Mycoplasma hyopneumoniae fusion protein as well as preparation method and application thereof

Abstract

The invention 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 amino acid sequence of the mycoplasma hyopneumoniae fusion protein comprises a mycoplasma hyopneumoniae P46 protein, a mycoplasma hyopneumoniae P65 protein and a rigid connecting peptide; the amino acid sequence of the mycoplasma hyopneumoniae P46 protein is as shown in SEQ ID NO.7; the amino acid sequence of the mycoplasma hyopneumoniae P65 protein is as shown in SEQ ID NO. 8; the amino acid sequence of the rigid connecting peptide is EAAAK. When the mycoplasma hyopneumoniae fusion protein provided by the invention is used for detection, the sensitivity is relatively good, the repeatability is good, the mycoplasma hyopneumoniae fusion protein does not generate cross reaction with other antibodies, the mycoplasma hyopneumoniae fusion protein has relatively strong specificity, and the accuracy is obviously higher than that of an 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 invention relates to the technical field of veterinary biological products, and in particular to a Mycoplasma hyopneumoniae fusion protein and a preparation method and application thereof. Background Art

[0002] At present, the key measure to effectively prevent and control Mycoplasmal pneumoniae of swine (MPS) is vaccination. This method has a more significant effect on alleviating lung lesions caused by Mycoplasma pneumoniae (Mhp) infection and increasing daily weight gain and feed conversion rate. It also plays a very important role in controlling infection in pigs in the same pen and reducing the bacterial carrier rate in sows.

[0003] Effective immune assessment will help implement the improved Mhp prevention and control program mentioned above, but the current Mhp ELISA antibody detection method has low sensitivity and the test results are inconsistent with the expected antibody levels, which is not conducive to the immune assessment of Mhp in pig farms.

[0004] Prior art CN 113588946 B provides a recombinant protein for indirect ELISA detection of Mycoplasma hyopneumoniae antibodies, the amino acid sequence of the recombinant protein includes sequentially connected label carrier amino acids, Mycoplasma hyopneumoniae P46 protein amino acids, spacer protein amino acids and Mycoplasma hyopneumoniae P36 protein amino acids. The recombinant protein provided by the invention can be used as a coating antigen to establish an indirect ELISA detection method that is simple to operate, low in cost, and can effectively identify positive and negative serum. The method has a high compliance rate and good specificity and repeatability, providing new ideas and methods for the comprehensive prevention and treatment 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 test is 78.8%, indicating that the method established by the technology has low sensitivity.

[0005] Prior art CN 103018442 B obtains two recombinant Escherichia 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 ELISA plate coated with the mutated Mycoplasma hyopneumoniae membrane protein P46 and P65 gene expression proteins as antigens and other core reagents. The invention discloses the cloning of Mycoplasma hyopneumoniae membrane protein P46 and P65 genes, site-directed mutagenesis, and the expression and purification methods of P46 and P65 proteins. It also discloses an indirect ELISA antibody detection method for Mycoplasma hyopneumoniae. The indirect ELISA antibody detection kit prepared by the invention can be used for large-scale clinical detection and epidemiological investigation of Mycoplasma hyopneumoniae antibodies, and has broad market prospects. The method established by the technology has a compliance rate of 91.3% (84 / 92) with IDEXX. It shows that the consistency of the two methods is very high, but it has been proved that IDEXX is not very sensitive to positive serum, which also shows that the sensitivity of this method will not be high. The positive rate of this technology was 47.8% (42 / 92), and the positive rate of IDEXX's Mhp ELISA kit was 45.7% (42 / 92), indicating that the positive rate of this method was higher than that of IDEXX, but basically the same. However, IDEXX's sensitivity to positive serum is not very high.

[0006] 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 10 μl concentrated washing solution, a color developing solution A, a color developing solution B and a stop solution. The detection plate of the kit is a detachable 96-well ELISA plate coated with a mutant Mycoplasma hyopneumoniae membrane protein P46 gene protein antigen, the enzyme conjugate working solution is a rabbit anti-swine antibody labeled with horseradish peroxidase, the positive control serum is taken from a pig that is positive through indirect hemagglutination and IDEXX ELISA detection kit detection, and has obvious Mycoplasma hyopneumoniae lesions in the lung after dissection, and the negative control serum is taken from a pig that is negative through indirect hemagglutination and IDEXX ELISA detection kit detection, and has no Mycoplasma hyopneumoniae lesions in the lung after dissection. The invention has the beneficial positive effects of: strong specificity, high sensitivity, simple operation, easy large-scale promotion and application, and has broad market prospects. However, the positive rate of P46-ELISA was 28.7% (41 / 143), and the positive rate of IDEXX's Mhp ELISA kit was 26.5% (38 / 143), indicating that the sensitivity of the detection method established by P46 protein is still low. This shows that the positive rate of this method is also slightly higher than that of IDEXX, but IDEXX's sensitivity to positive serum is not very high. Summary of the invention

