A recombinant Mh-DnaK protein and application thereof in detection of mycoplasma haemofelis

Abstract

The application discloses a recombinant Mh-DnaK protein and application thereof in detection of porcine hemophilic mycoplasma, and discloses the recombinant Mh-DnaK protein with an amino acid sequence as shown in SEQ ID NO. 1. The application further discloses a porcine hemophilic mycoplasma antibody indirect ELISA detection kit and a detection method, wherein the above-mentioned recombinant Mh-DnaK protein is used as a coated antigen. The application establishes a porcine hemophilic mycoplasma antibody indirect ELISA detection method based on the recombinant Mh-DnaK protein, which is used for detecting porcine hemophilic mycoplasma antibody level, does not cross-react with part of other pig disease serum, has good specificity and repeatability, and provides an effective means for clinical diagnosis, epidemiological study and immune detection of Mycoplasma suis, Mycoplasma parvum and Mycoplasma haemosuis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology. More particularly, it relates to a recombinant Mh-DnaK protein and its application in detecting Mycoplasma haemofelis. BACKGROUND

[0002] Hemotropic mycoplasma (HM, also known as eperythrozoon) is a group of mycoplasma that parasitizes on the surface of red blood cells and bone marrow of various mammals, belonging to the class of Mollicutes and the genus of Mycoplasma (Lu Chengping, editor. Veterinary microbiology[M]. Sixth edition. Beijing: China Agriculture Press, 2021: 223-225). HM destroys host red blood cells, causing hemolysis and infectious anemia, leading to stillbirth of sows, and skin pale and mucous membrane yellowing and emaciation of piglets and growing pigs caused by fever and anemia (Hoelzle L E, Felder K M, Hoelzle K. Porcine eperythrozoonosis: from Eperythrozoon suis to Mycoplasma suis[J]. Tierarztl Prax Ausg G Grosstiere Nutztiere. 2011, 39(4): 215-220.).Three HMs are prevalent in Chinese pig populations, Mycoplasma suis (M. suis formerly known as Eperythrozoon suis), M. parvum, and M. haemosuis reported in China in 2017 (Zhang H, Xie M, Zhang J, et al. Sequence determination and phylogenetic analysis of the 16S rRNA gene of Eperythrozoon suis. Chinese Journal of Animal and Veterinary Sciences. 2005(06):596-601. 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 Vet Med Sci. 2011, 73(12): 1657-1661. Fu Y, Shi T Y, Xu L H, et al. Identification of a novel Hemoplasma species from pigs in Zhejiang province, China. J Vet Med Sci. 2017, 79(5): 864-870), and a fourth hemoplasma infecting pigs was reported in Thailand in 2022, which has not been reported in China yet (Thongmeesee K, Kamkong P, Thanee S, Wattanapansak S, Kaewthamasorn M, Tiawsirisup S. Molecular detection and genetic analysis of porcine haemoplasmas in commercial pig farms from Thailand reveal a putative novel species. Transbound Emerg Dis. 2022 Mar 26. doi: 10.1111 / tbed.14537. PubMed PMID: 35338759.).

[0003] Epidemiological investigation by fluorescent quantitative PCR method found that M. haemosuis was prevalent in Zhejiang, Jiangsu, Henan and other places in China, mixed infection with other two pig-derived HMs often occurred, and the infection rate of sows was higher (Fu Y, Shi T Y, Xu L H, et al. Identification of a novel Hemoplasma species from pigs in Zhejiang province, China [J]. J Vet Med Sci. 2017, 79(5): 864-870.; Fu Y, Shi T Y, Xu L H, et al. Establishment and application of a triple TaqMan probe fluorescent quantitative PCR method for porcine hemoplasma. Acta Agricultrurae Biodiversitatis Sinica, 2022. 30(6)), M. haemosuis was also detected in pig populations in South Korea, Germany and Thailand (Thongmeesee K, Kamkong P, Thanee S, Wattanapansak S, Kaewthamasorn M, Tiawsirisup S. Molecular detection and genetic analysis of porcine haemoplasmas in commercial pig farms from Thailand reveal a putative novel species. Transbound Emerg Dis. 2022 Mar 26. doi: 10.1111 / tbed.14537. PubMed PMID: 35338759.; Seo M G, Kwon O D, Kwak D. 2019. Prevalence and phylogenetic analysis of hemoplasma species in domestic pigs in Korea. Parasites Vectors, 12(1): 378.; Stadler J, Ade J, Ritzmann M, et al. 2020. Detection of a novel haemoplasma species in fattening pigs with skin alterations, fever and anaemia. Vet Record, 187(2): 66.), and there have been reports of M. suis and M. parvum serum antibody detection methods (Amit Kadam, Wang Jinxiu, Xie Gaokun, et al. Serological and molecular epidemiological investigation reveals that Mycoplasma suis / Eperythrozoon is the main pig hemoplasma on Hainan Island.Parasit Vectors. 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]. Microbes Infect. 2007, 9(4): 466-474.) and lack of antibody detection methods for M. haemosuis.

[0004] Molecular chaperone proteins play an important role in assisting the folding, assembly and transport of newly synthesized polypeptides. DnaK is the central hub in the E. coli chaperone network (Calloni G, Chen T, Schermann SM, Chang HC, Genevaux P, Agostini F, Tartaglia GG, Hayer-Hartl M, Hartl FU. DnaK functions as a central hub in the E. coli chaperone network. Cell Rep. 2012 Mar 29;1(3):251-64. pii: S2211-1247(11)00017-9. doi: 10.1016 / j.celrep.2011.12.007. PubMed PMID: 22832197.) and is also an important molecular chaperone protein of Mycoplasma, belonging to the heat shock protein 70 (HSP70) family. Mycoplasma DnaK plays a key role in the pathway of DNA damage recognition and repair. The protein binds to poly (ADP-ribose) polymerase (PARP)-1, reduces its catalytic activity, and also affects the stability and anti-tumor function of the host cell p53 gene (Benedetti F, Cocchi F, Latinovic OS, Curreli S, Krishnan S, Munawwar A, Gallo RC, Zella D. Role of Mycoplasma Chaperone DnaK in Cellular Transformation. Int J Mol Sci. 2020 Feb 15;21(4) pii: ijms21041311. doi: 10.3390 / ijms21041311. PubMed PMID: 32075244.). At present, there is no report on the prokaryotic expression of DnaK protein of M. haemosuis.

