Mycoplasma hyopneumoniae P65 recombinant protein as well as preparation method and application thereof

Abstract

The invention discloses expression preparation and application of a truncated mycoplasma hyopneumoniae P65 protein. An original protein sequence of the full-length mycoplasma hyopneumoniae P65 protein is a sequence 2 in a sequence table, a coding gene of the full-length mycoplasma hyopneumoniae P65 protein is a sequence 1 in the sequence table, a truncated protein sequence is a sequence 4 in the sequence table, and a coding gene of the full-length mycoplasma hyopneumoniae P65 protein is a sequence 3 in the sequence table. The invention relates to preparation and application of a truncated mycoplasma hyopneumoniae P65 protein, which comprises the following steps: 1) transforming competence of escherichia coli BL21 (DE3) by a recombinant vector for expressing full-length and truncated mycoplasma hyopneumoniae P65 recombinant protein to respectively obtain recombinant bacteria for expressing the full-length and truncated mycoplasma hyopneumoniae P65 recombinant protein; (2) carrying out induced expression on the recombinant bacteria, collecting thalli, crushing, and collecting supernatant; dissolving and renaturating the inclusion body protein; (4) purifying inclusion body protein and supernatant protein; and 5) application of the truncated mycoplasma hyopneumoniae P65 recombinant protein in vaccine and antibody detection. The truncated mycoplasma hyopneumoniae P65 protein is expressed by adopting an escherichia coli expression system, the protein expression quantity is large, the stability is extremely good, the purification method is simple, the production cost is greatly reduced, and the promotion of subsequent large-scale production and application is facilitated.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a truncated Mycoplasma hyopneumoniae P65 recombinant protein and a preparation method and application thereof. Background Art

[0002] Mycoplasma pneumonia of swine (MPS), commonly known as swine panting disease or endemic pneumonia, is caused by Mycoplasma hyopneumoniae (Mhp). The Mhp pathogen is a small (0.2–0.4 μm) amorphous prokaryotic organism (lacking a cell wall). It is a chronic respiratory infection of swine, characterized by panting and coughing. The infection rate is high in susceptible animals, including suckling piglets, nursery pigs, fattening pigs, boars, and sows. Mhp disrupts the mucosal clearance system by destroying the cilia on the epithelial surface and also affects the respiratory immune system. Once infected, Mhp strains persist in the animal's body for at least 12 weeks, meaning the infected animal's immune response cannot rapidly clear it from the respiratory tract. Consequently, Mhp predisposes animals to co-infection with other pathogens, including bacteria, parasites, and viruses.

[0003] According to statistics, the global prevalence of MPS in swine herds is approximately 70% to 80%, with a morbidity rate exceeding 40%. MPS is characterized by postmortem findings of "meat-like" or "shrimp-like" changes in the apical and cardiac lobes of the lungs, also known as "pancreatic changes." Affected pigs also experience adverse outcomes such as poor growth and development and a high feed-to-meat ratio, significantly increasing production costs and reducing pork quality. Therefore, research on effective prevention and control of this disease is of great practical significance.

[0004] Numerous studies have been published domestically and internationally on Mhp vaccines, detection, and diagnostic methods. Currently, the most widely used commercial MPS vaccines are primarily live attenuated and inactivated vaccines. These vaccines typically consist of one or two injections and can be mixed or combined with porcine circovirus type 2 (PCV-2) and Haemophilus parasuis for immunization. However, some studies have shown that traditional commercial vaccines often provide incomplete protection against typical Mhp-induced clinical symptoms and lesions, fail to prevent pathogen colonization, and lack a significant immune response. In the early stages of MPS genetically engineered vaccine development, efforts focused on the expression of a single antigen to achieve simplicity and effective protection. As research progressed, it became clear that vaccines composed of a single antigen were insufficient to induce adequate immune protection. Therefore, the development of chimeric vaccines composed of multiple antigens is needed to achieve a relatively optimal immune response. Regarding detection methods, ELISA is the most commonly used serological test. However, Mhp exhibits close relationships with strains such as Mycoplasma hyopneumoniae, often resulting in cross-reactivity, resulting in low specificity for ELISAs using whole-cell proteins as coating antigens. Therefore, establishing specific diagnostic methods based on the study of specific proteins has become one of the solutions to this problem.

[0005] The main immunogenic proteins of Mhp are currently known, including P36, P46, P65, P74, and P97. In particular, P65, a membrane protein carrying genus-specific epitopes, is the primary immunogenic protein of Mhp. It elicits early immune responses in weaned and growing pigs and exhibits no cross-reactivity with other mycoplasmas. Furthermore, its antiserum can inhibit the growth of mycoplasmas in vitro. Therefore, P65 protein could be used as a diagnostic tool for Mhp and a candidate subunit protein vaccine. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a method for preparing a recombinant protein of Mycoplasma hyopneumoniae P65 by expressing it in an Escherichia coli system, and to make the protein have good biological activity and immunogenicity.

