CN114350636A

CN114350636A - Recombinant Mh-PGK protein and application thereof in detection of swine haemophilus mycoplasma

Info

Publication number: CN114350636A
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: 2022-04-15
Anticipated expiration: 2041-12-06
Also published as: CN114350636B

Abstract

The invention discloses a recombinant Mh-PGK protein and application thereof in detecting 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 using the recombinant Mh-PGK protein as an ELISA plate of a coating antigen. The invention establishes the swine haemophilus antibody indirect ELISA detection method based on the recombinant Mh-PGK protein, is used for detecting the level of the swine haemophilus antibody, has no cross reaction with partial other swine 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 haemosus.

Description

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

Technical Field

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

Background

Mycoplasma haemophilus (HM, also known as eperythrozoon) is a type of mycoplasma that infects many mammals, parasitizes on the surface of erythrocytes and in the bone marrow (landlocked, master edition. veterinary microbiology [ M ]. fourth edition. beijing: chinese agriculture press 2010: 240-. Disruption of red blood cells by HM results in haemolysis, infectious anemia in the host, which can lead to miscarriage, anemia in piglets, jaundice and wasting in sows (Hoelzle L E, Felder K M, Hoelzle K. Portone enterozoonosis: from Eperythrozoon suis to Mycoplasma suis [ J ]. Tierarztl Prax Ausg G Grossigerer Nutztier.2011, 39(4): 215; 220. Messick, J.B., Hemotrophic mycoplasmas: a review and new insights into viral potential.V.t.Clin Patholol, 2004.33(1): p.2-13.). Three kinds of HM prevail in Chinese swine herds, including early-identified Mycoplasma hyoscyami (M.suis, old called Eperythrozoon suis) (Zhaoji, Yangmeng, Zhao \394411, etc.. sequence determination and phylogenetic analysis of 16S rRNA gene of Eperythrozoon suis [ J ]. animal veterinary science, 2005(06): 596. RTM.), Mycoplasma parvum (M.parvum) identified at the 2011 molecular biology level (Watanabe Y, Fujihara M, Obara H, et al. o genetic clusterings in porcine genetic expressed by genes of the 16S rRNA and RNP RNA genes [ J ]. Vet. Sci.Sci., 73: 1667. 1651. and New Pieau. Ito.2011.7. Italy.Y, (C., blood of pig blood of Shi. Italy, Shi. Italy et al (blood of Shisane, blood of Shi. Italy et al, Shi et al., blood strain, Shi et al, Shi. Italy et al, Shi et al, china [ J ] J Vet Med Sci.2017,79(5): 864-870).

The epidemiological investigation of the PCR method found that M.haemosuis is prevalent in Zhejiang, Jiangsu, Henan, etc. China, and mixed infection with HM of two other swine sources occurs frequently, the infection rate of sows is high, and M.haemosuis (Seo M G, Kwon O D, Kwak D.2019. Preference and genetics analysis of bioplasma species in biomedical species in Korea. parasites Vectors,12(1): 378.; Staer J, Ade J, Ritzmann M, et al.2020.detection of a novel haemostasis species in fatty tissues with tissues, very and animal species (6): 187). M.suis and M.parvum serum antibody detection methods have been reported (Amit Kadam, Kingjin Xiu, Sheer Hibiscus, et al. serological and molecular biology epidemiological investigations revealed that Mycoplasma parvum/Eperythrozoon is the major Hainan island Mycoplasma hyophilus [ J ] parasite and medical insect journal 2018,25(03): 140. 147. Hsu F S, Liu M C, Chou S M, et al. evaluation of an enzyme-linked immunological assay for detection of Epythrozoon antibodies in small genes [ J ] Am J vector Res.1992,53(3): 352. 354.; Hoelzle L E, Hoelzle K, Helbling M, et al Gf 1, a surface-protein ] 2007. detection of Mycoplasma J.2007. 9. detection of Mycoplasma J.466. detection method.

The ELISA method has the advantages of sensitivity, rapidness, convenience, easy standardization and the like, Hoelzle and the like prove that the first adhesion protein MSG1 of M.suis has the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the ELISA method established by the recombinant protein has better specificity and is more sensitive and effective than a thalli antigen ELISA (Hoelzle K, Doser S, Ritzmann M, et al. conservation with the Mycoplasma suis recombinant protein MSG1 peptides a strain animal protein kinase to detect in samples [ J ]. Vaccine.2009,27(39):5376-5382.), the detection method of M.vprum truncated protein mp-tGAPDH is established by the aid of the ELISA method, the positive rate of M.parvum serum antibody is 36.69%, the positive rate of the serum antibody is 91/248%, the Hongjiang super-ELISA method is established based on the agricultural Hongjiang super protein ELISA method, 2021,33(05):809-815.).

Phosphoglycerate kinase (Phosphoglycerate kinase PGK) is an important enzyme of the glycolytic pathway of energy metabolism and an essential enzyme for the survival of organisms. PGK participates in the second stage of glycolysis, catalyzing the conversion of 1, 3-diphosphoglycerate to 3-phosphoglycerate, producing ATP. Mycoplasma haemophilus relies on host blood to provide energy, and glycolysis is its primary source of energy supply. PGK is also involved in cell homeostasis, pathogenic effects caused by Candida albicans adhering to host cells, nucleic acid interactions, tumorigenesis, cell death, and viral replication (Fu Q, Yu Z. phosphoglycerate kinase 1(PGK1) in cancer: A promoting 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: 32479953.; Rojas-Pirella M, Andre-Alvi ad D, Rojas V, Kemmerling U, C center AJ, Michels PA, Conci n JL,

W.Phosphoglycerate kinase:structural aspects and functions,with special emphasis on the enzyme from Kinetoplastea.Open Biol.2020 Nov; 10(11) 200302.doi 10.1098/rsob 200302.PubMed 33234025.). The crystal structure of PGK of pig, horse and yeast has been analyzed, it is a monomeric enzyme, and it is composed of two spherical structures, and the C-terminal peptide segment is very important in maintaining the stability of the structure (MasMT, developer ZE. Structure-function relationships in 3-phosphoglycerate kinase: roll of the carbon-terminal peptide. proteins. 1988; 4(1):56-62.doi:10.1002/prot.340040108. PubMed: 3054872. Shang Ice, Qijinjing, Wang Yun et al]The Chinese journal of zoonosis 2021,29(03): 71-79.). The PGK related research of mycoplasma haemophilus only has prokaryotic expression of the PGK protein of mycoplasma canicola, and has immunogenicity (Songqi, Qianlimin, Su Miao, in the early morning, Qin Shuang, Gong Yue. Canine eperythrozoon PGK prokaryotic expression, purification and polyclonal antibody preparation [ J]The proceedings of Tianjin academy of agriculture, 2018,25(03): 42-46.). However, the research on prokaryotic expression of the PGK protein of the swine mycoplasma hyopneumoniae is not reported.

