Mycoplasma hyopneumoniae fusion protein, and preparation method and application thereof

Abstract

The present application relates to the technical field of veterinary biological products, in particular to a mycoplasma hyopneumoniae fusion protein and a preparation method and application thereof.The mycoplasma hyopneumoniae fusion protein has an amino acid sequence comprising a mycoplasma hyopneumoniae P46 protein, a mycoplasma hyopneumoniae P65 protein and a rigid linker peptide;the amino acid sequence of the mycoplasma hyopneumoniae P46 protein is shown as SEQ ID NO.7;the amino acid sequence of the mycoplasma hyopneumoniae P65 protein is shown as SEQ ID NO.8;and the amino acid sequence of the rigid linker peptide is EAAAK.When the mycoplasma hyopneumoniae fusion protein is used for detection, the sensitivity is good, the repeatability is good, no cross reaction with other antibodies is generated, the specificity is strong, and the accuracy is obviously higher than that of the IDEXX method.The mycoplasma hyopneumoniae fusion protein can be used for clinical sample detection and pig herd immune level monitoring.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of veterinary biological products, in particular to a Mycoplasma hyopneumoniae fusion protein and a preparation method and application thereof. BACKGROUND

[0002] At present, the key measure to effectively prevent and control Mycoplasma pneumoniae of swine (MPS) is vaccination, which has a relatively significant effect on the reduction of lung lesions caused by Mycoplasma pneumoniae (Mhp) infection, the increase of daily weight gain and feed conversion rate, and plays a very important role in the control of infection in the same pen and the reduction of sow carrier rate.

[0003] Effective immune evaluation is helpful for the implementation of the improved Mhp prevention and control scheme, but the current Mhp ELISA antibody detection method has low sensitivity, and the detection result is inconsistent with the expected antibody level, which is not conducive to the immune evaluation of Mhp in pig farms.

[0004] The prior art CN 113588946 B provides a recombinant protein for indirect ELISA detection of Mycoplasma hyopneumoniae antibody, and the amino acid sequence of the recombinant protein comprises a tag carrier amino acid, a Mycoplasma hyopneumoniae P46 protein amino acid, a spacer protein amino acid and a Mycoplasma hyopneumoniae P36 protein amino acid connected in sequence. The recombinant protein provided by the application can be used as a coating antigen, and then an indirect ELISA detection method with simple operation, low cost and effective identification of positive and negative serum is established. The method has high coincidence rate, good specificity and repeatability, and provides a new idea and method for the comprehensive prevention and control of Mycoplasma hyopneumoniae. However, the positive detection rate of the method established by the technology is 65.4%, while the positive rate of the parallel IDEXX detection is 78.8%, indicating that the sensitivity of the method established by the technology is relatively low.

[0005] The prior art CN 103018442 B obtains two strains of recombinant E. coli pGEX-KG-46 and pGEX-KG-65 expressing Mycoplasma hyopneumoniae p46 protein and p65 protein through genetic engineering recombination technology. The kit of the invention includes an enzyme-labeled plate coated with mutant Mycoplasma hyopneumoniae membrane protein P46 and P65 gene expression proteins as antigens and other core reagents. The invention discloses the cloning, site-directed mutagenesis of Mycoplasma hyopneumoniae membrane protein P46 and P65 genes, and the expression and purification method of P46 and P65 proteins. It also discloses a Mycoplasma hyopneumoniae indirect ELISA antibody detection method. The indirect ELISA antibody detection kit prepared by the invention can be used for clinical large-scale detection and epidemiological investigation of Mycoplasma hyopneumoniae antibodies, and has a broad market prospect. The coincidence rate of the method established by the technology with IDEXX reaches 91.3% (84 / 92). It shows that the consistency of the two methods is very high, but it has been proved that the sensitivity of IDEXX to positive serum is not very high, which also shows that the sensitivity of the method will not be high. The positive rate of the technology is 47.8% (42 / 92), and the positive rate of the Mhp ELISA kit of IDEXX is 45.7% (42 / 92), indicating that the positive rate of the method is higher than that of IDEXX, but basically the same. But the sensitivity of IDEXX to positive serum is not very high.

[0006] The prior art CN 101236206 A discloses a Mycoplasma hyopneumoniae recombinant antigen ELISA detection kit. The kit is provided with an antibody detection plate, an enzyme conjugate working solution, a positive control, a negative control, a sample diluent, a 10x concentrated washing solution, color developing solution A, color developing solution B and a stop solution. The detection plate of the kit is a detachable 96-hole enzyme-labeled plate coated with mutant Mycoplasma hyopneumoniae membrane protein P46 gene protein antigen. The enzyme conjugate working solution is horseradish peroxidase-labeled rabbit anti-pig antibody. The positive control serum is taken from a pig with obvious Mycoplasma hyopneumoniae lesions in the lung after dissection, which is detected as positive by indirect hemagglutination and the ELISA detection kit of IDEXX. The negative control serum is taken from a pig without Mycoplasma hyopneumoniae lesions in the lung after dissection, which is detected as negative by indirect hemagglutination and the ELISA detection kit of IDEXX. The invention has the beneficial effects of high specificity, high sensitivity, simple operation, easy large-scale popularization and application, and broad market prospect. But the positive rate of P46-ELISA of the technology is 28.7% (41 / 143), and the positive rate of the Mhp ELISA kit of IDEXX is 26.5% (38 / 143), which shows that the sensitivity of the detection method established by P46 protein is still low. It shows that the positive rate of the method is slightly higher than that of the method of IDEXX, but the sensitivity of IDEXX to positive serum is not very high. SUMMARY

[0007] The application aims to provide a mycoplasma hyopneumoniae P46-P65 fusion protein with good sensitivity, and a preparation method and application thereof.