[0005] Therefore, cloning the gene sequence of DnaK protein of M. haemosuis of porcine hemophilic Mycoplasma and constructing an indirect ELISA detection method for porcine hemophilic Mycoplasma based on the protein are of great significance for the clinical diagnosis, epidemiological study and immune detection of porcine hemophilic Mycoplasma. SUMMARY

[0006] The first object of the present application is to provide a recombinant DnaK protein (referred to as recombinant Mh-DnaK protein) of M. haemosuis of porcine hemophilic Mycoplasma for detecting the antibody level of porcine hemophilic Mycoplasma and epidemiological monitoring, and evaluating the role of the disease in the prevalence of porcine hemophilic Mycoplasma disease.

[0007] A second object of the present application is to provide an application of the recombinant Mh-DnaK protein in detecting Mycoplasma haemosuis and / or in detecting Mycoplasma haemosuis antibody.

[0008] A third object of the present application is to provide a Mycoplasma haemosuis antibody indirect ELISA detection kit.

[0009] A fourth object of the present application is to provide a Mycoplasma haemosuis antibody indirect ELISA detection method.

[0010] To achieve the above objects, the present application adopts the following technical solutions:

[0011] In a first aspect, the present application provides a Mycoplasma haemosuis recombinant DnaK protein, referred to as a recombinant Mh-DnaK protein, the amino acid sequence of the recombinant Mh-DnaK protein is shown in SEQ ID NO. 1.

[0012] The recombinant Mh-DnaK protein of the present application is prepared by the following method:

[0013] The recombinant Mh-DnaK protein is obtained by constructing a recombinant plasmid with the optimized mh-dnaK gene shown in SEQ ID NO. 2 at positions 4-1812 and expressing it through an expression system; wherein the optimized mh-dnaK gene is codon-optimized for the mh-dnaK gene shown in SEQ ID NO. 3 (encoding the Mh-DnaK protein with the amino acid sequence shown in SEQ ID NO. 1 at positions 21-622), and the three TGA are optimized to the codon TGG that can encode tryptophan in E. coli and part of the codons are optimized to the E. coli preferred codon.

[0014] In a specific embodiment of the present application, the recombinant plasmid is pET28-mh-dnaK. The pET28-mh-dnaK is a recombinant plasmid obtained by replacing the DNA fragment between the NdeI enzyme and XhoI enzyme recognition sequences of the pET-28a(+) plasmid with the optimized mh-dnaK gene shown in SEQ ID NO. 2 at positions 4-1812 in the sequence listing, and the recombinant plasmid can express the recombinant Mh-DnaK protein shown in SEQ ID NO. 1 in the sequence listing; wherein the amino acid sequence encoded by the DNA sequence on the pET-28a(+) plasmid is SEQ ID NO. 1 at positions 1-20, and the Mh-DnaK protein sequence is SEQ ID NO. 1 at positions 21-622.

[0015] Further, the expression system is an E. coli expression system.

[0016] In a specific embodiment of the present application, the expression is that pET28-mh-dnaK is transformed into E. coli BL21 (DE3) to induce expression of recombinant Mh-DnaK protein.

[0017] In a second aspect, the present application provides the above-mentioned recombinant Mh-DnaK protein for use in any one of the following:

[0018] 1) detecting whether the serum of the animal to be tested contains porcine hemophilic mycoplasma antibody;

[0019] 2) in the preparation of products for detecting whether the serum of the animal to be tested contains porcine hemophilic mycoplasma antibody;

[0020] 3) detecting whether the animal to be tested is infected or has been infected with porcine hemophilic mycoplasma;

[0021] 4) in the preparation of products for detecting whether the animal to be tested is infected or has been infected with porcine hemophilic mycoplasma.

[0022] Further, the porcine hemophilic mycoplasma is Mycoplasma suis, Mycoplasma parvum and / or Mycoplasma haemosuis.

[0023] In a third aspect, the present application provides a porcine hemophilic mycoplasma antibody indirect ELISA detection kit, which comprises an enzyme-labeled plate with the above-mentioned recombinant Mh-DnaK protein as a coating antigen.

[0024] Further, the coating concentration of the recombinant Mh-DnaK protein is 0.44 μg / mL.

[0025] Further, the kit further comprises one or more of coating solution, blocking solution, washing solution, M. haemosuis positive serum, M. haemosuis negative serum, enzyme-labeled secondary antibody, substrate developing solution and stop solution.

[0026] Further, the coating solution is a pH 9.6 0.01M carbonate buffer (1.59g Na2CO3 and 2.93g NaHCO3 are added to deionized water to 1000mL).

[0027] Further, the blocking solution is 2.5% skimmed milk powder (100mL PBST added with 2.5g skimmed milk powder), 5% skimmed milk powder (100mL PBST added with 5g skimmed milk powder), 7.5% skimmed milk powder (100mL PBST added with 7.5g skimmed milk powder); preferably 5% skimmed milk powder.

[0028] Further, the washing solution is PBST (8.0 g NaCl, 0.2 g KCl, 2.86 g Na2HPO4·12H2O, 0.27 g KH2PO4 and 0.5 mL Tween-20, and deionized water to 1000 mL).

[0029] Further, the enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-swine IgG (IgG-HRP) (purchased from KPL Company).

[0030] Further, the dilution of the enzyme-labeled secondary antibody is 1:12500.

[0031] Further, the substrate developing solution is TMB substrate developing solution (purchased from Shanghai Bioengineering Co., Ltd., item number E661007).

[0032] Further, the termination solution is 0.5M H2SO4 (13.6 mL of concentrated sulfuric acid is slowly added into 400 mL of deionized water, and the volume is made up to 500 mL).

[0033] The application of the above-mentioned porcine hemophilic mycoplasma antibody indirect ELISA detection kit in detecting porcine hemophilic mycoplasma antibody is also within the protection scope of the present application.