[0007] The purpose of the present invention is 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 object, the technical solution of the present invention is as follows:

[0009] A Mycoplasma hyopneumoniae fusion protein, the amino acid sequence of which includes 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:

[0011] GQTESGSTSDSKPQAETLKHKVSNDSIRIALTDPDNPRWISAQKDIISYVDETEAATSTITKNQDAQNNWLTQQANLSPAPKGFIIAPENGSGVGTAV NTIADKGIPIVAYDRLITGSDKYDWYVSFDNEKVGELQGLSLAAGLLGKEDGAFDSIDQMNEYLRSHMPQETISFYTIAGSQDDNNSQYFYNGAMKVL KELMKNSQNKIIDLSPEGENAVYVPGWNYGTAGQRIQSFLTINKDPAGGNKIKAIGSKPASTFKGFLAPNDGMAEQAITKLKLEGFDTKKIFVTGQDYNDKAKTFIKDGDQNMTIYKPDKVLGKVAVEVLRVLIAKKNSATKDDVEMELKSKLPSISFKYDNKTYKSQNKIINTILVSPVVVTKANVDNPDA(SEQ ID NO.7).

[0012] The amino acid sequence of the Mycoplasma hyopneumoniae P65 protein is:

[0013] NDSDKNLMAKNFDFHPSIQGYKKIAHQLLLKLTLDQEEKDDSNAEELKNTTNFD DFDENKPTYSKVIDLSVFAKSNKEFLEKLNENKQTSEFIAQKSTFDTDQEAAIKDDKRTFGNIVREIVSLPIFDNFDFRELIPVKNPFVKAIINSYLGKPAGSLIKDIEQLENKVKDYARPNIKIFDTIIDSFI RKMVAFFAELNTDQEIKEFKMSPQILFLTLRNAILSPFDLTKLKDSATFKILMNLKPEQILTLLGLSKTPSVPKPEKPKDQSSKPQTDTSSQKQESGTSSTDSTKATTENQKPAEQTDSSEQSSTEPKSN(SEQ ID NO.8).

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

[0015] Other suitable rigid linking peptides can also be used as linkers without strict limitation.

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

[0017] P65 protein is a lipoprotein with species-specific epitopes and good immunogenicity on the surface of Mhp. It is a major immune protein of Mhp. Its main immunogenic region is located at the C-terminus, on the outside of the cell membrane. Its N-terminus has esterase activity and can decompose fats and fatty acids.

[0018] P46 and P65 are two important membrane proteins of Mhp, both of which have the ability to stimulate animals to produce strong immune responses. The present invention 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. The widely used IDEXX ELISA method and the Mhp metabolic inhibition test confirm that the method established by the present invention is more sensitive, more accurate, and has better specificity than the ELISA antibody detection system of the individual P46 and P65 proteins and the IDEXX ELISA method, which benefits from the synergistic effect between P46 and P65. However, replacing them with other similar proteins lacks this synergistic effect and cannot improve the accuracy.

[0019] In a preferred embodiment, the amino acid sequence of the Mycoplasma hyopneumoniae fusion protein is shown in SEQ ID NO.1.

[0020] SEQ ID NO.1:

[0021] GQTESGSTSDSKPQAETLKHKVSNDSIRIALTDPDNPRWISAQKDIISYVDETEAATSTITKNQDAQNNWLTQQANLSPAPKGFIIAPENGSGVGTAVNTIADKGIPIVAYDRLITGSDKYDWYVSFDN EKVGELQGLSLAAGLLGKEDGAFDSIDQMNEYLRSHMPQETISFYTIAGSQDDNNSQYFYNGAMKVLKELMKNSQNKIIDLSPEGENAVYVPGWNYGTAGQRIQSFLTINKDPAGGNKIKAVGSKPASIF KGFLAPNDGMAEQAITKLKLEGFDTQKIFVTGQDYNNKAKTFIKDGDQNMTIYKPDKVLGKVAVEVLRVLIAKKNKASRSEVENELKAKLPNISFKYDNQTYKVQGKNINTILVSPVIVTKANVDNPDA EAAAKNDSDKNLMAKNFDFHPSIQGYKKIAHQLLLKLTLDQEEKDDFNAEELKNTTNFDDFDDNKPTYSKVIDLSVFAKSNKEFLEKLNENKQTSEFIAQKSTFDTNQEAAIKDDKRTFGNIVREIVSLP IFDNFDFRELIPVSNPFVKAIINSYLGKPAGSLIKDIEQLENKVKDYARPNIKIFDTIIDSFIRKMVAFFAELNTDQEIKEFKMSPQILFLTLRNAILSPFDLTKLKDSATFKILMNLKPEQILTLLGLSKTPSVPKPEKPKDQGSKPQTDTSSQKQESGTSSTDSTKATTENQKPAEQTDSSEQSSTEPKSN.