[0008] In order to achieve the above-mentioned purpose, the technical scheme of the application is as follows:

[0009] A mycoplasma hyopneumoniae fusion protein, wherein the amino acid sequence comprises a mycoplasma hyopneumoniae P46 protein, a mycoplasma hyopneumoniae P65 protein and a rigid connecting peptide.

[0010] The amino acid sequence of the mycoplasma hyopneumoniae P46 protein is as follows:

[0011] GQTESGSTSDSKPQAETLKHKVSNDSIRIALTDPDNPRWISAQKDIISYVDETEAATSTITKNQDAQNNWLTQQANLSPAPKGFIIAPENGSGVGTAVNTIADKGIPIVAYDRLITGSDKYDWYVSFDNEKVGELQGLSLAAGLLGKEDGAFDSIDQMNEYLRSHMPQETISFYTIAGSQDDNNSQYFYNGAMKVLKELMKNSQNKIIDLSPEGENAVYVPGWNYGTAGQRIQSFLTINKDPAGGNKIKAIGSKPASTFKGFLAPNDGMAEQAITKLKLEGFDTKKIFVTGQDYNDKAKTFIKDGDQNMTIYKPDKVLGKVAVEVLRVLIAKKNSATKDDVEMELKSKLPSISFKYDNKTYKSQNKIINTILVSPVVVTKANVDNPDA (SEQ ID NO. 7).

[0012] The amino acid sequence of the mycoplasma hyopneumoniae P65 protein is as follows:

[0013] NDSDKNLMAKNFDFHPSIQGYKKIAHQLLLKLTLDQEEKDDSNAEELKNTTNFDDFDENKPTYSKVIDLSVFAKSNKEFLEKLNENKQTSEFIAQKSTFDTDQEAAIKDDKRTFGNIVREIVSLPIFDNFDFRELIPVKNPFVKAIINSYLGKPAGSLIKDIEQLENKVKDYARPNIKIFDTIIDSFIRKMVAFFAELNTDQEIKEFKMSPQILFLTLRNAILSPFDLTKLKDSATFKILMNLKPEQILTLLGLSKTPSVPKPEKPKDQSSKPQTDTSSQKQESGTSSTDSTKATTENQKPAEQTDSSEQSSTEPKSN (SEQ ID NO. 8).

[0014] The amino acid sequence of the rigid linker peptide is EAAAK.

[0015] Other suitable rigid linker peptides can also be used as the linker without strict limitations.

[0016] The P46 protein is a major preferred protein in the surface adhesion protein of Mycoplasma hyopneumoniae, has strong specificity and immunogenicity, and is an antigen that can detect a strong immune response reaction in the early stage of Mhp infection.

[0017] The P65 protein is a lipoprotein on the surface of Mhp, has a species-specific epitope and good immunogenicity, and is a major immune protein of Mhp. The main immunogenic region is located at the C-terminal end on the outside of the cell membrane. The N-terminal end has esterase activity and can decompose fat and fatty acids.

[0018] The P46 and P65 are two important membrane proteins of Mhp, both of which have the ability to stimulate animals to produce strong immune response reactions. The present application realizes the fusion expression of P46-P65 in Escherichia coli, and establishes an ELISA antibody detection method using the fusion recombinant protein as an antigen. It is confirmed by the widely used IDEXX ELISA method and the metabolic inhibition test of Mhp that the method established in the present application is more sensitive, more accurate, and has good specificity than the ELISA antibody detection system of P46 and P65 proteins alone and the IDEXX ELISA method, benefiting from the synergistic effect between P46 and P65. However, when it is replaced by other similar proteins, the synergistic effect is lacking, and the accuracy cannot be improved.

[0019] In one preferred embodiment, the Mycoplasma hyopneumoniae fusion protein has an amino acid sequence as shown in SEQ ID NO. 1.

[0020] SEQ ID NO. 1:

[0021] GQTESGSTSDSKPQAETLKHKVSNDSIRIALTDPDNPRWISAQKDIISYVDETEAATSTITKNQDAQNNWLTQQANLSPAPKGFIIAPENGSGVGTAVNTIADKGIPIVAYDRLITGSDKYDWYVSFDNEKVGELQGLSLAAGLLGKEDGAFDSIDQMNEYLRSHMPQETISFYTIAGSQDDNNSQYFYNGAMKVLKELMKNSQNKIIDLSPEGENAVYVPGWNYGTAGQRIQSFLTINKDPAGGNKIKAVGSKPASIFKGFLAPNDGMAEQAITKLKLEGFDTQKIFVTGQDYNNKAKTFIKDGDQNMTIYKPDKVLGKVAVEVLRVLIAKKNKASRSEVENELKAKLPNISFKYDNQTYKVQGKNINTILVSPVIVTKANVDNPDAEAAAKNDSDKNLMAKNFDFHPSIQGYKKIAHQLLLKLTLDQEEKDDFNAEELKNTTNFDDFDDNKPTYSKVIDLSVFAKSNKEFLEKLNENKQTSEFIAQKSTFDTNQEAAIKDDKRTFGNIVREIVSLP IFDNFDFRELIPVSNPFVKAIINSYLGKPAGSLIKDIEQLENKVKDYARPNIKIFDTIIDSFIRKMVAFFAELNTDQEIKEFKMSPQILFLTLRNAILSPFDLTKLKDSATFKILMNLKPEQILTLLGLSKTPSVPKPEKPKDQGSKPQTDTSSQKQESGTSSTDSTKATTENQKPAEQTDSSEQSSTEPKSN.

[0022] In the amino acid sequence shown as SEQ ID NO. 1, the amino acids at positions 1-388 are the amino acid sequence of M. hyopneumoniae P46, the amino acids at positions 389-393 are the amino acid sequence of a rigid protein linker, and the amino acids at positions 394-711 are the amino acid sequence of M. hyopneumoniae P65.

[0023] Based on the same inventive concept, the present application also claims a nucleic acid molecule encoding the M. hyopneumoniae fusion protein, the nucleotide sequence of which is shown as SEQ ID NO. 2.

