) plasmid with optimized mh-pgk genes shown in positions 7-1239 of SEQ ID No.2 in a sequence table.

4. Use of the recombinant Mh-PGK protein of any one of claims 1 to 3 for any one of:

1) The application of the kit in preparing a mycoplasma hyopneumoniae antibody product for detecting whether the serum of an animal to be detected contains swine blood;

2) The application of the kit in preparing the product for detecting whether the animal to be detected is infected or not or is infected by the mycoplasma hyopneumoniae.

5. The use according to claim 4, wherein the Mycoplasma hyopneumoniae is mycoprosma suis, mycoplasma parvum and/or Mycoplasma haemosuis.

6. An indirect ELISA detection kit for a mycoplasma hyopneumoniae antibody, which is characterized by comprising an ELISA plate taking the recombinant Mh-PGK protein as defined in any one of claims 1-3 as a coating antigen.

7. The indirect ELISA detection kit of mycoplasma hyopneumoniae antibodies of claim 6, wherein the kit further comprises one or more of a coating solution, a blocking solution, a washing solution, mycoplasma haemosuis positive serum, mycoplasma haemosuis negative serum, an enzyme-labeled secondary antibody, a substrate chromogenic solution, and a stop solution.

8. The indirect ELISA detection kit for mycoplasma hyopneumoniae antibodies of claim 7, wherein the recombinant Mh-PGK protein has a coating concentration of 0.31 μg/mL.

9. The kit for indirect ELISA detection of swine haemophilus influenzae antibodies according to claim 7, wherein the coating solution is a carbonate buffer solution with pH of 9.6.01M, PBS with pH of 4.0.01M or Tris-HCl with pH of 8.0.01M.

10. The indirect ELISA detection kit of mycoplasma hyopneumoniae antibodies of claim 7, wherein the blocking solution is 2.5% nonfat milk powder, 5% nonfat milk powder, 7.5% nonfat milk powder, 1% bsa, 2% bsa, or 3% bsa.

11. The indirect ELISA detection kit for mycoplasma hyopneumoniae antibodies of claim 7, wherein the wash solution is PBST.

12. The indirect ELISA detection kit for swine haemophilus parasuis antibodies according to claim 7, wherein the enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-swine IgG.

13. The indirect ELISA detection kit of mycoplasma hyopneumoniae antibodies of claim 7, wherein the dilution of the enzyme-labeled secondary antibody is 1:12500.

14. The indirect ELISA detection kit for mycoplasma hyopneumoniae antibodies of claim 7, wherein the substrate chromogenic solution is TMB substrate chromogenic solution.

15. The indirect ELISA kit for a swine haemophilus influenzae antibody according to claim 7, wherein the stop solution is 0.5. 0.5M H ₂ SO ₄ 。