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

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

[0024] SEQ ID NO.2:

[0025] ggacagacag aatcaggttc gactctgat tctaaaccac aagccgagac tctaaaaacataagtaagtaagta atgatctat tcgaatagca ctaaccgatc cagataatcc tcgattc tcgattc agtgcccaaagatattat tcttagtt gatcaaaaccag aggcaggagga ttcacaaaccta ctcactcagc aagctaattt

[0026] aagcccccg

[0027] ccaaaaggat ttattattgc ccctgaaaat ggaagtggag ttggaactgc tgttaatacaattgctgata aaggaattcc gattgttgcc tatgatcgac taattactgg atctgataaa tatgattggtatgtttcttt tgataatgaa aaagttggcg aattacaagg tctttcaggtgcttagcgagggata gcttttgatt caattgatca aatgaatgaa tatctaagat cacatatgcc ccaagagacaatttcttttt atacaatcgc gggttcccaa gatgataata attcccaata ttttataat ggcgcaatgaaagtacttaa agaattaatg aaaaattcgc aaaataaaat aattgattgaatta ggttagttatc ccaggatgga attatggaac tgccggtcaa agaatccaat cttttctaac aattaacaaagatccagcag gtggtaataa aataaaagct gttggttcaa aaccagcttc tattttcaaa ggatttcttgcaccaaatga tggaatggcc gaacaagcaa tcaccaaatt aaaacttgaatttactactagaatttacttac ggtcaagatt ataataataa agccaaaact tttatcaaag acggcgatca aaatatgacaatttataaac ctgataaagt tttaggaaaa gttgctgttg aagttcttcg ggttttaatt gcaaagaaaaataaagcatc tagatcagaa gtcgaaaacg aactaaataaccaattac ttaaatatgataatcaaca tataaagtgc aaggtaaaaa tattaataca attttagtaa gtccagtaat tgttacaaaagctaatgttg ataatcctgatgccgaggcc gccgccaaga atgattctga taaaaattta atggcgaaaaatttgactt ccacccttca attcaaggtt ataaaaaaat tgctcaccaa cttttgttaa aattaactcttgaccaagaa gaaaaagatg attttaatgc tgaagagtta aaaaatacta caaatttcga tgattttgatgataataaac cgacctattc caaagttat gacctaagtg tttttgcaaa atcaaataaa gaatttcttgaaaaattaaa cgaaataag caaactagtg aatttattgc tcaaaaatcc acttttgaca ccaatcaagaagctgcaatc aaagacgaca aacgcacttt tggaaatata gttcgagaaa ttgtatcttt accaatcttcgataattttg attttagaga gttaatacct gtttcaaatc cgtttgtaaa agcaattat aacagctatttagggaaacc agctggttct cttataaaag atatcgaaca actcgaaaat aaaagtgaaag attacgcaagacctaatatc aagattttcg atacaattat tgactcattc ataagaaaaa tggtagcatt tttgctgaattaaacactg atcaagaaat aaaagaattc aaaatgtcac ctcaaaatact atttctgaca ctaagaaatgcaatactaag tccatttgat ttaactaaat taaaaagacag tgctacaattt aaaattttaa tgaatctcaaaccagaacaa atttaactc 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, bp 1-1164 is the Mycoplasma hyopneumoniae P46 gene sequence, bp 1165-1179 is the rigid protein linker gene sequence; bp 1180-2133 is the Mycoplasma hyopneumoniae P65 gene sequence.

[0029] Based on the same inventive concept, the present invention also claims protection for a nucleic acid vector carrying the above-mentioned nucleic acid molecule encoding the Mycoplasma hyopneumoniae fusion protein.

[0030] Based on the same inventive concept, the present invention also claims to protect a method for preparing the Mycoplasma hyopneumoniae fusion protein, comprising:

[0031] Transforming the nucleic acid vector into competent Escherichia coli cells to obtain expression engineering bacteria;

[0032] The expression engineering bacteria are induced to express and purified to obtain the Mycoplasma hyopneumoniae fusion protein.

[0033] Based on the same inventive concept, the present invention also claims to protect the use of the Mycoplasma hyopneumoniae fusion protein in the preparation of a reagent for detecting Mycoplasma hyopneumoniae.

[0034] In one of the preferred embodiments, 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 invention also claims protection for an ELISA detection kit for Mycoplasma hyopneumoniae antibodies, wherein the kit comprises an ELISA enzyme labeling 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 of the preferred embodiments, in the kit, the optimal coating concentration of the protein is 1.4-2.1 μg / mL.

[0038] In one preferred embodiment, the kit further includes a diluent, a blocking solution, a washing solution, an enzyme-labeled goat anti-pig antibody, a substrate color development solution and a stop solution.

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

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

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

[0042] In a preferred embodiment, the stop solution is a sulfuric acid solution.