[0007] Based on the above purpose, the present invention provides a Mycoplasma hyopneumoniae P65 recombinant protein and a truncated protein thereof. The sequence of the Mycoplasma hyopneumoniae P65 recombinant protein is Sequence 2 in the sequence listing; the truncated protein of the Mycoplasma hyopneumoniae P65 recombinant protein is Sequence 4 in the sequence listing.

[0008] The present invention also provides the gene encoding the Mycoplasma hyopneumoniae P65 recombinant protein; preferably, the gene encoding the Mycoplasma hyopneumoniae P65 recombinant protein is shown in Sequence 1 in the sequence listing, and the gene encoding the truncated protein of the Mycoplasma hyopneumoniae P65 recombinant protein is shown in Sequence 3 in the sequence listing.

[0009] The present invention also provides a recombinant vector for expressing the Mycoplasma hyopneumoniae P65 recombinant protein or its truncated protein, which is obtained by inserting the coding gene of the Mycoplasma hyopneumoniae P65 recombinant protein shown in Sequence 1 into a starting vector.

[0010] The starting vector is preferably a pET-22b vector.

[0011] Preferably, the coding genes of the full-length and truncated P65 proteins of Mycoplasma hyopneumoniae shown in Sequence 1 or Sequence 3 are inserted between the NdeⅠ and BamHI enzyme recognition sites of pET-22b.

[0012] The present invention also provides an engineered bacterium expressing the Mycoplasma hyopneumoniae P65 recombinant protein or a truncated protein thereof, which is obtained by transforming the recombinant vector expressing the Mycoplasma hyopneumoniae P65 recombinant protein or a truncated protein thereof into competent Escherichia coli BL21 (DE3), and screening the recombinant bacterium expressing the Mycoplasma hyopneumoniae P65 recombinant protein and a truncated protein thereof.

[0013] Another object of the present invention is to provide a method for preparing a recombinant protein of Mycoplasma hyopneumoniae P65, which comprises culturing the engineered bacteria and inducing protein expression using IPTG; collecting the supernatant and inclusion bodies after the recombinant bacteria expressing the truncated Mycoplasma hyopneumoniae P65 recombinant protein are crushed, and collecting the inclusion bodies after the recombinant bacteria expressing the full-length Mycoplasma hyopneumoniae P65 recombinant protein are crushed; and purifying to obtain the full-length Mycoplasma hyopneumoniae P65 recombinant protein or the truncated Mycoplasma hyopneumoniae P65 recombinant protein.

[0014] Preferably, the method comprises the following steps:

[0015] 1) transforming the recombinant vector expressing the Mycoplasma hyopneumoniae P65 recombinant protein or its truncated protein into competent Escherichia coli BL21 (DE3) to obtain a recombinant bacterium expressing the Mycoplasma hyopneumoniae P65 recombinant protein or its truncated protein;

[0016] 2) Inducing expression in the recombinant bacteria obtained in step 1), collecting and disrupting the bacteria, and collecting the supernatant and inclusion bodies;

[0017] 3) dissolving and renaturing the inclusion bodies obtained in step 2) to obtain a recombinant protein of Mycoplasma hyopneumoniae P65 or its truncated protein inclusion body;

[0018] 4) The truncated Mycoplasma hyopneumoniae P65 recombinant supernatant and inclusion body proteins are concentrated and purified, and the full-length Mycoplasma hyopneumoniae P65 recombinant inclusion body proteins are concentrated and purified.

[0019] Specifically, the method includes the following steps:

[0020] (1) Target gene synthesis: The full-length and truncated P65 protein gene sequences of Mycoplasma hyopneumoniae shown in Sequence 1 and Sequence 3 were inserted between the NdeⅠ and BamHI enzyme recognition sites of pET-22b to obtain a recombinant vector expressing the Mycoplasma hyopneumoniae P65 recombinant protein or its truncated protein, which was named pET-P65;

[0021] (2) Screening of recombinant strains: The plasmid pET-P65 obtained in step (1) was transformed into competent Escherichia coli BL21 (DE3); an engineered strain expressing the Mycoplasma hyopneumoniae P65 recombinant protein or its truncated protein was obtained, and the two strains were named E. coli-Mhp-P65-QC and E. coli-Mhp-P65-JD, respectively;

[0022] (3) Expression of recombinant protein: The strains E. coli-Mhp-P65-QC and E. coli-Mhp-P65-JD obtained in step (2) were cultured and induced to express protein using an appropriate concentration of IPTG;

[0023] (4) Obtaining the recombinant protein: The recombinant bacteria obtained in step (3) were collected by centrifugation, the bacteria were disrupted by ultrasonication, and the supernatant containing the truncated P65 recombinant protein of Mycoplasma hyopneumoniae was obtained by centrifugation. The bacterial mud on the upper layer of the precipitate was scraped off and the inclusion bodies were collected to obtain inclusion bodies containing the full-length and truncated P65 recombinant proteins of Mycoplasma hyopneumoniae;