[0024] SEQ ID NO. 2:

[0025] ggacagacag aatcaggttc gacttctgat tctaaaccac aagccgagac tctaaaacataaagtaagta atgattctat tcgaatagca ctaaccgatc cagataatcc tcgatggatt agtgcccaaaaagatattat ttcttatgtt gatgaaacag aggcagcaac ttcaacaatt acaaaaaacc aggatgcacaaaataactgg ctcactcagc aagctaattt

[0026] aagcccagcg

[0027] ccaaaaggat ttattattgc ccctgaaaat ggaagtggag ttggaactgc tgttaatacaattgctgata aaggaattcc gattgttgcc tatgatcgac taattactgg atctgataaa tatgattggtatgtttcttt tgataatgaa aaagttggcg aattacaagg tctttcactt gcggcgggtc tattaggaaaagaagatggt gcttttgatt caattgatca aatgaatgaa tatctaagat cacatatgcc ccaagagacaatttcttttt atacaatcgc gggttcccaa gatgataata attcccaata tttttataat ggcgcaatgaaagtacttaa agaattaatg aaaaattcgc aaaataaaat aattgattta tctcctgaag gcgaaaatgctgtttatgtc ccaggatgga attatggaac tgccggtcaa agaatccaat cttttctaac aattaacaaagatccagcag gtggtaataa aataaaagct gttggttcaa aaccagcttc tattttcaaa ggatttcttgcaccaaatga tggaatggcc gaacaagcaa tcaccaaatt aaaacttgaa ggatttgata cccaaaaaatctttgtaact ggtcaagatt ataataataa agccaaaact tttatcaaag acggcgatca aaatatgacaatttataaac ctgataaagt tttaggaaaa gttgctgttg aagttcttcg ggttttaatt gcaaagaaaaataaagcatc tagatcagaa gtcgaaaacg aactaaaagc aaaactacca aatatttcat ttaaatatgataatcaaaca tataaagtgc aaggtaaaaa tattaataca attttagtaa gtccagtaat tgttacaaaagctaatgttg ataatcctgatgccgaggcc gccgccaaga atgattctga taaaaattta atggcgaaaaattttgactt ccacccttca attcaaggtt ataaaaaaat tgctcaccaa cttttgttaa aattaactcttgaccaagaa gaaaaagatg attttaatgc tgaagagtta aaaaatacta caaatttcga tgattttgatgataataaac cgacctattc caaagttatt gacctaagtg tttttgcaaa atcaaataaa gaatttcttgaaaaattaaa cgaaaataag caaactagtg aatttattgc tcaaaaatcc acttttgaca ccaatcaagaagctgcaatc aaagacgaca aacgcacttt tggaaatata gttcgagaaa ttgtatcttt accaatcttcgataattttg attttagaga gttaatacct gtttcaaatc cgtttgtaaa agcaattatt aacagctatttagggaaacc agctggttct cttataaaag atatcgaaca actcgaaaat aaagtgaaag attacgcaagacctaatatc aagattttcg atacaattat tgactcattc ataagaaaaa tggtagcatt ttttgctgaattaaacactg atcaagaaat aaaagaattc aaaatgtcac ctcaaatact atttctgaca ctaagaaatgcaatactaag tccatttgat ttaactaaat taaaagacag tgctacattt aaaattttaa tgaatctcaaaccagaacaa atattaactc tactaggcct aagtaaaacc ccttcagttc ctaaacctgaaaaaccaaaagatcaaggtt cgaagccaca aacagatact tctagtcaaa aacaagaaag cggaacaagttcaacagatt caacaaaagc tacaactgaa aaccaaaaaccagctgagca aacagattct tctgagcaatcaagtaccga gcctaaatca aac。

[0028] In the gene sequence shown in SEQ ID No. 2, the 1-1164 bp is the Mycoplasma hyopneumoniae P46 gene sequence, the 1165-1179 bp is the rigid protein linker gene sequence, and the 1180-2133 bp is the Mycoplasma hyopneumoniae P65 gene sequence.

[0029] Based on the same inventive concept, the present application also claims a nucleic acid vector carrying the nucleic acid molecule encoding the Mycoplasma hyopneumoniae fusion protein.

[0030] Based on the same inventive concept, the present application also claims a preparation method of the Mycoplasma hyopneumoniae fusion protein, comprising:

[0031] transforming the nucleic acid vector into an E. coli competent cell to obtain an expression engineering bacteria;

[0032] inducing expression and purification of the expression engineering bacteria to obtain the Mycoplasma hyopneumoniae fusion protein.

[0033] Based on the same inventive concept, the present application also claims the application of the Mycoplasma hyopneumoniae fusion protein in preparing a reagent for detecting porcine mycoplasma pneumonia.

[0034] In one preferred embodiment, in the above application, the Mycoplasma hyopneumoniae fusion protein is used as a coating antigen in the detection reagent.

[0035] Based on the same inventive concept, the present application also claims an ELISA detection kit of a Mycoplasma hyopneumoniae antibody, which comprises an ELISA enzyme-labeled plate coated with the Mycoplasma hyopneumoniae fusion protein.

[0036] In one preferred embodiment, in the kit, the coating amount of the Mycoplasma hyopneumoniae fusion protein is 0.12-0.16 μg / well.

[0037] In one preferred embodiment, in the kit, the optimal coating concentration of the protein is 1.4-2.1 μg / mL.

[0038] In one preferred embodiment, in the kit, the diluent, blocking solution, washing solution, enzyme-labeled goat anti-pig antibody, substrate developing solution and termination solution are further included.

[0039] In one preferred embodiment, the diluent is a BSA solution.

[0040] In one preferred embodiment, the washing solution is PBST.

[0041] In one preferred embodiment, the substrate developing solution is TMB developing solution.

[0042] In one preferred embodiment, the termination solution is sulfuric acid solution.

[0043] In one preferred embodiment, the blocking solution is skimmed milk powder.