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

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

[0045] When the Mycoplasma hyopneumoniae fusion protein provided by the present invention is used for detection, when the positive serum is diluted to 6400 times at most, the detection result is still positive (OD450nm>0.30), indicating that the sensitivity of the method is good. The results of inter-batch and intra-batch detection of 4 positive and 4 negative sera show that the intra-batch coefficient of variation is between 0.49% and 4.07%, and the inter-batch coefficient of variation is between 1.74% and 4.96%, both less than 5%, indicating that the established method has good repeatability. The OD450nm values ​​of the detection results of 7 positive sera selected from the Mhp negative field, including CSFV, FMDV, PRRSV, PRV, PCV2, PEDV and APP, are all less than the critical value of 0.3, indicating that the antibody detection method established in this study does not cross-react with the above antibodies and has strong specificity. The serum positive rates of the method of the present invention and the IDEXX method are 62.4% and 73.8% respectively, and the total coincidence rate of the detection results of the two methods is 88.6%, and the accuracy is significantly higher than that of the IDEXX method.

[0046] In summary, the present invention has established an indirect ELISA detection method with high sensitivity, good repeatability, and the ability to effectively identify positive and negative sera. It can be used for clinical sample detection and monitoring of pig herd immunity levels, and provides ideas and methods for the comprehensive prevention and treatment of Mycoplasma hyopneumoniae. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is the PCR amplification result of the gene fragment of the recombinant protein Mhp P46 P65 of Mycoplasma hyopneumoniae of the present invention, wherein, M: DNA molecule marker; lane 1: Mhp P46 P65 PCR amplification product.

[0048] Figure 2SDS PAGE electrophoresis diagram and West blot diagram of the expression and purification of the Mycoplasma hyopneumoniae fusion protein Mhp P46 P65 antigen provided in the embodiment of the present invention; 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 1 is a graph showing the specificity test results of the Mycoplasma hyopneumoniae r P46-P65 ELISA detection method provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0050] In order to more clearly explain the overall concept of the application, the following is described in detail by way of embodiment. In the following description, a large amount of specific details are provided so as to provide a more thorough understanding of the application. However, it is obvious to those skilled in the art that the application can be implemented without one or more of these details. In other examples, in order to avoid confusion with the application, some technical features well known in the art are not described.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0052] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0053] Example 1 Obtaining fusion protein P46-P65

[0054] The present invention is based on the pET-28-P46 and pET-28-P65 (the construction methods of pET-28-P46 and pET-28-P65 refer to the prior art: MHP virulence and immunity-related protein bioinformatics analysis, cloning and expression, Yu Jinxin, 2022, Foshan University of Science and Technology) recombinant plasmids preserved in this laboratory, and the primers are designed with reference to the Mhp232 strain (NC_006360.1) to amplify the corresponding sequence. The amplified gene sequence is connected with the pET-28a (+) (the construction method of pET-28a (+) refer to the prior art: MHP virulence and immunity-related protein bioinformatics analysis, cloning and expression, Yu Jinxin, 2022, Foshan University of Science and Technology) vector preserved in the laboratory, and the sequence is 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, and the fusion protein was induced and expressed 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 of 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 the reference sequence. Four specific primers were designed by Primer Select according to the sequences of P46 and P65 coding genes of Mhp232 strain and the principle of overlap extension PCR (SOE-PCR). Primers Fp46 and Rp65 introduced BamHⅠ and XholⅠ restriction sites, 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); Rp65: 5'-CCGCTCGAGGTTTGATTTAGGCTCGGTACTT (SEQ ID NO.6).

[0058] use HSDNA Polymerase system was used for PCR amplification (PCR amplification system: P HSDNA Polymerase: 1μL; dNTP: 4μL; Buffer: 10μL; dH20: 29μL; Template: 2μL; 2μL of upstream and downstream primers. PCR amplification conditions: pre-denaturation 98℃2min; denaturation: 98℃10s; annealing: 55℃15s; extension: 68℃4min; total extension: 68℃10min; number of cycles: 35 cycles). The amplified product was identified by agarose electrophoresis and showed that the amplified length was 2118bp (such as Figure 1 The PCR product has no mixed bands and the size is as expected. Clean according to the instructions of the OMEGAPCR Cleaning Kit.

[0059] 1.2 Cloning and transformation of recombinant plasmid

[0060] The cleaned gene fragment and pET-28a(+) plasmid were double-digested with two restriction endonucleases, BamHⅠ and XholⅠ (37°C, 2h), respectively. The reaction system is shown in Table 1, and the digested products were cleaned with OMEGA PCR cleaning kit.

[0061] Table 1 Double enzyme digestion reaction system

[0062]

[0063] The target gene after restriction digestion and cleaning was connected to the plasmid after restriction digestion and cleaning using T4 ligase (22°C, 30min). The reaction system is shown in Table 2.