[0024] (5) Purification of recombinant protein: The supernatant containing the truncated P65 recombinant protein of Mycoplasma hyopneumoniae from step (4) was filtered through a 0.45 μm filter membrane and purified by His affinity chromatography column;

[0025] (6) Denaturation, renaturation and purification of recombinant inclusion body protein: The inclusion bodies containing the full-length and truncated P65 recombinant proteins of Mycoplasma hyopneumoniae in step (4) were dissolved in inclusion body solubilization solution, and the dissolved inclusion bodies were slowly added to the inclusion body renaturation solution for dilution and renaturation, stirred at 4°C for 48 hours, and the replacement solution was slowly added to the renaturation solution, concentrated at 4°C, and finally concentrated Mycoplasma hyopneumoniae P65 recombinant inclusion body protein was obtained.

[0026] Another object of the present invention is to use the full-length and truncated Mycoplasma hyopneumoniae P65 recombinant proteins or the Mycoplasma hyopneumoniae P65 recombinant proteins prepared using the genes encoding the full-length and truncated Mycoplasma hyopneumoniae P65 recombinant proteins. Such applications include, but are not limited to, use in the preparation of diagnostic reagents for Mycoplasma hyopneumoniae and in the preparation of vaccines for the prevention or treatment of Mycoplasma hyopneumoniae, which also fall within the scope of protection of the present invention.

[0027] The positive beneficial effects of the present invention are:

[0028] The present invention uses an E. coli expression system to express full-length and truncated Mycoplasma hyopneumoniae P65 recombinant proteins. The truncated protein expression yields high protein expression levels, excellent stability, and a simple purification method, significantly reducing production costs and facilitating subsequent large-scale production and application.

[0029] The present invention optimizes the coding sequence of the Mycoplasma hyopneumoniae P65 recombinant protein, increasing protein expression. The truncated P65 protein can be expressed in large quantities in both the supernatant and inclusion bodies. By treating the inclusion bodies with a specially formulated protein refolding solution and replacement solution, the resulting Mycoplasma hyopneumoniae P65 recombinant protein has good biological activity and protein stability.

[0030] Animal experiments have shown that immunizing mice and pigs with the truncated P65 recombinant protein prepared by the present invention can produce high levels of antibodies, laying the foundation for the preparation of vaccines.

[0031] ELISA results show that the method for detecting Mycoplasma hyopneumoniae antibodies established using the truncated P65 recombinant protein prepared by the present invention has a good consistency rate with the commercial kit, laying a foundation for the establishment of Mycoplasma hyopneumoniae detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The results of PCR identification of colonies expressing full-length and truncated P65 proteins of Mycoplasma pneumoniae in pigs; M: DNA 2KPlus maker; S1: full-length P65 protein gene; S2: truncated P65 protein gene.

[0033] Figure 2 These are the SDS-PAGE results of the supernatant and precipitate of the full-length and truncated Mycoplasma pneumoniae P65 proteins; among them, P1: supernatant of the truncated Mycoplasma pneumoniae P65 protein; P2: precipitate of the truncated Mycoplasma pneumoniae P65 protein; M: Protein Maker; P3: precipitate of the full-length Mycoplasma pneumoniae P65 protein; P4: supernatant of the full-length Mycoplasma pneumoniae P65 protein.

[0034] Figure 3 The SDS-PAGE results of the purification of full-length and truncated Mycoplasma pneumoniae P65 proteins from supernatants and inclusion bodies, where M: maker; C1: full-length inclusion body P65 protein; C2: inclusion body truncated P65 protein; C3: soluble supernatant truncated P65 protein.

[0035] Figure 4 The SDS-PAGE results after purification of truncated P65 protein; M: maker; 1: truncated P65 protein

[0036] Figure 5To verify the stability of full-length and truncated P65 recombinant proteins at 2-8°C

[0037] Figure 6 To verify the stability of full-length and truncated P65 recombinant proteins at room temperature and 37°C DETAILED DESCRIPTION

[0038] The methods in the following examples are all conventional methods unless otherwise specified.

[0039] Example 1. Preparation of full-length and truncated Mycoplasma hyopneumoniae P65 recombinant proteins

[0040] 1.1 Experimental Materials

[0041] 1.1.1 Primers were synthesized by Shanghai Jierui Bioengineering Co., Ltd., and competent Escherichia coli BL21 (DE3) was maintained in the laboratory;

[0042] 1.1.2 Main Reagents

[0043] Restriction endonucleases, T4 ligase, plasmid DNA mini-extraction kit, DNA gel recovery kit, and IPTG were purchased from Anolun (Beijing) Biotechnology Co., Ltd.; Tris buffer, PBS buffer, and SDS-PAGE protein gel were purchased from Albertson Biotechnology Co., Ltd.; peptone and yeast extract were purchased from Oxoid; and BSA protein standard was purchased from ThermoFisher Scientific.