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

Disclosure of Invention

The first purpose of the invention is to provide a new porcine haemophilus recombinant PGK protein (simply referred to as recombinant Mh-PGK protein) which is used for detecting the antibody level of porcine haemophilus and epidemiological monitoring, and evaluating the role of the disease in the swine haemophilus epidemiology.

The second purpose of the invention is to provide the application of the recombinant Mh-PGK protein in detecting the mycoplasma hyorhinis and/or the mycoplasma hyorhinis antibody.

The third purpose of the invention is to provide an indirect ELISA detection kit for swine mycoplasma hyorhinis antibodies.

The fourth purpose of the invention is to provide an indirect ELISA detection method for the swine mycoplasma hyorhinis antibody.

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

in a first aspect, the invention provides a novel recombinant Mycoplasma hyorhinis PGK protein, referred to as recombinant Mh-PGK protein for short, wherein 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 to 1239 th sites of SEQ ID NO.2, and expressing the recombinant plasmid by an expression system to obtain the recombinant Mh-PGK protein; wherein, the optimized Mh-PGK gene is obtained by performing codon optimization on the Mh-PGK gene (Mh-PGK protein with the coding amino acid sequence shown in 22 th to 431 th positions of SEQ ID NO. 1) shown in SEQ ID NO.3 and optimizing 3 TGAs into codon TGG which can code tryptophan in escherichia coli and partial codon which is optimized into escherichia coli preference codon.

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

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

In a specific embodiment of the invention, pET28-Mh-PGK is transformed into Escherichia coli BL21(DE3) to induce expression to obtain recombinant Mh-PGK protein.

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

1) detecting whether the serum of an animal to be detected contains the swine mycoplasma hyorhinis antibody;

2) the application in preparing the product for detecting whether the serum of an animal to be detected contains the swine mycoplasma hyorhinis antibody;

3) detecting whether the animal to be detected is infected or infected with swine mycoplasma hyopneumoniae;

4) the application in preparing the product for detecting whether the animal to be detected is infected or infected with the swine mycoplasma hyopneumoniae.

The swine haemophilus is Mycoplasma suis, Mycoplasma parvum and/or Mycoplasma haemosus.

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

Further, the coating concentration of the recombinant Mh-PGK protein is 0.31 mu g/mL.

Further, the kit also comprises one or more of coating solution, confining solution, washing solution, M.haemosuis positive serum, M.haemosuis negative serum, enzyme-labeled secondary antibody, substrate color development solution and stop solution.

Further, the coating solution was a carbonate buffer solution (1.59g Na) of pH9.60.01M₂CO₃And 2.93g NaHCO₃Deionized water to 1000 mL), PBS (8.0g NaCl, 0.2g KCl, 2.86g Na) at pH 7.40.01M₂HPO₄·12H₂O and 0.27g KH₂PO₄Adding deionized water to 1000 mL) or Tris-HCl (1M Tris-HCl is prepared by adding 12.11g Tris base into about 80mL deionized water, dissolving, adding concentrated HCl to adjust pH value to 8.0, and diluting to 100 mL; adding 1mL of 1M Tris-HCl into 99mL of deionized water to obtain Tris-HCl with the pH value of 8.00.01M); Tris-HCl, pH8.00.01M, is preferred.

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

Further, the washing solution was PBST (8.0g NaCl, 0.2g KCl, 2.86g Na)₂HPO₄·12H₂O、0.27g KH₂PO₄And 0.5mL Tween-20 was added to 1000 mL).

Further, the enzyme-labeled secondary antibody was horseradish peroxidase-labeled goat anti-porcine IgG (IgG-HRP) (purchased from KPL).

Further, the dilution of the enzyme-labeled secondary antibody is 1: 12500.

Further, the substrate color developing solution is a TMB substrate color developing solution (purchased from Shanghai bioengineering Co., Ltd., product No. E661007).

Further, the stop solution was 0.5M H₂SO₄(16.4mL of concentrated sulfuric acid was slowly dropped into 400mL of deionized water to make up the volume to 500 mL).

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

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

1) coating antigen: coating the enzyme label plate with the coating liquid by using the recombinant Mh-PGK protein as a coating antigen, and washing with a detergent;

2) and (3) sealing: adding sealing liquid, sealing, and washing with detergent;

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

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

5) TMB color development: adding a substrate color development solution for reaction;

6) and (3) terminating the reaction: adding a stop solution to stop the reaction;

7) detection of OD₄₅₀The value: measuring OD of serum of each animal to be measured with microplate reader₄₅₀A value;

8) and (4) interpretation of results: the determination criterion was OD₄₅₀Value of>0.505 is positive (that is, the serum of the animal to be tested is positive serum of swine haemophilus mycoplasma, that is, the serum of the animal to be tested contains antibody of swine haemophilus mycoplasma or the animal to be tested is infected or infected with swine haemophilus mycoplasma), OD₄₅₀Value of<0.397 is judged as negative (namely, the serum of the animal to be testedIs swine haemophilus negative serum, that is to say, the serum of the animal to be tested does not contain swine haemophilus antibody or the level of the swine haemophilus antibody is lower than the lower detection limit), OD is more than or equal to 0.397 and less than or equal to₄₅₀And (3) the value is less than or equal to 0.505, the animal is determined to be suspicious (namely the serum of the animal to be tested is suspected to be positive serum of the swine haemophilus mycoplasma, namely the serum of the animal to be tested is suspected to contain swine haemophilus mycoplasma antibodies or the animal to be tested is suspected to be infected or infected with the swine haemophilus mycoplasma).