[0044] The beneficial effects of the present application are as follows:

[0045] When the positive serum is diluted to 6400 times at most, the detection result is still positive (OD450nm>0.30), indicating that the method has good sensitivity. The batch and batch detection of 4 positive and 4 negative sera shows that the batch variation coefficient is between 0.49% and 4.07%, and the batch variation coefficient is between 1.74% and 4.96%, both less than 5%, indicating that the method has good repeatability. The detection of 7 positive sera of CSFV, FMDV, PRRSV, PRV, PCV2, PEDV and APP selected from Mhp negative fields shows that the OD450nm value of the detection result is less than the critical value 0.3, indicating that the antibody detection method established in the present research does not produce cross reaction with the above antibodies, and has strong specificity. The serum positive rates of the method and the IDEXX method are 62.4% and 73.8% respectively, the total coincidence rate of the detection results of the two methods is 88.6%, and the accuracy is obviously higher than that of the IDEXX method.

[0046] In summary, the present application establishes an indirect ELISA detection method with high sensitivity, good repeatability and effective identification of positive and negative sera, which can be used for clinical sample detection, pig population immune level monitoring, and provides ideas and methods for comprehensive prevention and control of mycoplasma hyopneumoniae. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 PCR amplification result of the Mhp P46 P65 gene fragment of the recombinant protein of mycoplasma hyopneumoniae of the present application, wherein M: DNA molecular mark; lane 1: Mhp P46 P65 PCR amplification product.

[0048] Figure 2SDS PAGE electrophoretogram and Westblot diagram of expression and purification of Mycoplasma hyopneumoniae fusion protein Mhp P46 P65 antigen provided by the embodiment of the present application; M: protein marker; Lane 1: pET28a / BL21 supernatant; Lane 2: pET28a / BL21 precipitate; Lane 3: pET28a-p46-p65 / BL21 supernatant; Lane 4: pET28a-p46-p65 / BL21 precipitate; Lane 5: purified r P46-P65; Lane 6: Western blot identification of r P45-P65.

[0049] Figure 3 is the specific test result diagram of the Mycoplasma hyopneumoniae r P46-P65 ELISA detection method provided by the embodiment of the present application. DETAILED DESCRIPTION

[0050] In order to more clearly illustrate the overall concept of the present application, the following will be described in the form of examples. In the following description, a large number of specific details are given in order to provide a more thorough understanding of the present application. However, it is obvious to those skilled in the art that the present application can be implemented without one or more of these details. In other examples, some technical features known in the art are not described in order to avoid obscuring the present application.

[0051] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0052] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0053] Example 1 Obtaining of fusion protein P46-P65

[0054] This invention is based on the recombinant plasmids pET-28-P46 and pET-28-P65 (for the construction methods of pET-28-P46 and pET-28-P65, see the prior art: Bioinformatics Analysis, Cloning and Expression of MHP Virulence and Immune-Related Proteins, by Jinxin Yu, 2022, Foshan University of Science and Technology). Primers were designed with reference to the Mhp232 strain (NC_006360.1) to amplify the corresponding sequence. The amplified gene sequence was ligated with the pET-28a(+) vector preserved in the laboratory (for the construction methods of pET-28a(+), see the prior art: Bioinformatics Analysis, Cloning and Expression of MHP Virulence and Immune-Related Proteins, by Jinxin Yu, 2022, Foshan University of Science and Technology). The sequence was confirmed to be correct by sequencing to obtain the recombinant expression plasmid pET-28-P46-P65. The recombinant plasmid pET-P46-P65 was transformed into the competent E. coli expression strain BL21 (DE3) to obtain the BL21 (pET-P46-P65) strain. The fusion protein was induced to express with IPTG, purified, and identified by SDS-PAGE and Western blot to obtain the corresponding recombinant protein. The specific steps are as follows:

[0055] 1. Construction of expression vector for Mycoplasma hyopneumoniae P46-P65 fusion gene

[0056] 1.1 Primer design and PCR amplification

[0057] The genome of Mhp232 strain (NC_006360.1) was used as a reference sequence. Four specific primers were designed using Primer Select according to the sequences of the P46 and P65 coding genes of Mhp232 strain and the principle of SOE-PCR. The BamHI and XholⅠ restriction sites were introduced into primers Fp46 and Rp65, respectively. The designed primers were as follows: Fp46: 5'-CGCGGATCCGGACAGACAGAATCAGGTTCG (SEQ ID NO.3); Rp46: 5'-AATCATTCTTG GCGGCGGCCTCGGCATCAGGATTATCAACATTAG (SEQ ID NO.4); Fp65: 5'-GATGC CGAGGCCGCCGCCAAGAATGATTCTGATAAAAATTTAATGGC (SEQ ID NO.5); and Rp65: 5'-CCGCTCGAGGTTTGATTTAGGCTCGGTACTT (SEQ ID NO.6).

[0058] use HSDNA Polymerase system was used for PCR amplification (PCR amplification system: P HSDNA Polymerase: 1 μL; dNTP: 4 μL; Buffer: 10 μL; dH20: 29 μL; template: 2 μL; upstream and downstream primers: 2 μL each. The PCR amplification conditions were: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s; annealing at 55°C for 15 s; extension at 68°C for 4 min; total extension at 68°C for 10 min; and 35 cycles. The agarose electrophoresis of the amplification product showed that a specific band of about 2118 bp (as shown in Figure 1 The PCR product was cleaned according to the OMEGA PCR Clean-Up Kit instruction.

[0059] 1.2 Cloning and transformation of recombinant plasmid

[0060] The cleaned gene fragment and pET-28a(+) plasmid were double-digested with BamH I and Xho I (37°C, 2 h), respectively, and the reaction system is shown in Table 1. The digested products were cleaned using the OMEGA PCR Clean-Up Kit.