[0044] 1.2 Construction of full-length and truncated Mycoplasma hyopneumoniae P65 recombinant protein expression plasmids

[0045] The Mycoplasma hyopneumoniae P65 gene sequence was derived from the Mycoplasma hyopneumoniae epidemic strain isolated in our laboratory. Sequence analysis and comparison of the P65 gene sequence revealed three rare codons in the first 900 bp, so the front segment of the gene was synthesized by gene synthesis and codon optimized. After codon optimization, the target gene was synthesized by Nanjing Jierui Biological Co., Ltd. The synthesized gene was constructed into the pET-22b vector (which was preserved by our laboratory), and the 5' and 3' ends of the P65 gene were inserted into the NdeⅠ and BamHⅠ restriction sites, respectively. The P65 gene sequence is shown in Sequence 2 in the sequence table, and the encoded P65 protein sequence is shown in Sequence 1 in the sequence table.

[0046] According to AlphaFold prediction, the 581-600aa of P65 protein is relatively flexible, and pLDDT<50 (see attached figure). Therefore, the P65 protein was truncated during the construction and only 1-580aa was expressed. The specific gene sequence is shown in sequence 4 in the sequence table, and the encoded truncated P65 protein sequence is shown in sequence 3 in the sequence table.

[0047] 1.3 Inducible expression of recombinant Mycoplasma hyopneumoniae P65 protein

[0048] The P65 gene sequence or the truncated recombinant P65 gene sequence shown in SEQ ID NO: 1 was inserted between the NdeⅠ and BamHI enzyme recognition sites of pET-22b (purchased from Sangon Biotech Co., Ltd., catalog number: B540181-0001), respectively, to obtain recombinant vectors expressing the recombinant Mycoplasma hyopneumoniae P65 recombinant protein or its truncated protein, which were named pET-P65-QC and pET-P65-JD.

[0049] The above two expression plasmids pET-P65-QC and pET-P65-JD were transformed into Escherichia coli BL21 (DE3) competent cells, respectively. 1 μL of expression plasmid was added to 100 μL of competent cells, gently shaken to mix, ice bathed for 30 minutes, heat shocked in a 42°C water bath for 90 seconds, and continued ice bathing for 3 minutes. 800 μL of antibiotic-free LB medium was added, and the cells were cultured at 37°C, 200 rpm for 1 hour. Centrifuged at 6000 rpm for 5 minutes, 750 μL of supernatant was discarded, and the bacteria were resuspended with the remaining culture medium, evenly spread on a screening plate containing the corresponding antibiotics, and cultured at 37°C overnight.

[0050] Individual colonies were selected for PCR identification. The total PCR amplification reaction volume was 25 μl, containing 1 μl of upstream primer Mhp-F-1, 1 μl of downstream primer Mhp-R-1, 12.5 μl of KODPCR Mastermix, 9.5 μl of sterile distilled water, and 1 μl of template DNA. The cycling parameters were: initial denaturation at 94°C for 2 minutes, followed by 30 cycles of denaturation at 94°C for 10 seconds, annealing at 58°C for 10 seconds, and extension at 68°C for 1 minute; and a final extension at 68°C for 5 minutes.

[0051] The PCR products were detected by electrophoresis on 1% agarose gel (see Figure 1 ).Depend on Figure 1 It can be seen that a band of about 1800 bp and a band of about less than 1800 bp were amplified, which are consistent with the expected amplified fragment size.

[0052] Two positive single colonies were cultured overnight at 37°C and 200 rpm as seed solution. The next day, the seed solution was transferred to fresh LB medium at a ratio of 1:100 and cultured at 37°C and 250 rpm. When the bacterial solution OD 600 =0.6, add IPTG inducer to a final concentration of 0.5 mM, and continue induction culture at 37°C or 18°C ​​for 16-18 h.

[0053] 1.4 Bacteria collection and disruption

[0054] The bacteria were centrifuged at 5000 rpm for 10 min, collected, and resuspended in PBS. The recombinant E. coli was concentrated 10 times, that is, 50 mL of bacterial solution was centrifuged and resuspended in 5 mL of PBS. The bacteria were disrupted by ultrasound. The parameters of the ultrasound disruptor were set as follows: power 190 W, working for 5 s, off for 10 s, and ultrasound for 10 min to ensure that the bacteria were completely disrupted.

[0055] 1.5 Purification of inclusion bodies

[0056] Centrifuge the sonicated bacterial suspension at 12,000 rpm for 10 min, collect the supernatant, and use it for purification in step 1.8. Use a cell scraper to remove the gray bacterial sludge on the upper layer. Resuspend the inclusion bodies in inclusion body washing solution and centrifuge at 12,000 rpm for 10 min. Discard the supernatant and scrape the bacterial sludge again to obtain preliminarily purified inclusion bodies.

[0057] The formula of the inclusion body washing solution is as follows: 0.5% Triton X-100, 50 mM Tris-HCl, 300 mM NaCl, and the pH is adjusted to 8.0.