[0034] In a fourth aspect, the present application provides a porcine hemophilic mycoplasma antibody indirect ELISA detection method, comprising the following steps:

[0035] 1) Coating antigen: the above-mentioned recombinant Mh-DnaK protein is used as a coating antigen to coat an enzyme-labeled plate with a coating solution, and a washing agent is used for washing;

[0036] 2) Blocking: a blocking solution is added for blocking, and a washing agent is used for washing;

[0037] 3) Adding serum: the serum of an animal to be tested is added for reaction, and a washing agent is used for washing;

[0038] 4) Adding enzyme-labeled secondary antibody: an enzyme-labeled secondary antibody is added for reaction, and a washing agent is used for washing;

[0039] 5) TMB developing: a substrate developing solution is added for reaction;

[0040] 6) Reaction termination: a termination solution is added to terminate the reaction;

[0041] 7) Detecting OD 450 value of the serum of each animal to be tested by using an enzyme-labeled instrument; 450

[0042] 8) Result interpretation: the judgment standard is OD 450 ​A value >0.358 is considered positive (i.e., the serum of the test animal is positive for Haemophilus suis, meaning the serum of the test animal contains Haemophilus suis antibodies or the test animal has been infected with or has been infected with Haemophilus suis). OD 450 A value <0.275 is considered negative (i.e., the serum of the tested animal is negative for Haemophilus influenzae from pigs, meaning the serum of the tested animal does not contain Haemophilus influenzae from pigs or the level of Haemophilus influenzae from pigs is below the detection limit), and 0.275 ≤ OD 450 A value ≤0.358 is considered suspicious (i.e., the serum of the animal being tested is suspected to be positive for Haemophilus suis, meaning that the serum of the animal being tested is suspected to contain Haemophilus suis antibodies or the animal being tested is suspected to be infected with or has been infected with Haemophilus suis).

[0043] In this invention, the optimal coating concentration, blocking solution, blocking time, optimal serum dilution, serum reaction time, enzyme-labeled secondary antibody reaction time, and color development time were optimized. The optimal coating concentration was 0.44 μg / mL, the optimal blocking solution was 5% skim milk powder, the optimal blocking time was 1 h, the optimal serum dilution was 1:40, the optimal serum reaction time was 1 h, the optimal enzyme-labeled secondary antibody reaction time was 0.5 h, and the optimal substrate color development time was 7 min.

[0044] In a preferred embodiment of the present invention, the indirect ELISA detection method for porcine Haemophilus influenzae antibodies includes the following steps:

[0045] 1) Coating antigen: The above recombinant Mh-DnaK protein was used as the coating antigen. The coating concentration was 0.44 μg / mL in 0.01M carbonate buffer (pH 9.6), 100 μL / well. The plate was incubated at 37°C for 1 h and then placed at 4°C overnight to coat the microplate. The plate was washed 3 times with PBST for 3 min each time.

[0046] 2) Blocking: Add 250 μL of 5% skim milk powder, block at 37°C for 1 hour, and wash once with PBST;

[0047] 3) Add serum: Add 100 μL of serum from the animal to be tested at a dilution of 1:40, react at 37°C for 1 h, and wash 3 times with PBST;

[0048] 4) Add enzyme-labeled secondary antibody: Add horseradish peroxidase-labeled goat anti-pig IgG at a dilution of 1:12500, 100 μL / well, react at 37℃ for 0.5 h, and wash 3 times with PBST;

[0049] 5) TMB color development: Add 100 μL of TMB substrate to each well and react for 7 min;

[0050] 6) Reaction termination: 0.5M H2SO4, 50μL / well, to terminate the reaction;

[0051] 7) Detection OD 450 value: use the enzyme marker to measure the OD 450 value of each animal serum to be tested;

[0052] 8) Result interpretation: the determination criteria is OD 450 value > 0.358 is positive (i.e. the serum of the animal to be tested is Mycoplasma suis positive serum, that is, the serum of the animal to be tested contains Mycoplasma suis antibody or the animal to be tested is infected or infected with Mycoplasma suis), OD 450 value < 0.275 is negative (i.e. the serum of the animal to be tested is Mycoplasma suis negative serum, that is, the serum of the animal to be tested does not contain Mycoplasma suis antibody or the level of Mycoplasma suis antibody is lower than the detection lower limit), 0.275≤OD 450 value ≤ 0.358 is suspicious (i.e. the serum of the animal to be tested is suspected to be Mycoplasma suis positive serum, that is, the serum of the animal to be tested is suspected to contain Mycoplasma suis antibody or the animal to be tested is suspected to be infected or infected with Mycoplasma suis).

[0053] In the present application, the Mycoplasma suis is early identified Mycoplasma suis (M.suis), Mycoplasma parvum (M.parvum) and / or Mycoplasma haemosuis (M.haemosuis). The animal to be tested is a pig.

[0054] The beneficial effects of the present application are as follows:

[0055] The present application establishes a Mycoplasma suis antibody indirect ELISA detection method based on recombinant Mh-DnaK protein for detecting Mycoplasma suis antibody level, which 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 study and immune detection of Mycoplasma suis, Mycoplasma parvum and Mycoplasma haemosuis. BRIEF DESCRIPTION OF DRAWINGS

[0056] The specific embodiments of the present application will be further described in detail below with reference to the accompanying drawings.

[0057] Figure 1SDS-PAGE analysis of recombinant Mh-DnaK protein expression in E. coli BL21; wherein, M1, M2 represent Protein Marker; 1 represents the expression of recombinant protein in recombinant E. coli pET28 / BL21(DE3); 2 represents the expression of recombinant protein in uninduced recombinant E. coli pET28-mh-dnaK / BL21(DE3); 3 represents the expression of recombinant protein in IPTG-induced recombinant E. coli pET28-mh-dnaK / BL21(DE3); 4 represents the SDS-PAGE detection results of purified recombinant Mh-DnaK protein.

[0058] Figure 2 Western-blot analysis of the immunoreactivity of recombinant Mh-DnaK protein; wherein, M represents pre-stained Protein Marker; 1 represents the Western Blot reaction of mouse anti-recombinant Mh-DnaK protein serum and recombinant Mh-DnaK protein, and the arrow indicates the reaction band; 2 represents the Western Blot reaction of mouse negative serum and recombinant Mh-DnaK protein. DETAILED DESCRIPTION

[0059] In order to more clearly illustrate the present application, the present application will be further described below in conjunction with preferred embodiments and the accompanying drawings. Like components are denoted by the same reference numerals in the drawings. Those skilled in the art should understand that the specific descriptions below are illustrative rather than limiting, and should not limit the protection scope of the present application.

[0060] The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial channels unless otherwise specified; wherein, the main sources of materials, reagents and instruments are as follows:

[0061] E. coli BL21(DE3) competent cells were purchased from Beijing Zixingjin Biotechnology Co., Ltd.;

[0062] pET-28a(+) plasmid was purchased from Shanghai Bioengineering Co., Ltd., with the order number B540183;

[0063] M. haemosuis negative serum was imported from breeding pig serum, provided by Zhejiang Entry-Exit Inspection and Quarantine Bureau Inspection and Quarantine Technology Center, and was negative by PCR detection, a total of 148 portions.