In the invention, by optimizing the coating solution, the coating concentration, the sealing solution, the sealing time, the serum dilution, the serum reaction time, the enzyme-labeled secondary antibody dilution and the enzyme-labeled secondary antibody action time, the optimal Tris-HCl with the pH value of 8.00.01M is obtained, the optimal coating concentration is 0.31 mu g/mL, the optimal sealing solution is 5% skimmed milk powder, the optimal sealing time is 1h, the optimal serum dilution is 1:8, the optimal reaction time of 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 1 h.

In a preferred embodiment of the invention, the indirect ELISA detection method for the swine mycoplasma hyorhinis antibody comprises the following steps:

1) coating antigen: taking the recombinant Mh-PGK protein as a coating antigen, incubating the recombinant Mh-PGK protein with Tris-HCl of pH8.00.01M at the coating concentration of 0.31 mu g/mL and 100 uL/hole at 37 ℃ for 1h, placing the incubated product on an overnight coated ELISA plate at 4 ℃, and washing the coated ELISA plate 3 times with PBST for 3min each time;

2) and (3) sealing: adding 250 μ L of 5% skimmed milk powder, sealing at 37 deg.C for 1h, and washing with PBST for 1 time;

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

4) adding an enzyme-labeled secondary antibody: adding horse radish peroxidase labeled goat anti-pig IgG with the dilution of 1:12500, reacting at the temperature of 37 ℃ for 1h in 100 mu L/hole, and washing for 3 times by PBST;

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

6) and (3) terminating the reaction: 0.5M H was added₂SO₄Stop the reaction at 50. mu.L/well;

7) detection of OD450 value: measuring the OD450 value of the serum of each animal to be measured by using a microplate reader;

8) and (4) interpretation of results: the determination criterion was OD₄₅₀Value of>0.505 is positive (that is, the serum of the animal to be tested is positive serum of swine haemophilus mycoplasma, that is, the serum of the animal to be tested contains antibody of swine haemophilus mycoplasma or the animal to be tested is infected or infected with swine haemophilus mycoplasma), OD₄₅₀Value of<The animal is judged to be negative by 0.397 (namely the serum of the animal to be tested is the negative serum of the mycoplasma hyohaemophilus, namely the serum of the animal to be tested does not contain the mycoplasma hyohaemophilus antibody or the level of the mycoplasma hyohaemophilus antibody is lower than the lower detection limit), and OD is more than or equal to 0.397₄₅₀And (3) the value is less than or equal to 0.505, the animal is determined to be suspicious (namely the serum of the animal to be tested is suspected to be positive serum of the swine haemophilus mycoplasma, namely the serum of the animal to be tested is suspected to contain swine haemophilus mycoplasma antibodies or the animal to be tested is suspected to be infected or infected with the swine haemophilus mycoplasma).

In the present invention, the swine-derived Mycoplasma hyohemophilus is a Mycoplasma hyopneumoniae (m.suis), a Mycoplasma micromeritium (m.parvum), and/or a Mycoplasma hyohemophilus (m.haemophilus) that is identified at an early stage. The animal to be detected is a pig.

The invention has the following beneficial effects:

the invention establishes the swine haemophilus antibody indirect ELISA detection method based on the recombinant Mh-PGK protein, is used for detecting the level of the swine haemophilus antibody, has no cross reaction with partial other swine 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 haemosus.

Drawings

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

FIG. 1 is a SDS-PAGE analysis of the expression of recombinant Mh-PGK protein in E.coli BL 21; wherein, M1 and M2 represent Protein Marker; 1 represents the expression condition of recombinant protein in recombinant Escherichia coli pET28/BL21(DE 3); 2 represents the expression condition of the recombinant protein in the non-induced recombinant Escherichia coli pET28-mh-pgk/BL21(DE 3); 3 represents the expression condition of recombinant protein in IPTG-induced recombinant Escherichia coli pET28-mh-pgk/BL21(DE 3); 4 represents the SDS-PAGE detection result of the purified recombinant Mh-PGK protein.

FIG. 2 shows the immunoreactivity of the recombinant Mh-PGK protein analyzed by Western-blot; wherein, M represents a prestained protein Marker; 1 represents the Western Blot reaction of mouse anti-recombinant Mh-PGK protein serum and recombinant Mh-PGK protein, and the arrow indicates the 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 invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures 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 is not to be taken as limiting the scope of the invention.

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

escherichia coli Transetta (DE3) competent cells were purchased from Beijing Quanjin Biotechnology Ltd;

the pET-28a (+) plasmid is purchased from Shanghai bioengineering, Inc., and has the product number B540183;

and M.haemosuis negative serum is imported swine serum, is provided by the inspection and quarantine technical center of exit and entry inspection and quarantine bureau of Zhejiang province, and is negative by PCR (polymerase chain reaction) detection, and accounts for 135 parts in total.

The M.haemosuis positive serum is 25 parts of serum collected by sows identified as positive by a qPCR method;

after three swine haemophilus (M.suis, M.parvum and M.haemosuis) are all positive in pathogen identification by a PCR method, whole-bacterium antigen ELISA identifies antibody positive serum and then is stored;

after the positive serum of the pig toxoplasma is identified by the PCR method by the research laboratory to be pathogenic positive, the positive serum is identified by the toxoplasma IHA diagnostic kit of Lanzhou veterinary institute to be antibody positive and then is stored; the positive foot-and-mouth disease serum is preserved after the positive antibody is identified by a foot-and-mouth disease indirect hemagglutination antibody kit of Lanzhou veterinary research institute;

the positive sera of the porcine circovirus disease, the swine fever, the porcine pseudorabies and the porcine reproductive and respiratory syndrome are all detected, identified and stored by an ELISA detection kit corresponding to the IDEXX company in a swine disease research laboratory of a livestock institute of a farm institute of the agricultural department of Zhejiang province;

peroxidase goat anti-pig IgG-HRP was purchased from KPL;

Ni-Argrose affinity chromatography nickel beads were purchased from Invitrogen;

the TMB substrate developing solution is purchased from Shanghai Biotechnology engineering service, Inc., and has a cargo number E661007;

the microplate reader is Spectra Max M5, a product of Molecular Devices of America;

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

Example 1 preparation of Mycoplasma hyorhinis antibody Indirect ELISA antigen and detection of antigenicity