[0058] 1.6 Denaturation and renaturation of inclusion bodies

[0059] Weigh the inclusion bodies and dissolve them in the dissolving solution at a ratio of 30 mg / mL. Stir thoroughly overnight to completely dissolve the inclusion bodies. Take 2 mL of the inclusion body solution and slowly add it to 200 mL of the refolding solution. Stir and refold at 4°C for 48 hours. Concentrate to 50 mL using a concentration cup.

[0060] The formula of inclusion body solubilization solution is as follows: 8 M urea, 50 mM Tris-HCl, 100 mM NaCl, 10% glycerol, 10 mM EDTA, and the pH is adjusted to 8.0.

[0061] The formula of the refolding solution is as follows: 50mM Tris-HCl, 400mM L-arginine, and the pH is adjusted to 8.0.

[0062] 1.7 Concentration and replacement of refolding solution

[0063] Slowly add the replacement solution to the refolding solution to 1 L, stir at 4°C and concentrate to 50 mL to obtain the refolded Cap protein.

[0064] The replacement solution formula is as follows: 20mM Tris-HCl, 50mM NaCl, pH adjusted to 8.0.

[0065] 1.8 Concentration and purification of supernatant protein

[0066] Centrifuge the bacterial suspension after ultrasonic disruption in step 1.4 in a low-temperature centrifuge (12,000 rpm, 30 min, 4°C) and retain the supernatant. Filter through a 0.45 μm filter membrane and purify the protein using a His affinity chromatography column. The steps are as follows:

[0067] 1) Wash with 5 column volumes of deionized water to remove air and 20% ethanol; 2) Equilibrate the column with 5-10 column volumes of Buffer A (50 mM Tris, 150 mM NaCl, pH 8.0); 3) Pass the sample through the His column at 0.25 ml / min; 4) Equilibrate the column with Buffer A; 5) Elute with 50 mM, 200 mM, and 500 mM imidazole. Analyze the eluted samples on SDS-PAGE gels for the presence of the target protein.

[0068] 1.9 Protein qualitative analysis

[0069] The expression results of full-length and truncated P65 recombinant proteins were detected by SDS-PAGE, and the corresponding purity and concentration were calculated. Figure 2 、 Figure 3 and Figure 4 .

[0070] Example 2: Stability Study of Full-Length and Truncated Mycoplasma hyopneumoniae P65 Recombinant Proteins

[0071] The purified full-length and truncated P65 recombinant protein solutions were quantified to 0.1 mg / ml and then aliquoted into 0.5 mL EP tubes, 8 μL per aliquot, and stored at 2-8°C. Three aliquots were taken out monthly for SDS-PAGE grayscale determination of recombinant protein concentration for a total of 12 months. The purified full-length and truncated P65 recombinant protein solutions were then aliquoted into 0.5 mL EP tubes, 8 μL per aliquot, and stored at 25°C and 37°C, respectively. Three aliquots were taken out every 12 hours for a total of 14 times.

[0072] The experimental results show that the full-length and truncated P65 recombinant proteins can be stably stored at 2-8°C, with the full-length expression protein losing no more than 20% after 12 months and the truncated expression protein losing no more than 10%. At 25°C, the full-length expression protein did not change much within 96 hours, with a decrease of no more than 20%, and then showed a significant decrease in concentration, with a decrease of up to 50% after 144 hours; the truncated expression protein did not change much within 120 hours, and decreased by about 20% after 144 hours. At 37°C, the full-length expression protein did not change much within 72 hours, and decreased by up to 60% within 96 hours; the truncated expression protein did not change much within 84 hours, and decreased by about 30% after 144 hours. For details, see Figure 5 and Figure 6 .

[0073] Example 3. Application of full-length and truncated Mycoplasma hyopneumoniae P65 recombinant proteins in Mycoplasma hyopneumoniae subunit vaccines and inactivated vaccines

[0074] 3.1 Vaccine preparation

[0075] The vaccine was formulated by mixing filter-sterilized P65 protein with SEPPIC's 206 adjuvant in a 1:1 mass ratio, achieving the final concentrations of the components shown in Table 1. The emulsification conditions were: the adjuvant and aqueous phase were each warmed to 31°C, the aqueous phase was slowly added to the adjuvant, stirred at 350 rpm for 5 minutes, and then cooled at 20°C for 1 hour, avoiding movement and stirring. After complete mixing, the vaccine composition was obtained, and the emulsified quality of the vaccine met national quality standards.

[0076] Table 1

[0077]

[0078] 3.2 Piglet Safety Test

[0079] Thirty 2-3-week-old piglets, double-negative for Mycoplasma hyopneumoniae antigens and antibodies, were screened for vaccine safety testing. Twenty-five piglets were randomly selected and divided equally into five groups, each containing five pigs. Group 1 received 4 ml of a subunit vaccine containing 150 μg / ml of truncated P65 protein; Group 2 received 4 ml of a subunit vaccine containing 150 μg / ml of full-length P65 protein; Group 3 received 4 ml of a vaccine containing inactivated Mycoplasma antigen (provided by our laboratory); Group 4 received 4 ml of a mixed vaccine containing truncated P65 protein and inactivated Mycoplasma antigen; and Group 5 received 4 ml of a mixed vaccine containing full-length P65 protein and inactivated Mycoplasma antigen. The remaining five pigs served as the control group and received a 4 ml intramuscular injection of PBS into the neck. Body temperature was measured daily from day 0 to day 7 after immunization. The pigs' appetite and mental state were observed for 14 consecutive days. The injection site was observed and palpated for the presence of lumps.