[0064] M. haemosuis positive serum was serum collected from sows identified as positive by qPCR method, a total of 25 portions;

[0065] Three kinds of pig-derived hemophilic mycoplasma (M. suis, M. parvum, M. haemosuis) were identified by PCR method after the pathogen positive, and the whole bacterial antigen ELISA was identified after the antibody positive serum was stored;

[0066] The positive serum of pig toxoplasma was identified by PCR method after the pathogen positive by the laboratory of the present study, and the antibody positive serum was stored after identifying by Lanzhou Veterinary Research Institute toxoplasma IHA diagnostic kit;

[0067] The positive serum of pig toxoplasma was identified by PCR method after the pathogen positive by the laboratory of the present study, and the antibody positive serum was stored after identifying by Lanzhou Veterinary Research Institute toxoplasma IHA diagnostic kit;

[0068] Peroxidase goat anti-pig IgG-HRP was purchased from KPL company;

[0069] Affinity chromatography nickel beads Ni-Argrose was purchased from Invitrogen company;

[0070] TMB substrate color developing liquid was purchased from Shanghai Shengwo Biological Engineering Technology Service Co., Ltd., and the article number was E661007;

[0071] The enzyme-labeled instrument was Spectra Max M5, which was the product of American Molecular Devices company;

[0072] The protein electrophoresis instrument was the product of Bio-Rad company.

[0073] Example 1 Preparation of pig-derived hemophilic mycoplasma antibody indirect ELISA antigen and detection of antigenicity

[0074] 1. Preparation of antigen

[0075] 1) Optimization of gene

[0076] According to the dnaK gene (referred to as mh-dnaK gene, the nucleotide sequence of which is shown in SEQ ID NO. 3, which consists of 1809 nucleotides, and encodes the M. haemosuis DnaK protein (referred to as Mh-DnaK protein) whose amino acid sequence is shown in SEQ ID NO. 1, 21-622) amplified from the local strain M. haemosuis ZJ1102 in Zhejiang, three tryptophan-encoding codons TGA (due to the particularity of the genetic coding system of mycoplasma, TGA encodes a tryptophan non-stop codon) are optimized to the tryptophan-encoding codon TGG in E. coli and part of the codons are optimized to the E. coli preferred codon, to obtain the optimized mh-dnaK gene (the nucleotide sequence of which is shown in SEQ ID NO. 2, 4-1812, which consists of 1809 nucleotides, and the amino acid sequence of the obtained Mh-DnaK protein is unchanged), which is synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0077] 2) Induction expression of recombinant protein

[0078] A CAT is added to the 5' end of the optimized mh-dnaK gene to form an NdeI enzyme cleavage site (5' catatg 3') with the ATG itself, and an XhoI enzyme cleavage site (5' ctcgag 3') is added to the 3' end (the sequence is shown in SEQ ID NO. 2, which is synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.), and the NdeI enzyme and XhoI enzyme are used for double enzyme digestion, and the pET-28a(+) plasmid is also double enzyme digested with NdeI enzyme and XhoI enzyme. The digested optimized mh-dnaK gene and pET-28a(+) plasmid are ligated with DNA ligase to construct the recombinant plasmid pET28-mh-dnaK, which is transformed into E. coli Top10, and the plasmid is extracted and sequenced by Shanghai Bioengineering Co., Ltd. to determine that the inserted sequence is correct. The pET28-mh-dnaK is a recombinant plasmid obtained by replacing the DNA fragment between the NdeI enzyme and XhoI enzyme recognition sequences of the pET-28a(+) plasmid with the optimized mh-dnaK gene shown in SEQ ID NO. 2, 4-1812 in the sequence table, and the recombinant plasmid can express the fusion protein shown in SEQ ID NO. 1 in the sequence table, i.e. the M. haemosuis recombinant DnaK protein, referred to as recombinant Mh-DnaK protein; wherein SEQ ID NO. 1, 1-20 in the sequence table is the amino acid sequence encoded by the DNA sequence on the pET-28a(+) plasmid, and SEQ ID NO. 1, 21-622 is the Mh-DnaK protein sequence.

[0079] The recombinant plasmid pET28-mh-dnaK with correct sequence was transformed into E. coli BL21(DE3) to obtain recombinant E. coli pET28-mh-dnaK / BL21(DE3). The recombinant E. coli pET28-mh-dnaK / BL21(DE3) was induced for expression with 0.8 mM IPTG (37°C, 4 h), and the bacterial liquid was collected before induction and 4 h after induction. The above experiment was repeated with pET28a instead of pET28-mh-dnaK to obtain recombinant E. coli pET28 / BL21(DE3), and the bacterial liquid was collected 4 h after induction. 100 μL of each of the three bacterial liquids was centrifuged at 8000 rpm for 5 min at 4°C to precipitate the bacterial cells, resuspended in 40 μL double distilled water, added with 10 μL 5x SDS loading buffer and boiled, and used for SDS-PAGE to detect the expression of recombinant protein in the recombinant E. coli pET28-mh-dnaK / BL21(DE3) before induction and after induction with IPTG, and the expression of recombinant protein in the recombinant E. coli pET28 / BL21(DE3). The results are shown in Figure 1 Figure 1, and the recombinant E. coli pET28-mh-dnaK / BL21(DE3) after induction with IPTG showed a protein band at about 66.2 kD, which was consistent with the expected 67.4 kD, indicating that the recombinant Mh-DnaK protein was expressed; and the recombinant E. coli pET28 / BL21(DE3) and the recombinant E. coli pET28-mh-dnaK / BL21(DE3) before induction showed no expression of corresponding protein.

[0080] 3) Purification of recombinant protein

[0081] The IPTG-induced recombinant E. coli pET28-mh-dnaK / BL21(DE3) bacterial solution was centrifuged, the precipitate was resuspended with PBS, and was broken by ultrasonic wave. The precipitate was centrifuged again for 10 min, was washed with PBS (pH 7.4) twice, was resuspended with 19.7 mL of buffer A (Tris-HCl 6.06 g, EDTA 0.186 g, NaCl 2.92 g, glycerol 50 mL, and sterile deionized water to 1 L) and 0.3 mL of 20% sodium dodecyl sulfate (SKL) stock solution, was allowed to stand overnight, was centrifuged again for 10 min, the precipitate was discarded, the supernatant was taken, 20% PEG4000 (2 g of PEG4000 was dissolved in 10 mL of sterile deionized water) was added to the supernatant to a final concentration of 0.2%, 50 mmol / L of oxidized glutathione (0.153 g of oxidized glutathione was dissolved in 5 mL of deionized water) was added to a final concentration of 1 mmol / L, and 100 mmol / L of reduced glutathione (0.153 g of reduced glutathione was dissolved in 5 mL of deionized water) was added to a final concentration of 2 mmol / L, and was allowed to stand for 30 min to 2 h, was dialyzed against 0.01 M PBS (pH 7.4) at 4°C for 2 days, and the extracted inclusion bodies were obtained.