[0061] Table 1 Double-digestion reaction system

[0062]

[0063] The digested and cleaned target gene was ligated with the digested and cleaned plasmid using T4 ligase (22°C, 30 min), and the reaction system is shown in Table 2.

[0064] Table 2 Ligation system

[0065]

[0066] The ligation product was transformed into the E. coli expression strain BL21(DE3) competent cells. The specific steps of transformation were as follows: 100 μL of prepared competent cells were taken from the -80°C ultra-low temperature freezer, thawed in ice water, and then 10 μL of the ligation product was added to the competent cells, which were subjected to ice bath for 30 min. The ice-bathed competent cells were subjected to 42°C water bath heat shock for 90 s, and then immediately subjected to ice bath for 2 min. 600 μL of sterile LB liquid medium was added, and the mixture was incubated in a 37°C water bath constant temperature shaker at 180 rpm for 45 min. The bacterial solution was centrifuged at 5000 rpm for 2 min, and the liquid was discarded. 100 μL of sterile LB liquid medium was added to resuspend the bacteria, which were then spread on an LB solid plate containing 50 μg / mL kanamycin, and incubated in a 37°C incubator overnight. Thus, the BL21(pET-P46-P65) strain was obtained.

[0067] The single transformant on the plate was used as a template for PCR amplification with T7 universal primer of pET-28a(+) plasmid (Shanghai Baishengyue Biotechnology Co., Ltd.) (94°C pre-denaturation for 5 min, 94°C denaturation for 5 min, 55°C annealing for 30 s, 72°C extension for 1 min, 35 cycles, 72°C full extension for 10 min). The amplification system is shown in Table 3.

[0068] Table 3 Amplification system

[0069]

[0070]

[0071] Amplification verifies whether the ligation product is successfully transferred into E. coli. The successful transfer into E. coli is called a positive transformant.

[0072] The positive transformant verified successfully after amplification is inoculated into 10 mL sterile LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C, 180 rpm overnight. The plasmid is extracted according to the OMEGA plasmid extraction instruction, and 10 μL of the plasmid is sent to Beijing Qikang Biotechnology Co., Ltd. for sequencing. The sequencing result is completely consistent with the nucleotide sequence of the target gene registered in the GenBank database, which proves that the obtained is BL21 (pET-P46-P65) strain.

[0073] 2. Expression, purification and identification of recombinant protein P46-P65 (rP64-P65)

[0074] 2.1 Expression of recombinant protein P46-P65

[0075] (1) Under sterile operation, the rejuvenated bacterial liquid is added into LB liquid medium containing 100 mg / ml kanamycin at a ratio of 1 / 100 (volume ratio), and cultured at 37°C, 180 rpm. The bacteria are grown to the logarithmic phase (OD600=0.6-1.0), and then 1% volume of isopropyl thiogalactoside (final concentration of 1 mmol / L) is added. The culture is incubated at 37°C, 180 rpm for 5 h;

[0076] (2) The induced bacteria are centrifuged at 6000 rpm, 4°C for 5 min, and the supernatant is discarded. The bacteria are resuspended with 20 times concentrated ultrapure water;

[0077] (3) The resuspended bacterial slurry is placed in an ice water bath for ultrasonic crushing (amplitude rod 6, power 35%, working time 10 min, ultrasonic on 5 s and off 5 s). The bacterial slurry is centrifuged at 12000 rpm, 4°C for 20 min to separate the supernatant and the precipitate;

[0078] (4) Equal volume of resuspended pellet, respectively, 30 μL of whole bacteria, 30 μL of supernatant, 30 μL of precipitate sample, 10 μL of 4x Loading Buffer, boiling water bath for 5 min, 12000 rpm centrifugation for 5 min, 12% SDS-PAGE electrophoresis was performed on the prepared sample.

[0079] (5) Finally determined as the recombinant protein is soluble expression (see Figure 2 A).

[0080] 2.2 Purification and Western-bolt identification of recombinant protein P46-P65

[0081] (1) Sample processing: after ultrasonic crushing, the bacterial liquid was centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was filtered with a 0.44 μm filter membrane.

[0082] (2) Assemble gravity column: use Huyi biological Ni ion affinity chromatography medium to purify soluble protein, take 2 mL medium and add it to the protein purification column, pad the gasket, and dry the column with 20% ethanol;

[0083] (3) Water washing: add 10 mL of pure water to wash away the residual ethanol, and wash 2-3 times.

[0084] (4) Equilibrium: add 10 mL of equilibrium buffer (0.02M PB) and equilibrate 2-3 times.

[0085] (5) Sample loading: add the treated protein sample to the equilibrated purification column, control the flow rate at 0.2 mL / min, collect the flow-through liquid, and resample the collected flow-through liquid, a total of 3 times.

[0086] (6) Washing: sequentially wash with 50 mM and 100 mM imidazole containing 0.02M PB, each 10 mL, control the flow rate at 0.5 mL / min during washing, and collect each washing liquid at 10 mL / tube.

[0087] (7) Elution: elute with 500 mM imidazole containing 0.02M PB, control the flow rate at 0.2 mL / min, elution volume is 10 mL, and collect the eluate in 5 tubes.

[0088] (8) Medium cleaning and preservation: add 10 mL of eluent to wash the purification column, 10 mL of pure water to wash the column, wash 4-5 times, and finally add 20% ethanol to store the purification column at 4°C.

[0089] (9) The collected eluate was detected by SDS-PAGE, and the results are shown in 100 ku (results see Figure 2 A), the obtained r P46-P65 has less impurity protein concentration, indicating good purification effect.

[0090] (10) Western-bolt identification of the purified protein using mouse hyperimmune serum as the primary antibody. Western blot identification of the purified rP46-P65 using Mhp positive serum as the primary antibody (1:100) and goat anti-swine IgG-HRP as the secondary antibody (1:10 000). The specific process of Western blot identification is as follows:

[0091] (11) SDS-PAGE protein electrophoresis: add 8 μl rainbow mark, add 20 μl supernatant sample to each well, add 10 μl intracellular precipitate to each well, and electrophorese at 120 v for 70 min.