[0064] Table 2 Connection system

[0065]

[0066] The ligation product was transformed into the competent state of the Escherichia coli expression strain BL21 (DE3). The specific steps of transformation were as follows: 100 μL / tube of the prepared competent cells were taken out from the -80°C ultra-low temperature refrigerator, thawed in ice water, and then 10 μL of the ligation product was added to the competent cells and ice-bathed for 30 minutes. The competent cells after the ice bath were placed in a 42°C water bath for heat shock for 90S, and then immediately ice-bathed for 2 minutes, and 600 μL of sterile LB liquid culture medium was added, and placed in a water bath constant temperature oscillator at 37°C and 180rpm for 45 minutes; the bacterial liquid was centrifuged at 5000rpm for 2 minutes, and 100 μL of sterile LB liquid culture medium was added to resuspend the bacteria after discarding the liquid, and then it was spread on an LB solid plate containing 50 μg / mL kanamycin, and inverted and cultured overnight in a 37°C constant temperature box to obtain the BL21 (pET-P46-P65) strain.

[0067] A single transformant on the plate was used as a template and PCR amplification was performed using the T7 universal primer of the pET-28a(+) plasmid itself (Shanghai Baishengyue Biotechnology Co., Ltd.) (pre-denaturation at 94°C for 5 min, denaturation at 94°C for 5 min, annealing at 55°C for 30 s, extension at 72°C for 1 min, 35 cycles, and full extension at 72°C 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 has been successfully transformed into E. coli. Successfully transformed into E. coli is called a positive transformant.

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

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

[0074] 2.1 Expression of recombinant proteins P46-P65

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

[0076] (2) Centrifuge the induced cells at 6000 rpm and 4°C for 5 min, discard the supernatant, concentrate 20 times with ultrapure water, and fully resuspend the cells;

[0077] (3) The resuspended bacterial sludge was placed in an ice water bath for ultrasonic disruption (amplitude 6, power 35%, working time 10 min, ultrasonic on 5 s off 5 s), centrifuged at 12000 rpm, 4°C for 20 min, and the supernatant and precipitate were separated;

[0078] (4) Resuspend the precipitate in equal volumes, take 30 μL of whole bacterial solution, 30 μL of supernatant, and 30 μL of precipitate sample, add 10 μL of 4× Loading Buffer, boil in a boiling water bath for 5 min, centrifuge at 12000 rpm for 5 min, and perform 12% SDS-PAGE electrophoresis on the prepared samples.

[0079] (5) It was finally determined that the recombinant protein was expressed in a soluble form (see Figure 2 A).

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

[0081] (1) Sample treatment: The bacterial solution after ultrasonic disruption was centrifuged at 4°C and 12,000 rpm for 10 min, and the supernatant was filtered through a 0.44 μm filter membrane.

[0082] (2) Assembling a gravity column: Purify the soluble protein using Huiyan Bio Ni ion affinity chromatography medium, add 2 mL of the medium into the protein purification column, pad it with a gasket, and drain the 20% ethanol in the column;

[0083] (3) Washing with water: Add 10 mL of pure water to wash away the residual ethanol, and wash 2 to 3 times.

[0084] (4) Equilibration: Add 10 mL of equilibration buffer (0.02 M PB) and equilibrate 2 to 3 times.

[0085] (5) Loading: Add the treated protein sample to the equilibrated purification column, control the flow rate to 0.2 mL / min, collect the flow-through, and load the collected flow-through again, for a total of 3 times.

[0086] (6) Washing: Wash with 50 mM and 100 mM imidazole containing 0.02 M PB, respectively, with a washing volume of 10 mL each. During washing, the flow rate was controlled at 0.5 mL / min, and 10 mL / tube of each washing solution was collected.

[0087] (7) Elution: Use 500 mM imidazole containing 0.02 M PB for elution. Control the flow rate to 0.2 mL / min and the elution volume to 10 mL. Collect the eluate in 5 tubes.

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

[0089] (9) The eluate was collected and subjected to SDS-PAGE detection. The results showed that the target protein appeared at 100 ku (see the results). Figure 2 A), the obtained r P46-P65 has a lower concentration of impurity proteins, indicating a good purification effect.

[0090] (10) The purified protein was identified by Western blot using the high immune serum of mice immunized with Mhp vaccine as the primary antibody, and the purified rP46-P65 was identified by Western blot 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 of rainbow marker, 20 μl of supernatant sample to each well, and 10 μl of intracellular precipitate to each well, and perform electrophoresis at 120 V for 70 min.

[0092] (12) Transfer: Pretreatment of Millipore PVDF (0.45 μm) membrane: Soak in methanol for 1 min → Soak in transfer solution for 2 min. Transfer order: black side of clamp + sponge (polyethylene plate) + filter paper + gel + PVDF membrane (regardless of front and back) + filter paper + sponge + white side of clamp. Note that bubbles should be driven out with a glass rod after each layer is placed, and the gel should always be kept moist, otherwise the gel will be fragile. Bubbles must be driven out completely, otherwise a high background will be generated or protein transfer will be affected. Fix the sandwich in the transfer tank, with the black side of the clamp facing the black side of the transfer tank.