[0082] About 20 mL of the inclusion bodies were taken, 5 mL of Ni-Agarose that was previously equilibrated with pH 7.4 Binding buffer (purchased from Shanghai Sangon, item number C600303) was added, and was combined at room temperature for 30 min. During the combination, the mixture was shaken 3-5 times, was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the precipitate was washed with 20 mM, 30 mM, 40 mM, and 50 mM imidazole Binding buffer 4 times. 250 mM Elution buffer 5 mL / time was used for elution 3 times, the eluted Elution proteins were collected, were dialyzed against PBS pH 7.4 for 24 h, were concentrated to half of the volume, the protein solution was centrifuged at 10000 rpm for 10 min to take the supernatant, and the purified recombinant Mh-DnaK protein was obtained. The concentration of the purified recombinant Mh-DnaK protein was determined using a Shanghai Sangon BAC protein quantitative detection kit according to the instructions (the concentration of the purified recombinant Mh-DnaK protein was 1.4 mg / mL), and the purified recombinant Mh-DnaK protein was aliquoted and stored at -20°C for later use. The purified recombinant Mh-DnaK protein was detected by SDS-PAGE, and the results are shown in FIG. 2. The results show that only a single band appears at about 66.2 kD, indicating that the purification recovery is good, and the obtained recombinant Mh-DnaK protein has the amino acid sequence shown in SEQ ID NO. 1. Figure 1

[0083] 2. Antigenicity detection

[0084] 1) Preparation of mouse anti-recombinant Mh-DnaK protein serum: ​

[0085] BALB / c mice, 5 female, were selected to prepare the antibody according to the following steps:

[0086] First immunization: the purified recombinant Mh-DnaK protein (i.e. antigen) was dissolved in PBS and mixed with an equal volume of Freund's complete adjuvant by double-push method, and then subcutaneously injected into the back and muscles of the legs of 5 BALB / C mice in multiple points, with a dose of 40 μg per mouse; Second immunization: 14 days later, the same amount of antigen was mixed with incomplete Freund's adjuvant and then immunized in the same way, with a dose of 50 μg per mouse; 28 days later, the same amount of antigen was mixed with incomplete Freund's adjuvant and then immunized in the same way, with a dose of 50 μg per mouse, and the titer was determined by ELISA; after the last immunization, the serum titer was determined by tail bleeding, and if the desired purpose was achieved, the antiserum was collected. The mouse with high titer was selected for blood collection, and the serum was separated, thereby obtaining the mouse anti-recombinant Mh-DnaK protein serum.

[0087] 2) Western-blot: the purified recombinant Mh-DnaK protein was adjusted to a concentration of 365 μg / mL, and 10 μL per well was loaded for SDS-PAGE electrophoresis. After the electrophoresis, the gel strip was cut to an appropriate size, and the transfer buffer was equilibrated for 5 min x 3 times. The nitrocellulose (NC) membrane and filter paper were pre-cut to the same size as the gel strip and immersed in the transfer buffer for 10 min.

[0088] Electrotransfer: the positive electrode, filter paper, NC membrane, gel, filter paper, and negative electrode were placed in order, and the filter paper, NC membrane, and gel were aligned without air bubbles. According to the gel area, the current was connected at 0.65 mA-1.0 mA / cm2, and the electrotransfer was performed for 2 h.

[0089] Staining: the power was turned off, and each layer was lifted one by one. The gel was transferred to the ponceau red staining solution, and the protein band appeared on the membrane. After taking a photo, the nitrocellulose filter membrane was rinsed with deionized water until no protein band was observed.

[0090] Blocking: the transferred nitrocellulose filter membrane was blocked with PBST containing 1% BSA at room temperature for 2 h, and the blocking solution was discarded.

[0091] Primary antibody and target protein binding: the primary antibody was diluted with PBST, and the mouse anti-recombinant Mh-DnaK protein serum was diluted at 1:500 as the first antibody. The nitrocellulose membrane with Mh-DnaK protein was placed on a shaker at 4 degrees overnight. The nitrocellulose filter membrane was washed with PBST for 5 min x 3 times.

[0092] Secondary antibody binding: PBST dilute 1:3000 horseradish peroxidase (HRP) labeled rabbit anti-mouse secondary antibody, and nitrocellulose membrane was placed on a rocking bed at room temperature for 2h. The nitrocellulose filter was washed with PBST for 5min x 3 times. Add chemiluminescent reagent for color development: place the nitrocellulose filter into 1mL ECL luminescent reagent (Shanghai Biotech, item number C510043), expose and take a picture.

[0093] The above mouse anti-recombinant Mh-DnaK protein serum was replaced with mouse negative serum to repeat the above Western-blot.

[0094] The mouse anti-recombinant Mh-DnaK protein serum was analyzed for immunoreactivity with recombinant Mh-DnaK protein, and the results are shown in Figure 2 The results showed that recombinant Mh-DnaK protein could bind to mouse anti-recombinant Mh-DnaK protein serum, but had no binding reaction with mouse negative serum, indicating that the protein had good antigenicity.