[0080] Observation continued for 14 days after immunization. No significant temperature elevation was observed in any of the immunized piglets. The pigs were eating normally and in good spirits. The injection site was painless and free of swelling 5 days after immunization (Table 2). This indicates that all vaccines were safe for the immunized piglets.

[0081] Table 2

[0082]

[0083] 3.3 Mycoplasma hyopneumoniae vaccine immune protection test

[0084] Thirty-five weaned piglets were randomly divided into seven groups of five pigs each. Five of the five groups received the five vaccines prepared in Table 1, each injected intramuscularly with a dose of 2 ml per pig. Groups 6 and 7 served as blank controls and received the same dose of sterile PBS intramuscularly. Twenty-eight days after immunization, the immunized and control groups were challenged with Mycoplasma hyopneumoniae, with each pig receiving an intratracheal injection of 0.2 g of diseased lung tissue (100 MID). The pigs in each group were housed in isolation and fed antibiotic-free feed. Twenty-eight days after challenge, the pigs were sacrificed, and lung lesions were observed and scored using a 28-point scoring system. The percentage of lung lesion reduction was calculated. Vaccines were considered qualified if the immunized group achieved a lung lesion reduction of 60% or more. The 28-point scoring system assesses the percentage of specific lesion area in each lobule (left apex, left cardiac lobe, left diaphragmatic lobe, right apex, right cardiac lobe, right diaphragmatic lobe, and accessory lobe) relative to the total area of ​​each lobule. No pneumonia lesions in any lobule are assigned a score of 0; lesions covering 1% to 25% of the lobular area are assigned a score of 1; lesions covering 26% to 50% of the lobular area are assigned a score of 2; lesions covering 51% to 75% of the lobular area are assigned a score of 3; and lesions covering more than 75% of the lobular area are assigned a score of 4. The maximum score for any lesion in any lobule is 4, and the maximum score for lesions in seven lobes is 28. If lesions are present on both the front and back of a lobe, the side with the larger lesion area is scored.

[0085] Specific test results are shown in Table 3. Judging from the challenge results, vaccines 1, 3, 4, and 5 all had good immune protection and met the requirements, indicating that both full-length and truncated P65 proteins can be used as suitable subunit vaccines and that the addition of P65 protein can improve the protective efficiency of inactivated vaccines.

[0086] Table 3

[0087] Group Lung lesion score Protection rate (%) Qualified Vaccine 1 3.8 76.5 yes Vaccine 2 6.4 60.5 yes Vaccine 3 4.2 74.1 yes Vaccine 4 3.2 80.2 yes Vaccine 5 3.8 76.5 yes challenge control group 16.2 / -- Blank control group 1.2 / --

[0088] Example 4: Application of the expressed Mycoplasma hyopneumoniae P65 recombinant protein in the detection of Mycoplasma hyopneumoniae antibodies

[0089] 4.1 Preparation of Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit

[0090] The Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit includes:

[0091] (1) Coating of 96-well detachable polystyrene enzyme-linked reaction plates expressing full-length and truncated Mycoplasma hyopneumoniae P65 antigens; 2×96 wells.

[0092] (2) Positive control serum: The serum collected from pigs after immunization with inactivated Mycoplasma hyopneumoniae CJX strain virus is used as the positive control serum of the kit (1 tube, 1.5 ml / tube).

[0093] (3) Negative control serum: Specific pathogen-free (SPF) porcine serum, used as the negative control serum for the kit (1 tube, 1.5 ml / tube).

[0094] (4) Enzyme-labeled secondary antibody: horseradish peroxidase-labeled rabbit anti-pig IgG (purchased from Sigma, product number A5670) was used as the stock solution and diluted 1:30,000, 2 bottles (12 ml / bottle).

[0095] (5) Sample diluent: 0.01 M phosphate buffer with a pH of 7.4 and containing 0.5% (g / 100 ml) casein, 1 bottle (24 ml / bottle).

[0096] (6) Substrate solution A: Citrate phosphate buffer solution containing 0.6 mg / ml urea hydrogen peroxide (1 bottle, 12 ml / bottle)

[0097] (7) Substrate solution B: 0.2 mg / ml tetramethylbenzidine (TMB) solution (1 bottle, 12 ml / bottle).

[0098] (8) Stop solution: 2 mol / L sulfuric acid solution (1 bottle, 12 ml / bottle).

[0099] (9) 20-fold concentrated washing solution: 0.01 M phosphate buffer with a pH of 7.4 and a concentration of 0.8% to 1.2% (ml / ml) of Tween-20 (50 ml / bottle, 2 bottles).