[0092] (12) Membrane transfer: Millipore PVDF (0.45 μm) membrane pretreatment: soak in methanol for 1 min → soak in membrane transfer solution for 2 min. Membrane transfer placement order: clip black side + sponge (polyethylene plate) + filter paper + gel + PVDF membrane (no distinction between front and back) + filter paper + sponge + clip white side, note that air bubbles are chased once for each layer, and it must be kept wet at all times, otherwise the gel is easy to break, make sure to chase the air bubbles out, otherwise high background or affect protein transfer will be produced; fix the sandwich in the membrane transfer groove, and the clip black side faces the membrane transfer groove black side.

[0093] (13) Membrane transfer conditions: constant current 280 mA, time 40 min, and low temperature membrane transfer.

[0094] (14) Blocking: block at 37 °C incubator for 1 h, or block overnight at 4 °C.

[0095] (15) Primary antibody incubation: use Mhp positive serum as the primary antibody (1:100), place the PVDF membrane in the diluted primary antibody using the blocking solution, and incubate at 37 °C on the shaking table for 2 hours at a speed of 80 rpm. Wash with TBST on the side swing shaking table for 3 times at a speed of 60 rpm, each time for 15 min.

[0096] (16) Secondary antibody incubation: use goat anti-swine IgG-HRP as the secondary antibody (1:10 000), place the PVDF membrane in the diluted secondary antibody using the blocking solution, and incubate at 37 °C on the shaking table for 1 hour with slow shaking at a speed of 80 rpm. Wash with TBST on the side swing shaking table for 3 times at a speed of 60 rpm, each time for 15 min.

[0097] (17) Color development: the results show that specific bands appear at 100 ku( Figure 2 B), which indicates that the recombinant protein has good reactogenicity with the positive serum.

[0098] Example 2: Establishment and optimization of indirect ELISA method for detection of Mycoplasma hyopneumoniae antibody using rP46-P65

[0099] The present embodiment establishes an indirect ELISA method for detecting Mycoplasma hyopneumoniae antibody using recombinant protein Mhp-P46-P65, and optimizes it. Among them, 298 sera from different pig farms in Hunan Province are detected by using commercial IDEXX Mycoplasma hyopneumoniae antibody detection kit, and the qualified positive and negative sera are screened out, and the known positive and negative sera are used for optimization of ELISA detection method. The specific steps are as follows:

[0100] 1. Establishment of Mycoplasma hyopneumoniae P46-P65 indirect ELISA method

[0101] 1.1 Specific operation steps of indirect ELISA method

[0102] (1) Coating: The purified recombinant protein Mhp-P46-P65 was diluted to a certain concentration with 0.05M carbonate buffer (pH 9.6) as coating antigen, 100μL / well, coated at 4℃ overnight;

[0103] (2) Blocking: 5% skimmed milk powder was used for blocking, and incubated in a 37℃ incubator for 30min;

[0104] (3) Drying: Discard the coating liquid and pat dry, incubate in a 37℃ incubator for 2h;

[0105] (4) Serum incubation: The serum to be tested was diluted 100 times with serum diluent (PBST diluted 5% skimmed milk powder, added with 5% E. coli lysate), then added to the coated enzyme labeled plate, 100μL / well, incubated in a 37℃ incubator for 30min;

[0106] (5) Washing: Discard the liquid in the enzyme labeled plate, add washing liquid PBST (containing 0.05% Tween-20, pH 7.4), 300μL / well, repeat washing 4 times, and pat dry after the last time;

[0107] (6) Secondary antibody incubation: Add goat anti-pig IgG goat anti-pig IgG (purchased from Sigma company), 100μL / well, incubate in a 37℃ incubator for 30min;

[0108] (7) Washing, repeat step 5;

[0109] (8) Substrate color development: Add TMB color developing liquid (purchased from Beijing Tiangeng Biochemical Technology Co., Ltd.), 50μL / well, incubate in a 37℃ incubator for 15min;

[0110] (9) Stop: 50 μL / well of stop solution (2 M H2SO4) was added to stop the reaction, 50 μL / well, OD was read 450nm Absorbance. The content of components in the kit can be different according to different detection quantities, which has no effect on the effect.

[0111] 2. Optimization of indirect ELISA method for Mycoplasma hyopneumoniae recombinant protein P46-P65

[0112] 2.1 Determination of optimal antigen concentration

[0113] The r P46-P65 obtained above was diluted to 0.7 μg / mL, 1.4 μg / mL, 2.1 μg / mL, 2.8 μg / mL with carbonate buffer (pH 9.6) as coating solution, 100 μL / well was coated, 4 positive sera and 4 negative sera were selected to perform experiments according to the indirect ELISA operation steps, the P / N value under each antigen coating concentration was calculated, and the antigen coating concentration with the maximum P / N value was selected as the optimal antigen coating concentration. The optimal coating concentration of the protein was finally determined to be 1.4 μg / mL, as shown in Table 4.

[0114] Table 4 Determination of optimal antigen coating concentration

[0115]

[0116] 2.2 Determination of optimal serum dilution and goat anti-swine IgG dilution

[0117] The 5% skimmed milk powder was diluted with PBST, 5% E. coli lysate was added as serum diluent, and goat anti-swine IgG was used as enzyme-labeled secondary antibody. The optimal serum dilution and goat anti-swine IgG dilution were determined by the method of square titration for the preliminarily established ELISA method. 4 positive sera and 4 negative sera were selected, and the sera were diluted by 1 / 50, 1 / 100, and 1 / 200 with the serum diluent. The P / N value was detected and calculated. The results showed that the P / N value was the largest when the serum was diluted by 1 / 100, as shown in Table 5. The goat anti-swine IgG was diluted by 1 / 10000, 1 / 20000, 1 / 30000, 1 / 40000, and 1 / 50000 for detection. The results showed that the P / N value was the largest when the goat anti-swine IgG was diluted by 1 / 20000, as shown in Table 6. The optimal serum dilution was finally determined to be 1 / 100, and the optimal goat anti-swine IgG dilution was 1 / 20000.