[0095] Example 2 Establishment of indirect ELISA detection method for Mycoplasma haemosuis antibody and optimization of conditions

[0096] 1. Determination of optimal coating concentration and optimal serum dilution

[0097] 1) Coating antigen: Using square array titration method, recombinant Mh-DnaK protein was diluted by 100μL / well at 7, 3.5, 1.75, 0.88, 0.44, 0.22, 0.11μg / mL, respectively, and 1 row for each concentration, 7 concentrations in total, with the last row as blank control. After incubation at 37℃ for 1h and overnight coating at 4℃, the enzyme-labeled plate was washed with washing solution (PBST, 8.0g NaCl, 0.2g KCl, 2.86g Na2HPO4·12H2O, 0.27g KH2PO4, and 0.5mL Tween-20 added to 1000mL of deionized water) for 3 times, 3min each time;

[0098] 2) Blocking: Add 250μL blocking solution (5% skimmed milk powder, 100mL PBST added with 5g skimmed milk powder), block at 37℃ for 1h, and wash with PBST once;

[0099] 3) Add serum: M. haemosuis positive serum (randomly selected one of the above-mentioned qPCR method identified as positive serum collected from sows) and M. haemosuis negative serum (randomly selected one of the above-mentioned 148 PCR detection negative M. haemosuis negative serum) were respectively 1:10 initial horizontal ratio dilution in the first 6 columns and the last 6 columns of the enzyme-labeled plate, 7 holes were repeated vertically for each dilution, 100 μL / hole, 37°C reaction for 1 h, PBST washing 3 times;

[0100] 4) Add enzyme-labeled secondary antibody: add diluted 1:12500 horseradish peroxidase labeled goat anti-pig IgG (IgG-HRP) (diluted with blocking solution), 100 μL / hole, 37°C reaction for 1 h, PBST washing 3 times;

[0101] 5) TMB color development: add substrate color development liquid (TMB substrate color development liquid), 100 μL / hole, room temperature reaction for 8 min;

[0102] 6) Reaction termination: add termination liquid (0.5M H2SO4, 13.6mL concentrated sulfuric acid slowly dropped into 400mL deionized water, and the volume was made up to 500mL), 50 μL / hole, to terminate the reaction;

[0103] 7) Detect OD 450 value: use the enzyme-labeled instrument to measure the OD value of M. haemosuis positive serum and M. haemosuis negative serum (i.e. positive serum OD 450 value and negative serum OD 450 value) at 450nm wavelength, and calculate the P / N value. The antigen concentration and serum dilution corresponding to the beginning of the large change of positive serum OD 450 value (P) and the basically unchanged negative serum OD 450 value (N) are taken as the optimal coating concentration of antigen and the optimal dilution of serum.

[0104] The results of the recombinant Mh-DnaK protein square array titration test show that the positive serum OD 450 value does not change significantly when the antigen concentration is between 0.700 μg / hole and 0.044 μg / hole, and the positive serum OD 450 value decreases significantly when the dilution is between 0.044 μg / hole and 0.011 μg / hole. Therefore, the antigen concentration (0.044 μg / hole, i.e. 0.44 μg / mL) when the positive serum OD 450 value begins to decrease significantly is selected as the optimal coating concentration of the recombinant Mh-DnaK protein.

[0105] The positive serum OD 450The value dropped significantly; when the antigen was diluted 1:40, the OD value of positive serum decreased. 450 Values ​​and negative serum OD 450 The ratio of values ​​(P / N) is the highest, and negative serum OD is the highest. 450 The values ​​remain essentially unchanged. Therefore, 1:40 is chosen as the optimal serum dilution.

[0106] 2. Selection of optimal sealing solution and sealing time

[0107] Following steps 1)-7), the blocking solution was replaced with 2.5% skim milk powder (obtained by adding 2.5g skim milk powder to 100mL PBST), 5% skim milk powder (obtained by adding 5g skim milk powder to 100mL PBST), and 7.5% skim milk powder (obtained by adding 7.5g skim milk powder to 100mL PBST) for blocking. Under the optimal conditions described above, the effect of each blocking solution on the P / N value was examined, with all other steps remaining unchanged. For each blocking solution, four M. haemosuis positive sera (randomly selected from 4 serum samples collected from sows identified as positive by the above qPCR method) and four M. haemosuis negative sera (randomly selected from 4 samples of 148 M. haemosuis negative sera detected by PCR) were tested, with each test repeated in duplicate. OD was measured. 450 Value, i.e., positive serum OD 450 Value (P) and negative serum OD 450 The P-value (N) is calculated for different sealing liquids, including the mean P, mean N, and P / N value. The sealing liquid with the highest P / N value is selected as the optimal sealing liquid.

[0108] The mean P, mean N, and P / N values ​​of different blocking solutions are shown in Table 1. It can be seen from the P / N values ​​that the P / N value is the highest when the blocking solution is 5% skim milk powder. Therefore, the optimal blocking solution is 5% skim milk powder.

[0109] Table 1. Optimization results of the best blocking solution for the indirect ELISA detection method.

[0110]

[0111] Following steps 1)-7), the blocking time was changed from 1 hour to 1 hour, 1.5 hours, and 2 hours. Under the optimal conditions described above, the effect of different blocking times on the P / N value was examined, while other steps remained unchanged. For each blocking time, five M. haemosuis positive sera (randomly selected from 5 sera collected from sows identified as positive by the above qPCR method) and five M. haemosuis negative sera (randomly selected from 5 sera randomly selected from 148 M. haemosuis negative sera detected by PCR) were tested, with each test repeated in duplicate. OD was measured. 450 Value, i.e., positive serum OD 450OD of positive serum 450 OD of negative serum 450 P value (P) and N value (N) were calculated, and the serum reaction time with the maximum P / N value was selected as the optimal serum reaction time.

[0112] P value, N value and P / N value of different serum reaction times were shown in Table 2. From the P / N value, it could be seen that the P / N value was the highest when the serum reaction time was 1 h and 1.5 h, therefore, the optimal serum reaction time was 1 h.

[0113] Table 2 Optimization results of serum reaction time of indirect ELISA detection method

[0114]

[0115] 3. Optimal reaction time of serum

[0116] According to steps 1)-7) in step 1, the serum reaction time was replaced by 0.5 h, 1 h and 1.5 h, and the influence of each serum reaction time on P / N value was detected under the above optimal conditions, and other steps were unchanged. Each serum reaction time was tested by 5 M. haemosuis positive sera (5 sera randomly selected from the above qPCR method identified positive sera of sows) and 5 M. haemosuis negative sera (5 sera randomly selected from the above 148 M. haemosuis negative sera detected by PCR), each repeated 2 wells, and OD 450 OD of positive serum 450 OD of negative serum 450 P value (P) and N value (N) were calculated, and the serum reaction time with the maximum P / N value was selected as the optimal serum reaction time.

[0117] P value, N value and P / N value of different serum reaction times were shown in Table 3. From the P / N value, it could be seen that the P / N value was close when the serum reaction time was 1 h and 1.5 h, therefore, the serum reaction time of 1 h was selected as the optimal serum reaction time.