[0100] If necessary, the kit may also include serum dilution plates (2 plates, 96 wells / plate) for diluting serum samples.

[0101] The preparation method of the 96-well detachable polystyrene enzyme-linked reaction plate coated with full-length and truncated expressed Mycoplasma hyopneumoniae P65 antigens is as follows: the full-length and truncated expressed Mycoplasma hyopneumoniae P65 antigens prepared in Example 1 are dissolved in a carbonate solution at pH 9.6, and then added to a 96-well polystyrene enzyme-linked reaction plate at 1 μg of P65 protein per well. The plate is placed at 2-8° C. for 8-12 hours to allow the recombinant antigen to fully bind to the enzyme-linked reaction plate. Then, 300 μl / well of PBS buffer containing 1% (g / ml) bovine serum albumin (BSA) at pH 7.4 is added, and the plate is blocked at 37° C. for 2-3 hours. After drying, the plate is dried and sealed and stored at 4° C.

[0102] 4.2 How to use the Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit

[0103] The method of using the Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit is as follows:

[0104] 1. Equilibration: Take the test kit out of the refrigerated environment and equilibrate it at room temperature for 30 minutes; mix the liquid reagents before use.

[0105] 2. Prepare the solution: dilute the concentrated wash solution 20-fold with distilled water or deionized water to obtain the wash buffer solution; 3. Set up: 2 negative control wells and 2 positive control wells, and the rest are test sample wells.

[0106] 4. Pre-dilution of the test specimen: Use sample diluent to dilute the test sample serum, negative control serum, and positive control serum at a ratio of 1:200.

[0107] 5. Sample addition: Add 100 μl of the pre-set diluted sample to each well. The time span of the sample addition process should be as short as possible.

[0108] 6. Incubation: Vortex to mix, place in a 37°C incubator, and react for 30 minutes.

[0109] 7. Wash the plate: discard the reaction solution, add 300 μl of diluted wash buffer to each well, soak for 15 seconds, discard the wash solution, wash the plate 4 times in a row and pat dry.

[0110] 8. Add enzyme: Add 100 μl horseradish peroxidase-labeled rabbit anti-pig IgG antibody to each well.

[0111] 9. Incubation: Place in a 37°C incubator and react for 30 minutes.

[0112] 10. Washing: Discard the reaction solution, add 300 μl of diluted washing buffer to each well, soak for 15 seconds, discard the washing solution, wash the plate 4 times in a row and pat dry.

[0113] 11. Color development: Add 100 μl of substrate working solution to each well (substrate working solution is prepared by mixing equal amounts of substrate solution A and substrate solution B, which should be prepared immediately before use), shake to mix, and place in a 37°C incubator in the dark for 15 minutes.

[0114] 12. Add 50 μl of color stop solution to each well and shake to stop the reaction.

[0115] 13. Measure the OD of each well 450nm Value (OD should be read within 15 minutes after adding the stop solution) 450nm value).

[0116] Determination of test results:

[0117] 1. Negative control OD 450nm The average value should be ≤ 0.15, otherwise it is invalid.

[0118] 2. Each test value of the positive control should be between 1.0 and 2.5, otherwise it will be invalid.

[0119] 3. Calculation of critical value: critical value = 0.18 × positive control OD 450nm The average value.

[0120] OD determination of serum to be tested 450nm The value ≥ the critical value was judged as positive; the serum to be tested was measured for OD 450nm Values ​​< critical value were considered negative.

[0121] 4.3 Sensitivity test of Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit

[0122] Using the Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit prepared according to the method in 3.1, sensitivity tests were performed on 30 sera from pigs known to be positive for Mycoplasma hyopneumoniae infection (animals with severe pneumonia symptoms, whose disease samples were identified as Mycoplasma hyopneumoniae positive by RT-PCR amplification) according to the method for using the Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit in 3.2 above. The experimental results are shown in Table 4. The sensitivity of the kit using the full-length P65 protein as the coating antigen for the 30 known positive sera was 100%, and the sensitivity of the kit using the truncated P65 protein as the coating antigen for the 30 known positive sera was 96.7%.

[0123] Table 4 Sensitivity test results of Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit

[0124] antigen Detection rate Sensitivity Truncated P65 protein 29 / 30 96.7％ Full-length P65 protein 30 / 30 100％

[0125] 4.4 Minimum Detection Limit Test of Mycoplasma hyopneumoniae P65 Protein ELISA Antibody Detection Kit

[0126] One portion of pig serum with strong positive antibodies to Mycoplasma hyopneumoniae was selected and diluted in multiples of 1:20, 1:40 to 1:2560. The Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit prepared in 3.1 was used to detect the diluted pig serum according to the method of using the Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit in 3.2 above. The results showed that the kit with truncated P65 protein as the coating antigen could detect positive serum diluted 1:640 times, and the full-length P65 protein could detect positive serum diluted 1:160 times. In Table 5, the positive control is the pig serum collected 14 days after three immunizations with the inactivated Mycoplasma hyopneumoniae vaccine at an interval of 14 days, which serves as the positive control serum of the kit (1 tube, 1.5 ml / tube). The negative control is the specific pathogen-free (SPF) pig serum, which serves as the negative control serum of the kit (1 tube, 1.5 ml / tube). The critical value (Cut-off value) = 0.18 × positive control OD 450 nm value average.