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

[0094] (14) Blocking: Block for 1 h in a 37°C incubator and overnight at 4°C.

[0095] (15) Primary antibody incubation: Mhp positive serum was used as the primary antibody (1:100), and the PVDF membrane was placed in the primary antibody diluted in the blocking solution and incubated on a shaker at 37°C for 2 h at 80 rpm. The membrane was then washed three times with TBST on a side-to-side shaker at 60 rpm, with each wash lasting 15 min.

[0096] (16) Secondary antibody incubation: Goat anti-swine IgG-HRP was used as the secondary antibody (1:10,000). The PVDF membrane was placed in the secondary antibody diluted in the blocking solution and incubated on a shaker at 37°C for 1 hour at 80 rpm. The membrane was then washed three times with TBST at 60 rpm on a side-to-side shaker for 15 min each time.

[0097] (17) Color development: The results show that at 100 ku ( Figure 2 B) showed a specific band, which indicated that the recombinant protein had good reactivity with the positive serum.

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

[0099] In this implementation, an indirect ELISA detection method for Mycoplasma hyopneumoniae antibodies using recombinant protein Mhp-P46-P65 was established and optimized. First, 298 sera from different pig farms in Hunan were tested using the commercial IDEXX Mycoplasma hyopneumoniae antibody detection kit to screen out the positive and negative sera that met the conditions, and then the known positive and negative sera were used to optimize the ELISA detection method. The specific steps are as follows:

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

[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 0.05 M carbonate buffer (pH 9.6) and added to a 96-well ELISA plate as a coating antigen, 100 μL / well, and coated overnight at 4°C;

[0103] (2) Blocking: Block with 5% skimmed milk powder and incubate in a 37°C incubator for 30 min;

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

[0105] (4) Serum incubation: The serum to be tested was diluted 100 times with serum diluent (5% skim milk powder diluted in PBST, with 5% E. coli lysis buffer added), added to the coated ELISA plate, 100 μL / well, and incubated in a 37°C incubator for 30 min;

[0106] (5) Washing: Discard the liquid in the ELISA plate, add PBST (containing 0.05% Tween-20, pH 7.4), 300 μL / well, and wash repeatedly for 4 times. Pat dry after the last wash.

[0107] (6) Secondary antibody incubation: add goat anti-swine IgG (purchased from Sigma), 100 μL / well, and incubate in a 37°C incubator for 30 min;

[0108] (7) Wash and repeat step 5;

[0109] (8) Substrate color development: Add TMB color development solution (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.), 50 μL / well, and incubate in a 37°C incubator for 15 min;

[0110] (9) Termination: Add 50 μL / well stop solution (2M H2SO4) to terminate the reaction, 50 μL / well, and read the OD 450nm Absorbance value. The content of the components in the kit can vary according to the number of tests, which has no effect on the effect.

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

[0112] 2.1 Determination of optimal antigen concentration

[0113] Using carbonate buffer (pH 9.6) as coating solution, the r P46-P65 obtained above was diluted to 0.7 μg / mL, 1.4 μg / mL, 2.1 μg / mL, and 2.8 μg / mL with coating solution, and coated at 100 μL / well. Four positive sera and four negative sera were selected to perform the experiment according to the indirect ELISA operation steps, and the P / N value at each antigen coating concentration was calculated. The antigen coating concentration with the largest P / N value was selected as the optimal antigen coating concentration. The optimal protein coating concentration 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 the optimal serum dilution and goat anti-swine IgG dilution

[0117] 5% skim milk powder was diluted with PBST, 5% Escherichia coli lysate was added as serum diluent, goat anti-pig IgG was used as enzyme-labeled secondary antibody, and the optimal serum dilution and goat anti-pig IgG dilution were determined by the standard titration method for the initially established ELISA method. Four positive sera and four negative sera were selected, and the serum was diluted with serum diluent at 1 / 50, 1 / 100, and 1 / 200. The P / N value was detected and calculated. The results showed that the serum was tested at a dilution of 1 / 100, and the P / N value was the largest. The results are shown in Table 5. Goat anti-pig IgG was diluted at 1 / 10000, 1 / 20000, 1 / 30000, 1 / 40000, and 1 / 50000 for detection. The results showed that the goat anti-pig IgG was tested at a dilution of 1 / 20000, and the P / N value was the largest. The results are shown in Table 6. Finally, the optimal serum dilution was determined to be 1 / 100, and the optimal goat anti-pig IgG dilution was 1 / 20000.