[0118] Table 5 Determination of optimal serum dilution

[0119]

[0120] Table 6 Determination of goat anti-swine IgG dilution

[0121]

[0122]

[0123] 2.3 Optimal incubation time determination

[0124] Take 4 copies of serum respectively negative and positive (serum with S / P value above 2.0 is positive serum) detected by IDEXX as reference serum, conduct experiment according to the optimal serum and goat anti-pig IgG dilution determined above, and incubate the serum to be detected for 20 min, 30 min, 40 min, 50 min and 60 min respectively for detection, incubate goat anti-pig IgG for 20 min, 30 min, 40 min, 50 min and 60 min respectively for detection, and incubate substrate for 10 min, 15 min, 20 min, 25 min and 30 min respectively for detection, and calculate P / N value. The results show that when serum is incubated for 30 min, P / N is the largest, as shown in Table 7; when goat anti-pig IgG is incubated for 30 min, P / N is the largest, as shown in Table 8; and when substrate is incubated for 15 min, P / N is the largest, as shown in Table 9. Therefore, the optimal serum and goat anti-pig IgG incubation time is determined to be 30 min, and the optimal substrate incubation time is determined to be 15 min.

[0125] Table 7 Determination of optimal serum incubation time

[0126]

[0127] Table 8 Determination of optimal goat anti-pig IgG incubation time

[0128]

[0129]

[0130] Table 9 Optimal substrate incubation time

[0131]

[0132] 2.4 Determination of positive and negative critical value

[0133] Under the optimal ELISA detection conditions determined above, 80 copies of serum which are negative identified by PCR on pig lung and whose corresponding serum is negative detected by IDEXX antibody kit are detected, and the results show that the OD 450nm Average value of negative serum is 0.135, and the standard deviation (S) is 0.056. According to the critical value calculation formula , the critical value of indirect ELISA method is calculated to be 0.3, that is, the detection value of sample to be detected ≥ 0.3 is positive, and < 0.3 is negative.

[0134] 2.5 Sensitivity experiment

[0135] Four Mhp positive serum samples with S / P value above 2.0 by IDEXX antibody detection kit were selected for dilution (1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800) and detection by the established ELISA method. The results showed (Table 10) that the detection results were still positive (OD450nm>0.30) when the positive serum was diluted to 6400 times, indicating that the method had good sensitivity.

[0136] Table 10 Sensitivity experiment results

[0137]

[0138] 2.6 Reproducibility experiment

[0139] The method was used to detect 4 positive and 4 negative serum samples between batches and within batches, and the results showed that the within-batch coefficient of variation was between 0.49% and 4.07%, and the between-batch coefficient of variation was between 1.74% and 4.96%, both less than 5%, and the results were shown in Table 11, indicating that the established method had good reproducibility.

[0140] Table 11 Indirect ELISA reproducibility test results

[0141]

[0142] 2.7 Specificity experiment

[0143] Seven positive serum samples of CSFV, FMDV, PRRSV, PRV, PCV2, PEDV and APP selected from Mhp negative fields were detected by the indirect ELISA method established in this study, and the detection results were shown in Table 13. Figure 3 The OD450nm values of the above detection results were all less than the critical value 0.3, indicating that the antibody detection method established in this study did not produce cross-reaction with the above antibodies and had strong specificity.

[0144] Example 3 Clinical sample detection

[0145] 3.1 Comparison of detection results of two methods

[0146] A total of 298 serum samples from clinical Mhp positive pig farms and Mhp negative fields were detected by two methods, and the detection results were shown in Table 12.

[0147] Table 12 Detection results of clinical samples

[0148]

[0149] The research showed that the 13 sera with suspicious results by IDEXX Mhp test were actually positive, which were proved by metabolic inhibition test. Therefore, the 13 sera with suspicious results were counted into the positive sera.

[0150] As shown in Table 12, there were 220 positive sera and 78 negative sera by the present method, and there were 173 positive sera, 13 suspicious sera and 112 negative sera by IDEXX method. The research showed that the 13 sera with suspicious results by IDEXX Mhp test were actually positive, which were proved by metabolic inhibition test. Therefore, the 13 sera with suspicious results were counted into the positive sera. There were 186 positive sera by IDEXX method, and all of the 186 sera were positive by the present method. There were 78 negative sera by the present method, and all of the 78 sera were negative by IDEXX method. However, there were 34 sera which were negative by IDEXX method but positive by the present method. The positive rates of the present method and IDEXX method were 62.4% and 73.8% respectively, and the total coincidence rate of the two methods was 88.6%.

[0151] 3.2 Metabolic inhibition test for the sera with inconsistent results by the two methods

[0152] The 34 sera with inconsistent results by the two methods were identified by color change test. The CCU of Mhp168 vaccine strain was 1 x 108 50 8 CCU / mL. The 168 strain was diluted 10 times by KM2 liquid medium to 10 -6 , 10 -7 , 10 - 8 ​CCU / mL, and 10% of the serum to be tested was added, incubated at 37°C, and the results were determined. The results showed that the culture medium of the control group turned yellow after 9-10 days of culture, and in the culture medium of 27 serum samples of the 34 serum samples with IDEXX detection results being negative and the method being positive, the culture medium did not change color after 14 days of culture, indicating that 27 serum samples were Mhp antibody positive, and only 7 serum samples were indeed negative, therefore, among the 298 serum samples, there were actually only 78+5=85 negative serum samples, not 112 of IDEXX, and the positive serum samples should be 186+27=213, not 173 of IDEXX (i.e., 40 positive serum samples of IDEXX were not detected), that is, the accuracy rate of the positive serum samples of the method of the present application was 96.8% (213 / 220), while the accuracy rate of IDEXX detection was only 81.2% (173 / 213); the accuracy rate of negative serum samples: the method was 91.8% (78 / 85), and IDEXX was 75.9% (85 / 112). The total accuracy rate of positive and negative serum samples of the method was 97.7% (291 / 298), and the total accuracy rate of IDEXX was 86.6% (258 / 298). The accuracy rate of the method of the present application, whether from negative, positive serum samples or the total, was significantly higher than that of the method of IDEXX, and the improvement degree was more than 10%.