[0118] Table 3 Optimization results of serum reaction time of indirect ELISA detection method

[0119]

[0120] 4. Determination of optimal reaction time of enzyme-labeled secondary antibody

[0121] According to steps 1)-7) in step 1, replace the reaction time of horseradish peroxidase labeled goat anti-swine IgG (IgG-HRP) with 0.5 h, 1 h and 1.5 h, detect the influence of the reaction time of each enzyme-labeled secondary antibody on P / N value under the above optimal conditions, and the other steps remain unchanged. Each enzyme-labeled secondary antibody reaction time is tested with 5 M. haemosuis positive sera (5 randomly selected from the above qPCR method identified as positive sera collected from sows) and 5 M. haemosuis negative sera (5 randomly selected from the above 148 PCR negative M. haemosuis negative sera), each repeated 2 wells, and the OD 450 value, i.e. the OD 450 value of positive serum (P) and the OD 450 value of negative serum (N) is determined, the P value, N value and P / N value of different enzyme-labeled secondary antibody reaction time are calculated, and the enzyme-labeled secondary antibody reaction time when the P / N value is maximum is selected as the optimal reaction time of enzyme-labeled secondary antibody.

[0122] The P value, N value and P / N value of different enzyme-labeled secondary antibody reaction time are shown in Table 4. From the P / N value, it can be seen that when the enzyme-labeled secondary antibody reaction time is 0.5 h, the P / N value is the highest, therefore, the optimal reaction time of enzyme-labeled secondary antibody is determined to be 0.5 h.

[0123] Table 4 Optimization results of enzyme-labeled secondary antibody reaction time of indirect ELISA detection method

[0124]

[0125] 5. Determination of optimal reaction time of substrate color developing solution (optimal color developing time)

[0126] According to steps 1)-7) in step 1, replace the reaction time of horseradish peroxidase labeled goat anti-swine IgG (IgG-HRP) with 0.5 h, 1 h and 1.5 h, detect the influence of the reaction time of each enzyme-labeled secondary antibody on P / N value under the above optimal conditions, and the other steps remain unchanged. Each enzyme-labeled secondary antibody reaction time is tested with 5 M. haemosuis positive sera (5 randomly selected from the above qPCR method identified as positive sera collected from sows) and 5 M. haemosuis negative sera (5 randomly selected from the above 148 PCR negative M. haemosuis negative sera), each repeated 2 wells, and the OD 450 value, i.e. the OD 450 value of positive serum (P) and the OD 450 value of negative serum (N) is determined, the P value, N value and P / N value of different enzyme-labeled secondary antibody reaction time are calculated, and the enzyme-labeled secondary antibody reaction time when the P / N value is maximum is selected as the optimal reaction time of enzyme-labeled secondary antibody.

[0127] The P mean value, N mean value and P / N value of the optimal reaction time of different substrate color developing solution are shown in Table 5. From the P / N value, it can be seen that the P / N value is the highest when the optimal reaction time of the substrate color developing solution is 7 min. Therefore, the optimal reaction time (i.e. the optimal color developing time) of the substrate color developing solution is determined to be 7 min.

[0128] Table 5 Optimization results of the reaction time of the substrate color developing solution of the indirect ELISA detection method

[0129]

[0130] 6. The indirect ELISA detection method for porcine Haemophilus-like mycoplasma antibodies:

[0131] According to the optimization test of the above steps 1-5, the optimized indirect ELISA detection method for porcine Haemophilus-like mycoplasma antibodies comprises the following steps:

[0132] 1) Coating antigen: using the recombinant Mh-DnaK protein as the coating antigen, coating the enzyme-labeled plate with the coating solution (0.01M carbonate buffer solution, pH 9.6) at a coating concentration of 0.44μg / mL, 100uL / well, incubating at 37℃ for 1h, and then placing it at 4℃ overnight for coating, and washing with PBST for 3 times, each time for 3min;

[0133] 2) Blocking: adding 250μL of blocking solution (5% skimmed milk powder), blocking at 37℃ for 1h, and washing with PBST once;

[0134] 3) Adding serum: adding the serum of the animal to be tested at a dilution of 1:40, 100μL / well, reacting at 37℃ for 1h, and washing with PBST for 3 times;

[0135] 4) Adding enzyme-labeled secondary antibody: adding the horseradish peroxidase-labeled goat anti-pig IgG (IgG-HRP) at a dilution of 1:12500 (diluted with the blocking solution), 100μL / well, reacting at 37℃ for 0.5h, and washing with PBST for 3 times;

[0136] 5) TMB color development: adding the substrate color developing solution (TMB substrate color developing solution), 100μL / well, reacting at room temperature for 7min;

[0137] 6) Reaction termination: adding the termination solution (0.5M H2SO4), 50μL / well, to terminate the reaction;

[0138] 7) Detecting OD 450 value: measuring the OD value of each serum of the animal to be tested at 450nm wavelength with the enzyme-labeled instrument.

[0139] 7. Determination of the positive critical value of the indirect ELISA detection method for porcine Haemophilus-like mycoplasma antibodies

[0140] The 148 samples of M. haemosuis negative serum were screened by PCR detection, and the indirect ELISA detection method of swine hemophilic mycoplasma antibody was used for indirect ELISA detection according to the optimized step 6, and the OD 450 value of M. haemosuis negative serum was determined, and the average value and standard deviation (SD) were calculated, and the value less than was negative, the value between and was suspicious, and the value greater than was positive. The average value was 0.108, and the standard deviation SD value was 0.083, so the determination standard was OD 450 value > 0.358 was positive (i.e. the serum of the animal to be tested was M. haemosuis positive serum, that is, the serum of the animal to be tested contained M. haemosuis antibody or the animal to be tested was infected or previously infected with M. haemosuis), OD 450 value < 0.275 was negative (i.e. the serum of the animal to be tested was M. haemosuis negative serum, that is, the serum of the animal to be tested did not contain M. haemosuis antibody or the level of M. haemosuis antibody was lower than the detection lower limit), and 0.275 ≤ OD 450 value ≤ 0.358 was suspicious (i.e. the serum of the animal to be tested was suspected to be M. haemosuis positive serum, that is, the serum of the animal to be tested was suspected to contain M. haemosuis antibody or the animal to be tested was suspected to be infected or infected with M. haemosuis).

[0141] 8. Swine hemophilic mycoplasma antibody indirect ELISA detection kit

[0142] The swine hemophilic mycoplasma antibody indirect ELISA detection kit comprises an enzyme-labeled plate coated with a recombinant Mh-DnaK protein as a coating antigen, a coating solution, a blocking solution, a washing solution, M. haemosuis positive serum, M. haemosuis negative serum, an enzyme-labeled secondary antibody, a substrate developing solution and a termination solution.