[0118] Table 5 Determination of optimal serum dilution

[0119]

[0120] Table 6 Determination of sheep anti-pig IgG dilution

[0121]

[0122]

[0123] 2.3 Determination of the optimal incubation time

[0124] Four sera that were negative and positive (the sera with S / P value above 2.0 were positive sera) in the IDEXX test were used as reference sera. The experiment was carried out according to the best serum and goat anti-pig IgG dilutions determined above. The serum to be tested was incubated for 20min, 30min, 40min, 50min and 60min for testing, goat anti-pig IgG was incubated for 20min, 30min, 40min, 50min and 60min for testing, and the substrate was incubated for 10min, 15min, 20min, 25min and 30min for testing, and the P / N value was calculated. The results showed that the P / N was the largest when the serum was incubated for 30min, as shown in Table 7; the P / N was the largest when the goat anti-pig IgG was incubated for 30min, as shown in Table 8; the P / N was the largest when the substrate was incubated for 15min, as shown in Table 9. Therefore, it was determined that the incubation time of the best serum and goat anti-pig IgG was 30min, and the incubation time of the best substrate was 15min.

[0125] Table 7 Determination of optimal serum incubation time

[0126]

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

[0128]

[0129]

[0130] Table 9 Optimal substrate incubation time

[0131]

[0132] 2.4 Determination of positive and negative critical values

[0133] The above conditions were used as the optimal ELISA test conditions. 80 sera that were negative by PCR identification of pig lungs and whose corresponding sera were negative by IDEXX antibody test kit were tested. The results showed that the OD of negative sera was 450nm average value is 0.135, the standard deviation (S) is 0.056, according to the critical value calculation formula The critical value of the indirect ELISA method was calculated to be 0.3, that is, the test value of the sample ≥ 0.3 was positive, and the value < 0.3 was negative.

[0134] 2.5 Sensitivity experiment

[0135] Four Mhp positive sera with S / P values ​​above 2.0 detected by IDEXX antibody test kit were selected and diluted in multiples (1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800) and tested by the established ELISA method. The results showed (Table 10) that when the positive serum was diluted up to 6400 times, the test result was still positive (OD450nm>0.30), indicating that the sensitivity of the method was good.

[0136] Table 10 Sensitivity test results

[0137]

[0138] 2.6 Repeatability Experiment

[0139] This method was used to perform inter-batch and intra-batch testing on 4 positive and 4 negative sera. The results showed that the intra-batch coefficient of variation was between 0.49% and 4.07%, and the inter-batch coefficient of variation was between 1.74% and 4.96%, both of which were less than 5%. The results are shown in Table 11, indicating that the established method has good repeatability.

[0140] Table 11 Indirect ELISA repeatability test results

[0141]

[0142] 2.7 Specificity test

[0143] The indirect ELISA method established in this study was used to detect seven positive sera selected from Mhp-negative fields, including CSFV, FMDV, PRRSV, PRV, PCV2, PEDV, and APP. The test results are shown in Figure 3 The OD450nm values ​​of the above test results were all less than the critical value of 0.3, indicating that the antibody detection method established in this study did not cross-react 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 sera from clinically Mhp-positive pig farms and Mhp-negative farms were tested using two methods. The test results are shown in Table 12.

[0147] Table 12 Test results of clinical samples

[0148]

[0149] *Studies have shown that sera that were suspicious by IDEXX Mhp were actually positive by metabolic inhibition, therefore, 13 sera that were suspicious were counted as positive.

[0150] As shown in Table 12, there are 220 positive sera and 78 negative sera in the test results of this method; there are 173 positive sera, 13 suspicious sera and 112 negative sera in the test results of IDEXX method. Studies have shown that the sera that are suspicious in the IDEXX Mhp test are actually positive. After verification by the metabolic inhibition test, 13 suspicious sera are indeed positive, that is, there are 186 positive sera in total in the IDEXX method, and all of these 186 sera are positive when tested by this method. There are 78 negative sera in the test results of this method, and all of these 78 sera are negative in the test results of IDEXX; however, 34 samples of sera that are negative in the IDEXX test are positive when tested by this method. The serum positivity rates of this indirect ELISA method and IDEXX method are 62.4% and 73.8% respectively, and the total coincidence rate of the test results of the two methods is 88.6%.

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

[0152] The 34 sera that were inconsistent with the two methods were identified by color change test. 50 The result is 1×10 8 CCU / mL. KM2 liquid medium was used to dilute the 168 strains 10-fold to 10 -6 , 10 -7 , 10 - 8CCU / mL, and add 10% of the serum to be tested, culture at 37℃ and determine the results. The results show that the culture medium of the control group turned yellow after 9-10 days of culture, while the culture medium of 27 sera in the culture medium with 34 pig sera that were negative in the IDEXX test and positive in this method did not change color after 14 days of culture, indicating that 27 sera were positive for Mhp antibodies and only 7 sera were negative. Therefore, it means that there are actually only 78+5=85 negative sera in 298 sera, not 112 as in IDEXX, and the positive sera should be 186+27=213, not 173 as in IDEXX. The accuracy of the positive serum of the method of the present invention is 96.8% (213 / 220), while the accuracy of the IDEXX test is only 81.2% (173 / 213); the accuracy of the negative serum: the accuracy of the method is 91.8% (78 / 85), and the accuracy of the IDEXX is 75.9% (85 / 112). The total accuracy of the positive and negative serum of the method is 97.7% (291 / 298), and the overall accuracy of the IDEXX is 86.6% (258 / 298). The accuracy of the method of the present invention is significantly higher than that of the IDEXX method in terms of negative and positive serum or overall accuracy, and the improvement is greater than 10%.