[0153] In addition, the present application also repeats part of the existing prior art and detects the results, which are as follows:

[0154] 1. The ELISA antibody detection method established in reference 1 (Li Shuguang, Zhang Di, Zhao Jialei, et al. Expression of Mycoplasma hyopneumoniae fusion protein MHP-P46-P36 and establishment of indirect ELISA antibody detection method [J]. Chinese Journal of Veterinary Medicine, 2022(007):042) was used for sample detection, and compared with the IDEXX method, the total coincidence rate was 71.2%, and the positive sample detection rate of the ELISA method established by rP46-P36 was not as good as the IDEXX method. The positive detection rate and coincidence rate of the method established in the present application are higher than those of the method established in reference 1. This result can indicate that the rP46-P65 indirect ELISA method established in the present research has better sensitivity.

[0155] Reference 2 (Shen Qingchun, Wang Fang, Han Mingyuan, et al. Prokaryotic expression of P46 gene of Mycoplasma hyopneumoniae and establishment of indirect ELISA method [J]. Chinese Journal of Animal and Veterinary Sciences, 2012, 43(3): 431-437.) uses the ELISA detection kit of IDEXX and rP46-ELISA reagent to compare and detect 143 pig sera, and the detection results show that the positive detection rate of P46-ELISA is 28.7% (41 / 143), and the positive detection rate of the Mhp ELISA kit of IDEXX is 26.5% (38 / 143), which shows that the detection method established by the P46 protein is not as sensitive as IDEXX. The positive rate of the method established in the application is 73.8% (220 / 298), and the positive rate of the antibody detection kit of IDEXX is 58.1% (173 / 298). And 40 positive sera are not detected by the method of IDEXX, and the detection results show that the positive detection rate of IDEXX is significantly lower than the method established in the application. The above results show that the method established in the application is more sensitive than the method established in the reference 2.

[0156] Reference 3 (Maojun, Liu, Gaimei, et al. Development of a blocking ELISA for detection of Mycoplasma hyopneumoniae infection based on a monoclonal antibody against protein P65. [J]. Journal of Veterinary Medical Science, 2016.) compares the results of each serum sample in the blocking ELISA and the commercial IDEXX ELISA, and finds that among the samples detected as positive by IDEXX, 147 / 155 (94.8%) are detected as positive by the method established by the P65 protein, and all the samples (173 / 173, 100%) detected as positive by IDEXX are detected as positive by the method, which shows that the method established in the application is more sensitive.

[0157] Reference 4 (Liu MJ, Ding ZY, Liu DX, et al. Expression of Mycoplasma hyopneumoniae DnaK gene and establishment of ELISA method [J]. Journal of Jinling Institute of Technology, 2011, 27(4): 79-84.) used the established ELISA antibody detection method to detect 132 serum samples, and the positive rate was 35.6%, while the positive rate detected by the Mhp antibody detection kit of IDEXX company was 11.4%, and the coincidence rate was 59.8%. The total coincidence rate of the method established in the application and the detection result of IDEXX was 88.6% (264 / 298), and the total coincidence rate was higher than that of the method in the compared reference 4, indicating that the method of the application has more advantages.

[0158] The above results show that the indirect ELISA method for detecting Mhp antibody established by the recombinant P46-P65 protein expressed by E. coli in the present application has good specificity and higher sensitivity than the method widely used in the clinic. Therefore, the method is expected to become a substitute method for Mhp serum epidemiological investigation and detection of immune response effect of vaccination.

[0159] Although the content of the present application is described in combination with the present embodiment, it cannot be considered as a limitation on the scope of the present application, and the protection scope of the present application is defined by the appended claims. In addition, those skilled in the art can make various modifications or modifications to the present application within the scope defined by the appended claims, and these modification or modification forms also fall within the protection scope of the present application.

Claims

1. A Mycoplasma hyopneumoniae fusion protein, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A nucleic acid molecule encoding the Mycoplasma hyopneumoniae fusion protein according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

2.

3. A nucleic acid vector, characterized in that It contains the nucleic acid molecule according to claim 2.

4. The method for preparing the Mycoplasma hyopneumoniae fusion protein according to claim 1, wherein: include: Transforming the nucleic acid vector according to claim 3 into competent Escherichia coli cells to obtain expression engineered bacteria; The expression engineering bacteria are induced to express and purified to obtain the Mycoplasma hyopneumoniae fusion protein.

5. Use of the Mycoplasma hyopneumoniae fusion protein according to claim 1 in the preparation of a reagent or kit for detecting Mycoplasma hyopneumoniae.

6. The use according to claim 5, characterized in that The Mycoplasma hyopneumoniae fusion protein according to claim 1 is used as a coating antigen in a detection reagent or a kit.

7. An ELISA detection kit for Mycoplasma hyopneumoniae antibodies, characterized in that: The kit comprises an ELISA plate coated with the Mycoplasma hyopneumoniae fusion protein according to claim 1.

8. The kit according to claim 7, characterized in that The coating amount of Mycoplasma hyopneumoniae fusion protein is 0.12-0.16 μg / well; the coating concentration of the protein is 1.4-2.1 μg / mL.

9. The kit according to claim 7, characterized in that The kit also includes a diluent, a blocking solution, a washing solution, an enzyme-labeled goat anti-pig antibody, a substrate color developing solution and a stop solution.

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