[0127] Table 5 Test results of the minimum detection limit of the Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit

[0128]

[0129] 4.5 Specificity test of Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit

[0130] Use the Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit prepared according to 3.1 and the Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit usage method of the above 3.2 to test 100 healthy pig sera, 2 swine foot-and-mouth disease type A positive sera, 2 swine foot-and-mouth disease type O positive sera, 2 porcine circovirus positive sera, 2 classical swine fever positive sera and 2 porcine reproductive and respiratory syndrome virus (PRRS) positive sera.

[0131] The specificity test results of the kit are shown in the following table (Table 6). The test results for 100 healthy pig sera using both full-length and truncated P65 proteins showed that the specificity of the kit was 100%. The test results for 2 sera positive for Asian foot-and-mouth disease type A, 2 sera positive for foot-and-mouth disease type O, 2 sera positive for classical swine fever, 2 sera positive for porcine circovirus, and 2 sera positive for porcine reproductive and respiratory syndrome virus (PRRS) were all negative. Therefore, the specificity of the kit for these 8 pig positive sera was 100%.

[0132] Table 6 Specificity test results of Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit

[0133]

[0134]

[0135] 4.6 Compliance test of Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit

[0136] The truncated Mycoplasma hyopneumoniae P65 protein ELISA antibody detection kit prepared in accordance with 3.1 was used to test 55 immune pig sera and 55 pig sera from different pig farms together with similar commercial antibody detection kits from home and abroad. See Table 4. The results showed that the test results of the three kits for the 55 immune pig sera were consistent and all were positive. The results of the 55 pig sera from different pig farms were slightly different. The overall consistency rate of this kit with domestic commercial kits was 91.7%, and the overall consistency rate with foreign commercial kits was 88.5%.

[0137] Table 7. Validity test results of Mycoplasma hyopneumoniae P65 protein ELISA antibody test kit

[0138]

[0139] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of the appended claims of the present invention.

Claims

1. A recombinant protein of Mycoplasma hyopneumoniae P65, whose amino acid sequence is sequence 2 in the sequence table.

2. The truncated protein of the Mycoplasma hyopneumoniae P65 recombinant protein according to claim 1, whose amino acid sequence is sequence 4 in the sequence listing.

3. A gene encoding the recombinant protein of Mycoplasma hyopneumoniae P65 or its truncated protein according to claim 1.

4. The coding gene according to claim 3, characterized in that The coding gene of the Mycoplasma hyopneumoniae P65 recombinant protein is shown in Sequence 1 in the sequence list, and the amino acid sequence of the truncated protein is shown in Sequence 3 in the sequence list.

5. A recombinant expression vector expressing the recombinant protein of Mycoplasma hyopneumoniae P65 according to claim 1 or the truncated protein according to claim 2, which is obtained by inserting the coding gene of the recombinant protein of Mycoplasma hyopneumoniae P65 shown in Sequence 1 or Sequence 3 into a starting vector.

6. The recombinant vector according to claim 5, characterized in that The starting vector is PET-22b; the Mycoplasma hyopneumoniae P65 gene sequence shown in sequence 1 or sequence 3 is inserted between the NdeⅠ and BamHI enzyme recognition sites of pET-22b.

7. An engineered bacterium expressing the recombinant protein of Mycoplasma hyopneumoniae P65 or a truncated protein thereof according to claim 1, characterized in that: The recombinant vector described in claim 5 or 6 is transformed into competent Escherichia coli BL21 (DE3), and the recombinant bacteria expressing the recombinant protein of Mycoplasma hyopneumoniae P65 or its truncated protein are obtained by screening.

8. A method for preparing a truncated Mycoplasma hyopneumoniae P65 recombinant protein by expression in an Escherichia coli system, comprising culturing the engineered bacteria according to claim 7, inducing protein expression using IPTG; collecting supernatant and inclusion bodies; and purifying to obtain soluble supernatant Mycoplasma hyopneumoniae P65 recombinant protein and inclusion body Mycoplasma hyopneumoniae P65 recombinant protein.

9. Use of the full-length and truncated Mycoplasma hyopneumoniae P65 recombinant proteins or their encoding genes as claimed in claim 1 in the preparation of diagnostic reagents for Mycoplasma hyopneumoniae and vaccines for the prevention or treatment of Mycoplasma hyopneumoniae.

10. Use of the recombinant vector according to any one of claims 4 to 6, or the engineered bacteria according to claim 7 in the preparation of a diagnostic reagent for Mycoplasma hyopneumoniae, or a vaccine for the prevention or treatment of Mycoplasma hyopneumoniae.

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