[0153] In addition, the present invention also repeats some of the existing prior art and tests 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. Compared with the IDEXX method, the total compliance rate was 71.2%, and the positive sample detection rate detected by the ELISA method established by r P46-P36 was not as good as that of the IDEXX method. The positive detection rate and compliance rate of the method established by the present invention are higher than those of the method established in reference 1. This result shows that the sensitivity of the r P46-P65 indirect ELISA method established in this study is better.

[0155] Reference 2 (Shen Qingchun, Wang Fang, Han Mingyuan, et al. Prokaryotic expression of Mycoplasma hyopneumoniae P46 gene and establishment of indirect ELISA method [J]. Acta Animal Husbandry and Veterinary Medicine, 2012, 43 (3): 431-437.) used IDEXX's ELISA test kit and rP46-ELISA reagent to compare 143 pig sera. The test results showed that the positive detection rate of P46-ELISA was 28.7% (41 / 143), and the positive detection rate of IDEXX's Mhp ELISA kit was 26.5% (38 / 143), which showed that the detection method established by P46 protein was not as sensitive as IDEXX. The positive rate of the method established by the present invention was 73.8% (220 / 298), and the positive rate of the IDEXX antibody detection kit was 58.1% (173 / 298). In addition, 40 positive sera could not be detected by the IDEXX method. The test results showed that the positive detection rate of IDEXX was significantly lower than that of the method established by the present invention. The above results show that the sensitivity of the method established in the present invention is higher than that of the method established in 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.) compared the results of each serum sample in the blocking ELISA and the commercial IDEXX ELISA, and found that among the samples that were positive in the IDEXX test, 147 / 155 (94.8%) were positive samples detected by the method established for the P65 protein, while all the samples that were positive in the IDEXX test by this method were positive (173 / 173, 100%), indicating that the method established by the present invention has better sensitivity.

[0157] Reference 4 (Liu Maojun, Ding Zhiyong, Liu Dongxia, et al. Expression of DnaK gene of Mycoplasma hyopneumoniae 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 with a positive rate of 35.6%, while the positive rate detected by the Mhp antibody detection kit of IDEXX was 11.4%, and the coincidence rate between the two was 59.8%. The total coincidence rate between the method established in the present invention and the IDEXX test results was 88.6% (264 / 298), which was higher than the method invented in the reference 4, indicating that the method of the present invention has more advantages.

[0158] The above results show that the indirect ELISA method for detecting Mhp antibodies established in this study using the recombinant P46-P65 protein expressed by E. coli as the antigen has good specificity and sensitivity compared with the method currently widely used in clinical practice. Therefore, this method is expected to become an alternative method for Mhp seroepidemiology surveys and detecting the immune response effect of vaccination.

[0159] Although the content of the present invention is described in conjunction with the present embodiment, it cannot be considered as limiting the scope of the present invention, and the protection scope of the present invention is defined by the attached claims. In addition, those skilled in the art may make various changes or modifications to the present invention within the scope defined by the attached claims, and these changes or modifications also fall within the protection scope of the present invention.

Claims

1. A Mycoplasma hyopneumoniae fusion protein, characterized in that: Its amino acid sequence includes Mycoplasma hyopneumoniae P46 protein, Mycoplasma hyopneumoniae P65 protein and a rigid connecting peptide; The amino acid sequence of the Mycoplasma hyopneumoniae P46 protein is shown in SEQ ID NO.7; The amino acid sequence of the Mycoplasma hyopneumoniae P65 protein is shown in SEQ ID NO.8; The amino acid sequence of the rigid linker peptide is EAAAK.

2. The Mycoplasma hyopneumoniae fusion protein according to claim 1, characterized in that: The amino acid sequence of the Mycoplasma hyopneumoniae fusion protein is shown in SEQ ID NO.

1.

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

2.

4. A nucleic acid vector, characterized in that It carries the nucleic acid molecule encoding the Mycoplasma hyopneumoniae fusion protein as claimed in claim 3.

5. The method for preparing the Mycoplasma hyopneumoniae fusion protein according to claim 1 or 2, characterized in that: include: Transforming the nucleic acid vector into competent Escherichia coli cells to obtain expression engineering bacteria; The expression engineering bacteria are induced to express and purified to obtain the Mycoplasma hyopneumoniae fusion protein.

6. Use of the Mycoplasma hyopneumoniae fusion protein according to claim 1 or 2 in the preparation of a reagent or kit for detecting porcine Mycoplasma pneumonia.

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

8. 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 or 2.

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

10. The kit according to claim 8, 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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