[0143] The coating solution is a 0.01M carbonate buffer solution with pH 9.6; the blocking solution is 5% skimmed milk powder; the washing solution is PBST; the enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-swine IgG (IgG-HRP); the substrate developing solution is TMB substrate developing solution; and the termination solution is 0.5M H2SO4.

[0144] Example 3 Specificity and sensitivity test of swine hemophilic mycoplasma antibody indirect ELISA detection method

[0145] 1. Specificity test

[0146] The indirect ELISA method for detecting porcine Haemophilus antibodies established in Example 2 was used to detect positive sera for seven common porcine diseases: M. suis, M. parvum, M. haemosuis, porcine circovirus disease (PCV), classical swine fever (CSFV), porcine reproductive and respiratory syndrome (PRRS), and porcine toxoplasmosis (PT). OD was measured. 450 The value was used to determine the specificity of the indirect ELISA method for detecting porcine Haemophilus influenzae antibodies in serum.

[0147] The specific detection results are shown in Table 6. The results show that there is no cross-reactivity with serum of common swine diseases (porcine circovirus disease, classical swine fever, porcine reproductive and respiratory syndrome and porcine toxoplasmosis), and three positive serums of porcine haemoplasma (M. suis, M. parvum, M. haemosuis) can be detected simultaneously.

[0148] Table 6 ELISA Specific Detection Results

[0149]

[0150] 2. Repeatability test

[0151] Six M. haemosuis-negative serum samples were randomly selected from 148 samples screened by PCR testing (each serum sample was tested in triplicate). The indirect ELISA method for detecting porcine Haemosuis antibodies, established in Example 2, was used. Three batches (0330, 0516, 0517) of recombinant Mh-DnaK protein were used to coat the ELISA plates. The detection was performed based on the OD value of each serum sample. 450 Calculate the average value separately Standard deviation (SD) and coefficient of variation (CV).

[0152] The results are shown in Table 7, which represent the OD values ​​of different batches of recombinant Mh-DnaK protein detected by ELISA. 450 The coefficient of variation ranged from 8.2% to 15.2%, indicating good batch-to-batch repeatability of the antigen.

[0153] Table 7. Coefficients of variation among batches of recombinant Mh-DnaK protein coating in indirect ELISA.

[0154]

[0155] Example 4: Application of the indirect ELISA method for detecting porcine Haemophilus influenzae antibodies

[0156] The 209 serum samples from 13 pig farms in Zhejiang were detected by the indirect ELISA method for detecting Haemophilus parasuis antibody established in Example 2, and the results are shown in Table 8. The results show that the positive rate of serum antibody of Haemophilus parasuis is 54.5% (114 / 209).

[0157] Table 8 indirect ELISA detection of serum antibody of Haemophilus parasuis

[0158]

[0159] The 209 clinical pig serum samples were detected by the indirect ELISA method for detecting Haemophilus parasuis antibody established in Example 2 (referred to as "DnaK-ELISA") and the previously established Mh-PGK ELISA method (patent publication No. CN114350636A), and the positive coincidence rate, negative coincidence rate and coincidence rate were calculated. The calculation formula is as follows:

[0160] Positive coincidence rate (%) = [number of positive samples / (number of positive samples + number of false negative samples)] x 100%;

[0161] Negative coincidence rate (%) = [number of negative samples / (number of negative samples + number of false positive samples)] x 100%;

[0162] Coincidence rate (%) = [(number of positive samples + number of negative samples) / total number of detections] x 100%.

[0163] The detection results are shown in Table 9. The total coincidence rate is 70.81%, of which the positive coincidence rate is 84.42% and the negative coincidence rate is 62.88%. The higher positive coincidence rate indicates that the DnaK-ELISA method is more sensitive than the PGK-ELISA method.

[0164] Table 9 DnaK-ELISA and PGK-ELISA coincidence rate test results

[0165]

[0166] Obviously, the above examples of the present application are only examples for clearly illustrating the present application, and are not limitations on the embodiments of the present application. For ordinary skilled in the art, other different forms of changes or variations can be made on the basis of the above description, and it is impossible to enumerate all the embodiments here. Any obvious changes or variations derived from the technical solutions of the present application still fall within the protection scope of the present application.

Claims

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

1.

2. The use of the recombinant Mh-DnaK protein according to claim 1 in the preparation of a product for detecting whether a test animal is infected with or has been infected with Haemophilus influenzae from pigs, wherein the Haemophilus influenzae from pigs is... Mycoplasma suis , Mycoplasma parvum and / or Mycoplasma haemosuis .

3. A porcine-derived Haemophilus influenzae antibody indirect ELISA detection kit, characterized in that, The kit includes an enzyme-labeled plate coated with the recombinant Mh-DnaK protein of claim 1 as the antigen; the porcine Haemophilus influenzae is... Mycoplasma suis , Mycoplasma parvum and / or Mycoplasma haemosuis .

4. The porcine-derived Mycoplasma haematobium antibody indirect ELISA detection kit according to claim 3, wherein the kit further comprises coating solution, blocking solution, washing solution, and... Mycoplasma haemosuis Positive serum, Mycoplasma haemosuis One or more of the following: negative serum, enzyme-labeled secondary antibody, substrate chromogenic solution, and stop solution.

5. The porcine-derived Mycoplasma haematobium antibody indirect ELISA detection kit according to claim 4, wherein the coating concentration of the recombinant Mh-DnaK protein is 0.44 μg / mL; And / or, the coating solution is a 0.01M carbonate buffer solution with a pH of 9.6; And / or, the sealing liquid is 2.5% skim milk powder, 5% skim milk powder, or 7.5% skim milk powder; And / or, the washing solution is PBST; And / or, the enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-pig IgG; And / or, the dilution of the enzyme-labeled secondary antibody is 1:12500; And / or, the substrate colorimetric solution is a TMB substrate colorimetric solution; And / or, the terminating solution is 0.5M H2SO4.

6. The use of the porcine Haemophilus influenzae antibody indirect ELISA detection kit according to any one of claims 3-5 in the preparation of products for detecting porcine Haemophilus influenzae antibodies; wherein the porcine Haemophilus influenzae is... Mycoplasma suis , Mycoplasma parvum and / or Mycoplasma haemosuis .

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