1. Preparation of antigens

1) Optimization of mh-pgk Gene

According to the preference of Escherichia coli codons, codon optimization is carried out on Mh-PGK gene (Mh-PGK protein for short) of ZJ1102 strain of new swine haemophilus (M.haemosis) of local strain in Zhejiang, which is obtained by amplification (the nucleotide sequence of the Mh-PGK gene is shown in SEQ ID NO.3 and consists of 1233 nucleotides, and the coding amino acid sequence of the PGK protein of new swine haemophilus (PGK protein for short) is shown in 22 nd to 431 th positions of SEQ ID NO.1, 3 TGAs (TGAs code tryptophan non-stop codons due to the specificity of the gene coding system of mycoplasma) are optimized into TGG codons capable of coding tryptophan in Escherichia coli and partial codons are optimized into Escherichia coli preference codons, so that the optimized Mh-PGK gene (the nucleotide sequence of which is shown in 7 th to 1239 th positions of SEQ ID NO.2 and consists of 1233 nucleotides, and the amino acid sequence of the Mh-PGK protein obtained by coding is not changed), synthesized by Suzhou Jinzhi Biotechnology, Inc.

2) Inducible expression of recombinant proteins

NdeI enzyme cutting site (5 'catatg 3') is added at the 5 'end of the optimized mh-pgk gene, XhoI enzyme cutting site (5' ctcgag3 ') is added at the 3' end (the sequence is shown in SEQ ID NO.2 and is synthesized by Jinzhi Biotechnology Ltd, Suzhou), double-enzyme cutting is carried out by NdeI enzyme and XhoI enzyme, and meanwhile, pET-28a (+) plasmid is double-enzyme cut by NdeI enzyme and XhoI enzyme. And connecting the optimized mh-pgk gene after enzyme digestion and pET-28a (+) plasmid by using DNA ligase to construct a recombinant plasmid pET28-mh-pgk, transforming the recombinant plasmid into escherichia coli Top10, extracting the plasmid, and determining that the insertion sequence is correct through sequencing identification by Shanghai bioengineering company. The pET28-Mh-PGK is a recombinant plasmid obtained by replacing a DNA fragment between NdeI enzyme recognition sequences and XhoI enzyme recognition sequences of pET-28a (+) plasmid with an optimized Mh-PGK gene shown as the 7 th to 1239 th sites of SEQ ID NO.2 in a sequence table, and the recombinant plasmid can express a fusion protein shown as SEQ ID NO.1 in the sequence table, namely a new mycoplasma hyorhinis recombinant PGK protein, which is simply referred to as a recombinant Mh-PGK protein; wherein, the 1 st to 21 st sites of SEQ ID NO.1 in the sequence table are the amino acid sequences coded by the DNA sequences on pET-28a (+) plasmids, and the 22 nd to 431 nd sites of SEQ ID NO.1 are Mh-PGK protein sequences.

The recombinant plasmid pET28-mh-pgk with the correct sequence is transformed into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli pET28-mh-pgk/BL21(DE 3). Recombinant Escherichia coli pET28-mh-pgk/BL21(DE3) was expressed by induction with 0.8mM IPTG (37 ℃, 4h), and the bacterial solutions were collected before and after induction for 4h, respectively. The above experiment was repeated with pET28a substituted for pET28-mh-pgk to obtain recombinant Escherichia coli pET28/BL21(DE3), and a bacterial solution after 4h of induction was collected. The three bacterial liquids are respectively taken to be 100 mu L, centrifuged and precipitated for 5min at 8000rpm at 4 ℃, resuspended in 40 mu L double distilled water, added with 10 mu L5 xSDS loading buffer and boiled, and are respectively used for detecting the expression condition of recombinant protein in non-induced and IPTG-induced recombinant Escherichia coli pET28-mh-pgk/BL21(DE3) and the expression condition of recombinant protein in recombinant Escherichia coli pET28/BL21(DE3) by SDS-PAGE. As shown in FIG. 1, the expression protein band appeared at about 45kD in IPTG-induced recombinant E.coli pET28-Mh-PGK/BL21(DE3), and the size of the expression protein band coincided with the expected size, indicating that the recombinant Mh-PGK protein is expressed; and the recombinant Escherichia coli pET28/BL21(DE3) and the non-induced recombinant Escherichia coli pET28-mh-pgk/BL21(DE3) have no expression of corresponding proteins.

3) Purification of recombinant proteins

The IPTG-induced recombinant Escherichia coli pET28-mh-pgk/BL21(DE3) bacterial liquid is centrifuged, and the precipitate is resuspended in PBS and ultrasonically broken. Centrifuging for 10min again, washing the precipitate with PBS (pH 7.4) for 2 times, adding 19.7mL buffer solution A (Tris-HCl 6.06g, EDTA 0.186g, NaCl 2.92g, glycerol 50mL, adding sterile deionized water to 1L) and 0.3mL 20% sodium dodecylsarcosinate (SKL) stock solution, resuspending the precipitate overnight, centrifuging for 10min, collecting supernatant, adding 20% PEG4000 (2g PEG4000 and sterile deionized water to 10mL) to a final concentration of 0.2%, adding 50mmol/L oxidized glutathione (0.153g oxidized glutathione deionized water to 5mL) to a final concentration of 1 mmol/L), adding 100mmol/L reduced glutathione (0.153g reduced glutathione deionized water to 5mL) to a final concentration of 2mmol/L, standing for 30min-2h, standing at 4 deg.C, dialyzing with 0.01MPBS (pH 7.4) for 2 days, obtaining the extracted inclusion body;

about 20mL of inclusion bodies were added to 5mL of Ni-Agarose previously equilibrated with pH7.4 Binding buffer (purchased from Shanghai Producer, cat. No. C600303) and combined for 30min at room temperature. During the process, the mixture was shaken 3-5 times at 4000rpm for 5min, the supernatant was discarded, and the pellet was washed 4 times with imidazole Binding buffer containing 20mM, 30mM, 40mM, and 50 mM. And eluting the 250mM Elution buffer for 3 times by 5 mL/time, collecting and combining the eluted Elution protein, dialyzing the mixture for 24 hours by PBS (phosphate buffer solution) pH7.4, concentrating the mixture to a half of the volume, centrifuging the protein solution at 10000rpm for 10min, and taking the supernatant to obtain the purified recombinant Mh-PGK protein. Using a Shanghai worker BAC protein quantitative detection kit to measure the concentration (the concentration of the purified recombinant Mh-PGK protein is 730 mug/mL) according to the instruction, and subpackaging at-20 ℃ for freezing storage 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 appears only at about 45kD, the purification and recovery result is good, and the obtained amino acid sequence is the recombinant Mh-PGK protein shown in 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 prepared antibodies according to the following procedure:

dissolving the purified recombinant Mh-PGK protein (namely antigen) in PBS, mixing the protein and an equivalent volume of Freund's complete adjuvant by double pushing, injecting 5 immunized BALB/C mice by back subcutaneous and leg muscle at multiple points with the dosage of 40 mug/mouse; and (2) avoiding: 14d, uniformly mixing the same amount of antigen and incomplete Freund's adjuvant by the same method, and immunizing at a dose of 50 mu g/mouse; after 28 days, the same amount of antigen and incomplete Freund's adjuvant are mixed uniformly and then are immunized, the dosage is 50 mu g/mouse, and the titer is measured by ELISA; after the last immunization, the blood is taken by cutting the tail to measure the titer of the serum, and if the expected purpose is achieved, the antiserum can be collected by bleeding. And selecting a mouse with high titer for blood collection, and separating serum to obtain the mouse anti-recombinant Mh-PGK protein serum.

2) Western-blot: adjusting the concentration of the purified recombinant Mh-PGK protein to 365 mug/mL, loading the recombinant Mh-PGK protein to 10 muL/hole, performing SDS-PAGE electrophoresis, cutting the adhesive tape to a proper size after the SDS-PAGE electrophoresis is finished, balancing the gel by electrotransformation for 5min multiplied by 3 times, pre-cutting a cellulose Nitrate (NC) membrane and filter paper to be the same as the size of the adhesive tape, and immersing the gel in the electrotransformation for balancing for 10 min.

Electric conversion: placing the positive electrode horizontally, placing the filter paper, placing the NC membrane, the gel, the filter paper and the negative electrode sequentially, aligning the filter paper, the NC membrane and the gel without bubbles, switching on current according to the area of the gel and 0.65-1.0 mA/cm2, and performing electrotransfer for 2 hours.

Dyeing: and (3) switching off the power supply, lifting off all layers one by one, transferring the gel into ponceau dyeing solution, dyeing until protein bands appear on the membrane, and taking a picture. The nitrocellulose filter was rinsed with deionized water until protein bands were absent.

And (3) sealing: the transferred nitrocellulose filter was blocked with 1% BSA in PBST for 2h at room temperature, and the blocking solution was discarded.

Binding of primary antibody to target protein: PBST dilution primary antibody, mouse anti recombinant Mh-PGK protein serum as the first antibody according to 1:500 dilution, transfer with Mh-PGK protein nitrocellulose membrane flat in the shaker 4 degrees overnight, nitrocellulose filter membrane PBST washing, 5min x 3 times.

And (3) binding of a secondary antibody: PBST 1:3000 diluted horseradish peroxidase (HRP) labeled rabbit anti-mouse secondary antibody, and with nitrocellulose membrane on a shaking bed at room temperature for 2 h. The nitrocellulose filter was washed with PBST, 5min X3 times. Adding a chemiluminescent reagent for color development: the nitrocellulose filter was placed in 1mL of ECL luminophore (Shanghai Producer, cat. C510043) and photographed by exposure.

And replacing the mouse anti-recombinant Mh-PGK protein serum with mouse negative serum to repeat the Western-blot.

The immunoreactivity 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 combined reaction with the mouse negative serum, which indicates that the protein has good antigenicity.

Example 2 establishment and Condition optimization of Indirect ELISA detection method for Mycoplasma hyorhinis antibody

1. Determination of optimal coating concentration and optimal serum dilution

1) Coating antigen: the recombinant Mh-PGK protein was coated with a coating solution (pH9.60.01M carbonate buffer, 1.59g Na)₂CO₃And 2.93g NaHCO₃Adding deionized water to 1000 mL), diluting at 100 μ L/well (antigen concentration of 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078 μ g/mL) for each concentration, coating 1 line with 7 concentrations, performing blank control in the last line, incubating at 37 deg.C for 1h, coating enzyme label plate at 4 deg.C overnight, taking out, and washing with washing solution (PBST, 8.0g NaCl, 0.2g KCl, 2.86g Na₂HPO₄·12H₂O、0.27g KH₂PO₄And 0.5mL Tween-20 plus deionized water to 1000 mL) for 3 times, each for 3 min;

2) and (3) sealing: adding 250 μ L of sealing solution (5% skimmed milk powder, 100mL PBST added with 5g skimmed milk powder), sealing at 37 deg.C for 1h, and washing with PBST for 1 time;

3) adding serum: respectively transversely diluting M.haemosis positive serum (one part is randomly selected from serum collected from sows identified as positive by the qPCR method) and M.haemosis negative serum (one part is randomly selected from 135 parts of M.haemosis negative serum detected as negative by PCR) in the first 6 columns and the last 6 columns of the enzyme label plate in a multiple ratio (the dilution ratio is 1:4, 1:8, 1:16, 1:32, 1:64 and 1:128 respectively), longitudinally repeating 7 holes in each dilution, repeating 100 mu L/hole, reacting at 37 ℃ for 1h, and washing with PBST for 3 times;

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

5) TMB color development: adding substrate developing solution (TMB substrate developing solution), 100 μ L/hole, reacting at room temperature for 8 min;

6) and (3) terminating the reaction: adding stop solution (0.5M H)₂SO₄16.4mL of concentrated sulfuric acid is slowly dropped into 400mL of deionized water, the volume is supplemented to 500 mL), 50 mu L/hole is formed, and the reaction is stopped;

7) detection of OD450 value: respectively measuring OD values of M.haemosuis positive serum and M.haemosuis negative serum (namely positive serum OD) at 450nm by using enzyme-labeling instrument₄₅₀Value and negative serum OD₄₅₀Value) and calculates the P/N value. As positive serum OD₄₅₀The value (P) began to vary greatly, and the corresponding negative serum OD₄₅₀The antigen concentration and serum dilution corresponding to the value (N) being substantially unchanged are used as the antigen optimal coating concentration and serum optimal dilution.

The result of the recombinant Mh-PGK protein matrix titration test shows that the OD of the positive serum is obtained when the antigen concentration is between 0.500 mu g/hole and 0.031 mu g/hole₄₅₀The change was not significant, the OD of positive serum was between 0.031. mu.g/well and 0.015. mu.g/well dilutions₄₅₀The change in value is greatly reduced. Thus, positive serum OD was selected₄₅₀The antigen concentration at which a large reduction in value begins to occur (0.031. mu.g/well, i.e., 0.31. mu.g/mL) was used as the optimal antigen coating concentration to coat the recombinant Mh-PGK protein.

Positive serum OD at serum dilutions between 1:8 and 1:128₄₅₀The value is greatly reduced, and positive serum OD is obtained when the antigen is diluted at 1:8₄₅₀Value and negative serum OD₄₅₀Maximum ratio of values (P/N), negative serum OD₄₅₀The value is substantially unchanged. Therefore, 1:8 was chosen as the optimal dilution of serum.

2. Optimal coating solution selection

According to 1) -7) of step 1), the carbonate buffer solution of pH9.60.01M of the coating solution was replaced with PBS (8.0g NaCl, 0.2g KC) of pH 7.40.01M, respectivelyl、2.86g Na₂HPO₄·12H₂O and 0.27g KH₂PO₄Adding deionized water to 1000 mL), and adding Tris-HCl (1M Tris-HCl is obtained by adding 12.11g Tris base into about 80mL deionized water, dissolving, adding concentrated HCl to adjust pH value to 8.0, and diluting to 100 mL. Adding 1mL of 1M Tris-HCl into 99mL of deionized water, namely Tris-HCl with pH of 8.00.01M) and carbonate buffer solution with pH of 9.60.1M to coat the ELISA plate, and detecting the influence of each coating solution on the P/N value under the optimal conditions, wherein other steps are unchanged. Each coating solution was tested in 5 parts of m.haemosis positive serum (5 parts of the serum collected from sows identified as positive by the qPCR method described above were randomly selected) and 5 parts of m.haemosis negative serum (5 parts of the 135 parts of m.haemosis negative serum tested as negative by PCR) and each was repeated for 2 wells to determine OD₄₅₀Value, i.e. positive serum OD₄₅₀Value (P) and negative serum OD₄₅₀And (N), calculating the P mean value, the N mean value and the P/N value of different coating solutions, and selecting the coating solution with the maximum P/N value as the optimal coating solution.

The P-mean, N-mean and P/N values of different coating solutions 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 solution is Tris-HCl with pH8.00.01M, i.e. the coating effect is optimal, therefore, the optimal coating solution is Tris-HCl with pH 8.00.01M.

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

3. Selection of optimal blocking fluid and blocking time

According to steps 1) -7) of step 1, the blocking solution is blocked by replacing 5% skimmed milk powder with 2.5% skimmed milk powder (obtained by adding 2.5g skimmed milk powder to 100ml PSST), 5% skimmed milk powder (obtained by adding 5g skimmed milk powder to 100ml PSST), 7.5% skimmed milk powder (obtained by adding 7.5g skimmed milk powder to 100ml PSST), 1% BSA 1% (obtained by adding 1g BSA to 100ml PSST), 2% BSA (obtained by adding 2g BSA to 100ml PSST) or 3% BSA (obtained by adding 3g BSA to 100ml PSST), respectively, and the detection is carried out under the above-mentioned optimal conditionsAnd (3) measuring the influence of each confining liquid on the P/N value, wherein other steps are not changed. Each blocking solution was tested with 5 parts of m.haemosuis positive serum (5 parts of the serum collected from sows identified as positive by the qPCR method described above) and 5 parts of m.haemosuis negative serum (5 parts of the 135 parts of m.haemosuis negative serum tested negative by PCR) and each was repeated for 2 wells to determine OD₄₅₀Value, i.e. positive serum OD₄₅₀Value (P) and negative serum OD₄₅₀And (N) calculating the P-mean value, the N-mean value and the P/N value of different confining liquids, and selecting the confining liquid with the maximum P/N value as the optimal confining liquid.

The P-average, N-average and P/N values of the different confining liquids are shown in Table 2, and it can be seen from the P/N values that the P/N value is highest when the confining liquid is 5% skimmed milk powder, and therefore, the best confining liquid is 5% skimmed milk powder.

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

And (3) replacing the sealing time from 1h to 0.5h, 1h and 1.5h according to 1) to 7) in the step 1, detecting the influence of different sealing times on the P/N value under the optimal condition, and keeping other steps unchanged. For each blocking period, 5 m.haemosuis positive sera (5 randomly selected out of the sera collected from sows identified as positive by the qPCR method) and 5 m.haemosuis negative sera (5 randomly selected out of the 135 m.haemosuis negative sera that were negative by the PCR assay) were tested, and each replicate 2 wells for OD determination₄₅₀Value, i.e. positive serum OD₄₅₀Value (P) and negative serum OD₄₅₀And (N) calculating the P mean value, the N mean value and the P/N value of different closing time, and selecting the closing time when the P/N value is maximum as the optimal closing time.

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

TABLE 3 optimization of blocking time for indirect ELISA detection method

4. Optimal reaction time of serum

And (3) according to 1) to 7) in the step 1), replacing the serum reaction time from 1h to 0.5h, 1h and 1.5h, and detecting the influence of each serum reaction time on the P/N value under the optimal condition, wherein the other steps are not changed. For each serum reaction time, 5 m.haemosuis positive sera (5 randomly selected out of sera collected from sows identified as positive by the qPCR method) and 5 m.haemosuis negative sera (5 randomly selected out of 135 m.haemosuis negative sera that were negative by the PCR assay) were tested, and each was repeated for 2 wells to determine OD₄₅₀Value, i.e. positive serum OD₄₅₀Value (P) and negative serum OD₄₅₀And (N) calculating the P mean value, the N mean value and the P/N value of different serum reaction times, and selecting the serum reaction time when the P/N value is maximum as the optimal serum 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 the largest when the serum reaction time is 1h, and thus, the optimal serum reaction time is determined to be 1 h.

TABLE 4 optimization of the serum reaction time of the indirect ELISA detection method

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

And (3) according to 1) -7) in the step 1), replacing the dilution of the horseradish peroxidase labeled goat anti-porcine IgG (IgG-HRP) from 1:10000 to 1: 7500, 1:10000, 1:12500 and 100 mu L/hole, and detecting the influence of the dilution of each enzyme labeled secondary antibody on the P/N value under the optimal condition, wherein the other steps are not changed. Dilution of each enzyme-labeled secondary antibody 5Duplicate 2 wells of each of the m.haemosuis positive serum (5 random samples of the serum collected from the sows identified as positive by the qPCR method described above) and 5 m.haemosuis 5 samples, and OD was determined₄₅₀Value, i.e. positive serum OD₄₅₀Value (P) and negative serum OD₄₅₀And (N), calculating the P mean value, the N mean value and the 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-mean, N-mean and P/N values of the dilutions of different enzyme-labeled secondary antibodies are shown in Table 5, and the highest P/N value can be seen from the P/N values when the dilution of the enzyme-labeled secondary antibodies is 1:12500, so that the optimal dilution of the enzyme-labeled secondary antibodies is determined to be 1: 12500.

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

And (3) according to the steps 1) to 7) in the step 1), replacing the action time of the horseradish peroxidase-labeled goat anti-porcine IgG (IgG-HRP) from 1h to 30min, 60min and 90min, detecting the influence of the reaction time of each enzyme-labeled secondary antibody on the P/N value under the optimal condition, and keeping other steps unchanged. For each enzyme-labeled secondary antibody, 5 parts of m.haemosuis-positive serum (5 parts of the serum collected from the sow identified as positive by the qPCR method were randomly selected) and 5 parts of m.haemosuis-negative serum (5 parts of the 135 parts of the m.haemosuis-negative serum detected as negative by the PCR method) were assayed, and 2 wells were repeated to determine OD₄₅₀Value, i.e. positive serum OD₄₅₀Value (P) and negative serum OD₄₅₀And (N), calculating the P mean value, the N mean value and the P/N value of the reaction time of different enzyme-labeled secondary antibodies, and selecting the reaction time of the enzyme-labeled secondary antibody with the maximum P/N value as the optimal reaction time of the enzyme-labeled secondary antibody.

The P mean value, the N mean value and the P/N value of the reaction time of different enzyme-labeled secondary antibodies are shown in Table 6, and the P/N value can be seen to be the 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 antibody is determined to be 60 min.

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

6. The indirect ELISA detection method of the swine haemophilus antibody comprises the following steps:

according to the optimization test of the steps 1-5, the optimized indirect ELISA detection method for the swine haemophilus antibody comprises the following steps:

1) coating antigen: recombinant Mh-PGK protein is used as coating antigen, the coating solution (Tris-HCl with pH8.00.01M) is incubated at 37 ℃ for 1h at a coating concentration of 0.31 mu g/mL and 100 uL/hole, and then placed on an overnight coated enzyme label plate at 4 ℃, and the plate is washed 3 times with PBST for 3min each time;

2) and (3) sealing: adding 250 μ L of blocking solution (5% skimmed milk powder), blocking at 37 deg.C for 1h, and washing with PBST for 1 time;

4) adding an enzyme-labeled secondary antibody: adding horse radish peroxidase labeled goat anti-pig IgG (IgG-HRP) with the dilution of 1:12500 (diluted by a blocking solution), reacting at the temperature of 37 ℃ for 1h, and washing by PBST for 3 times;

6) and (3) terminating the reaction: adding stop solution (0.5M H)₂SO₄) Stop the reaction at 50. mu.L/well;

7) detection of OD450 value: the OD value of the serum of each animal to be tested was measured at a wavelength of 450nm using a microplate reader.

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

Carrying out PCR detection and screening on 135 parts of M.haemosuis negative serum sample, and optimizing according to step 6 to obtain the swine mycoplasma hyorhinis antibody indirect ELISA detection methodIndirect ELISA detection method, determination of M.haemosuis negative serum OD₄₅₀Value, calculating the average value

And Standard Deviation (SD) of less than

Value as negative, between

And

the value in between is judged suspicious, is greater than

The result was positive. Calculated mean value

0.181 and a standard deviation SD of 0.107, and therefore, the judgment standard was OD₄₅₀Value of>0.505 is positive (that is, the serum of the animal to be tested is positive serum of swine haemophilus mycoplasma, that is, the serum of the animal to be tested contains antibody of swine haemophilus mycoplasma, or the animal to be tested is infected with or has been infected with swine haemophilus mycoplasma), OD₄₅₀Value of<The animal is judged to be negative by 0.397 (namely the serum of the animal to be tested is the negative serum of the mycoplasma hyohaemophilus, namely the serum of the animal to be tested does not contain the mycoplasma hyohaemophilus antibody or the level of the mycoplasma hyohaemophilus antibody is lower than the lower detection limit), and OD is more than or equal to 0.397₄₅₀And (3) the value is less than or equal to 0.505, the animal is determined to be suspicious (namely the serum of the animal to be tested is suspected to be positive serum of the swine haemophilus mycoplasma, namely the serum of the animal to be tested is suspected to contain swine haemophilus mycoplasma antibodies or the animal to be tested is suspected to be infected or infected with the swine haemophilus mycoplasma).

8. Indirect ELISA (enzyme-linked immuno sorbent assay) detection kit for swine mycoplasma hyorhinis antibody

The indirect ELISA detection kit for the swine haemophilus antibody comprises an ELISA plate, coating liquid, confining liquid, washing liquid, M.haemosuis positive serum, M.haemosuis negative serum, enzyme-labeled secondary antibody, substrate developing liquid and stop solution, wherein the recombinant Mh-PGK protein shown by SEQ ID NO.1 in a sequence table is used as a coating antigen;

wherein the coating solution is Tris-HCl with pH8.00.01M; the confining liquid is 5% of skimmed milk powder; the washing solution is PBST; the enzyme-labeled secondary antibody is horse radish peroxidase-labeled goat anti-pig IgG (IgG-HRP); the substrate color developing solution is a TMB substrate color developing solution; the stop solution is 0.5M H₂SO₄。

Example 3 specificity and sensitivity test of Indirect ELISA detection method for Mycoplasma hyorhinis antibody

1. Specificity test

M.suis, M.parvum, M.haemosuis, porcine circovirus disease (PCV), swine fever (CSFV), Porcine Reproductive and Respiratory Syndrome (PRRS), porcine toxoplasma gondii (PT) and Foot and Mouth Disease (FMD)8 common swine disease positive sera were detected by the indirect ELISA detection method for porcine haemophilus antibody established in example 2, and OD was determined₄₅₀And determining the specificity of the indirect ELISA detection method for the swine mycoplasma hyorhinis antibody for detecting the serum.

The specific detection results are shown in table 7, and the results show that the kit has no cross reaction with the serum of common swine diseases (porcine circovirus disease, swine fever, porcine reproductive and respiratory syndrome, swine toxoplasma and foot and mouth disease) and can simultaneously detect the positive serum of three porcine-derived mycoplasma hyopneumoniae (m.suis, m.parvum and m.haemosuis).

TABLE 7 ELISA specificity test results

2. Repeatability test

Batch to batch repeatability test: randomly selecting 7 parts of M.haemosis negative serum (each serum is provided with 3 times of repetition) from 135 parts of M.haemosis negative serum samples screened by PCR detection, coating an ELISA plate with 3 batches (0802, 0804 and 0808) of recombinant Mh-PGK protein by using the indirect ELISA detection method for swine mycoplasma haemophilus antibodies established in example 2, and respectively coating the ELISA plate with the OD (optical density) of each serum₄₅₀Calculate the mean values separately

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

The results are shown in Table 8, and the OD detected by ELISA for different batches of antigen recombinant Mh-PGK proteins₄₅₀The coefficient of variation of the value is between 2.3 and 8.9 percent, which shows that the repeatability among antigen batches is good.

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

Example 4 application of Indirect ELISA detection method for swine haemophilus antibodies

Collecting 795 parts of pig blood from 18 pig farms in Zhejiang, Henan, Anhui, Jiangsu, etc., and storing the separated serum in a laboratory at-20 deg.C for later use;

the detection of 795 sera was carried out by indirect ELISA detection method for Mycoplasma hyorhinis antibody established in example 2, and the results are shown in Table 9, which indicates that the seropositivity of Mycoplasma hyorhinis antibody is 37.9% (301/795).

25 parts of positive serum are randomly selected from 202 parts of positive serum, and the positive serum is detected to be positive through whole bacteria antigen ELISA, so that the positive coincidence rate of the invention is 100%.

TABLE 9 Indirect ELISA for detection of serum antibodies to swine-derived Mycoplasma hyorhinis

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.

SEQUENCE LISTING

<110> Zhejiang province academy of agricultural sciences

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

<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 sequence

<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> artificially synthesized 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 in SEQ ID NO. 1.

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

and (3) constructing a recombinant plasmid by using the optimized Mh-PGK gene shown in the 7 th to 1239 th sites of SEQ ID NO.2, and expressing the recombinant plasmid by using an expression system to obtain the recombinant Mh-PGK protein.

3. The recombinant Mh-PGK protein of claim 2, wherein the recombinant plasmid is pET 28-Mh-PGK; the pET28-mh-pgk is a recombinant plasmid obtained by replacing a DNA fragment between NdeI enzyme recognition sequences and XhoI enzyme recognition sequences of pET-28a (+) plasmid with an optimized mh-pgk gene shown in the 7 th to 1239 th sites of SEQ ID NO.2 in a sequence table.

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

5. The use according to claim 4, wherein the Mycoplasma hyorhinis is Mycoplasma suis, Mycoplasma parvum and/or Mycoplasma haemosynus.

6. An indirect ELISA detection kit for a swine mycoplasma hyorhinis antibody, characterized in that the kit comprises an ELISA plate using the recombinant Mh-PGK protein of any one of claims 1-3 as a coating antigen.

7. The indirect ELISA detection kit for Mycoplasma hyorhinis antibody according to claim 6, further comprising one or more of a coating solution, a blocking solution, a washing solution, Mycoplasma haemobiosis positive serum, Mycoplasma haemobiosis negative serum, an enzyme-labeled secondary antibody, a substrate developing solution and a stop solution.

8. The indirect ELISA detection kit for mycoplasma hyorhinis antibody according to claim 7, wherein the coating concentration of the recombinant Mh-PGK protein is 0.31 μ g/mL;

and/or, the coating solution is carbonate buffer solution with pH9.60.01M, PBS with pH7.40.01M or Tris-HCl with pH8.00.01M; preferably Tris-HCl of pH8.00.01M;

and/or the blocking solution is 2.5% skimmed milk powder, 5% skimmed milk powder, 7.5% skimmed milk powder, 1% BSA, 2% BSA or 3% BSA; preferably 5% skimmed milk powder;

and/or, the wash solution is PBST;

and/or the enzyme-labeled secondary antibody is horse radish peroxidase-labeled goat anti-pig IgG;

and/or the dilution of the enzyme-labeled secondary antibody is 1: 12500;

and/or the substrate color developing solution is a TMB substrate color developing solution;

and/or the stop solution is 0.5M H₂SO₄。

9. Use of the indirect ELISA detection kit for mycoplasma hyorhinis antibody of any one of claims 6-8 for detecting mycoplasma hyorhinis antibody.

10. An indirect ELISA detection method for a swine mycoplasma hyorhinis antibody is characterized by comprising the following steps:

1) coating antigen: coating the enzyme label plate with the recombinant Mh-PGK protein of any one of claims 1-3 by using a coating solution, and washing by using a detergent;

2) and (3) sealing: adding sealing liquid, sealing, and washing with detergent;

8) and (4) interpretation of results: the determination criterion was OD₄₅₀Value of>0.505 positive, OD₄₅₀Value of<The negative result was found when the OD was 0.397. ltoreq.OD₄₅₀The value is less than or equal to 0.505, and the person is judged to be suspicious;

preferably, the optimal coating solution is Tris-HCl with the pH value of 8.00.01M, the optimal coating concentration is 0.31 mu g/mL, the optimal blocking solution is 5% skimmed milk powder, the optimal blocking time is 1h, and the optimal dilution of serum is 1:8, the optimal reaction time of 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 1 h.