An immunogenic Mycoplasma ovipneumoniae HSP70-P113 fusion protein

Abstract

The invention discloses an immunogenic Mycoplasma ovipneumoniae HSP70-P113 fusion protein, which is composed of a Mycoplasma ovipneumoniae heat shock protein gene HSP70 and Mycoplasma ovipneumoniae adhesin gene P113 The spliced ​​fusion gene encodes an amino acid sequence shown in SQE ID NO. 2. Western Blot test proves that the Mycoplasma ovipneumoniae HSP70-P113 fusion protein has immunogenicity and can be used to prepare Mycoplasma ovipneumoniae subunit vaccine.

Description

Technical Field

[0001] The invention belongs to the technical field of recombinant proteins, and in particular relates to an immunogenic Mycoplasma ovipneumoniae HSP70-P113 fusion protein. Background Art

[0002] Mycoplasma ovipneumoniae ( Mycoplasma ovipneumoniae , Movi) is one of the main pathogens causing mycoplasma pneumonia in sheep and goats. It is distributed worldwide and has caused huge economic losses to the world's sheep farming industry. The treatment of Mycoplasma ovipneumoniae is mainly antibiotics. However, the drug resistance caused by antibiotics makes the treatment of Mycoplasma ovipneumoniae unsatisfactory, so Mycoplasma ovipneumoniae should be mainly prevented. However, the current domestic Movi vaccine is mainly inactivated vaccine, mainly inactivated by whole bacterial antigen. However, mycoplasmas prevalent in different places have genetic diversity, low cross-protection between strains, and Movi in ​​vitro culture yield is relatively low and time-consuming. Whole bacterial antigens have safety risks, so the development of efficient and safe new Movi vaccines has been a trend in recent years. Subunit vaccines have the advantages of high safety, no nucleic acid, no toxicity, no re-strengthening, and a single antigen component, high purity, and high vaccine yield, which has become a trend in vaccine development. However, there is no commercial subunit vaccine for Mycoplasma ovipneumoniae (Movi). At present, many studies focus on screening proteins with good immunogenicity through antigen epitope identification for the preparation of subunit vaccines. P113 is an adhesin, which is an important virulence factor of mycoplasma and the most important immunogen. It is the main protein that causes the body to produce an immune response. Heat shock protein HSP70 is a highly conserved biological molecule in the body and an important membrane protein of Movi. It has immunogenicity and immune adjuvant effect. Its C-terminal antigenicity advantage is obvious, and it can induce the body to produce humoral immunity and cellular immune response. The present invention is screened HSP70 Gene and MO ATCC 29419 isolate P113 The dominant antigen region in the gene was optimized for rare codons in the gene sequences of the two dominant antigen regions, and a linker (GGGGSGGGGS) was added between the two gene sequences, and then copied to the pET-30a(+) prokaryotic expression vector, named HSP70-P113- pET-30a(+). After induced expression, protein purification, and immunization of mice, the Western Blot test proved that the HSP70-P113 recombinant protein was immunogenic, which can provide a basis for the development of Mycoplasma ovis pneumonia subunit vaccine. Summary of the invention

[0003] The purpose of the present invention is to provide an immunogenic Mycoplasma ovipneumoniae HSP70-P113 fusion protein.

[0004] To achieve the above object, the present invention adopts the following technical solution:

[0005] The invention discloses an immunogenic Mycoplasma ovipneumoniae HSP70-P113 fusion protein, wherein the amino acid sequence of the Mycoplasma ovipneumoniae HSP70-P113 fusion protein is the sequence shown in SQE ID NO.2.

[0006] A fusion gene encoding the above-mentioned Mycoplasma ovipneumoniae HSP70-P113 fusion protein, wherein the fusion gene is formed by splicing the Mycoplasma ovipneumoniae heat shock protein gene HSP70 and the Mycoplasma ovipneumoniae adhesin gene P113 using a linker (GGGGSGGGGS), and its nucleotide sequence is the sequence shown in SQE ID NO.1.

[0007] An E. coli expression plasmid vector is constructed by inserting the above fusion gene between the Nde I and BamH I sites of plasmid pET-30a, and the E. coli is BL21 (DE3).

[0008] An Escherichia coli strain for producing the Mycoplasma ovipneumoniae HSP70-P113 fusion protein, the Escherichia coli strain contains the plasmid vector, and the Escherichia coli is BL21 (DE3).

[0009] A method for preparing the above-mentioned Mycoplasma ovipneumoniae HSP70-P113 fusion protein comprises the following steps:

[0010] 1) Insert the fusion gene with the sequence shown in SQE ID NO.1 between the Nde I and BamH I sites of the E. coli plasmid pET-30a, transform the plasmid into E. coli to obtain a strain containing HSP70-P113-pET-30a(+), inoculate and culture, and induce expression with IPTG;

[0011] 2) After the expression bacteria were lysed by centrifugation, the inclusion bodies were harvested, resuspended in PBS buffer, centrifuged, and the supernatant was subjected to Ni-NTA affinity chromatography to obtain the Mycoplasma ovipneumoniae HSP70-P113 fusion protein;

[0012] Wherein, the Escherichia coli is BL21 (DE3).

[0013] Application of the Mycoplasma ovipneumoniae HSP70-P113 fusion protein in the preparation of Mycoplasma ovipneumoniae subunit vaccine.

[0014] The significant advantage of the present invention is that: the present invention selects HSP70 with immunogenicity and immune adjuvant effect and adhesin P113 with good immunogenicity for the first time, and successfully expresses HSP70-P113 recombinant protein by selecting the dominant antigenic regions of the two genes for codon optimization, and the HSP70-P113 recombinant protein has strong immunogenicity, providing a basis for the development of Mycoplasma ovipneumoniae subunit vaccine. In addition, the HSP70-P113 recombinant protein has immunogenicity and can react with Mycoplasma ovipneumoniae positive serum, and at the same time provides antigens for the establishment of serological methods such as the indirect ELISA method of Mycoplasma ovipneumoniae, and is the core component of the ELISA kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 for HSP70-P113 -Plasmid map of pET-30a(+).

[0016] Figure 2 The soluble expression and inclusion body expression of HSP70-P113 fusion protein. Lane M: Protein marker; Lane PC 1 :BSA (1μg); Lane PC 2 :BSA (2μg); Lane NC: uninduced whole cell sample; Lane 1: whole cell sample induced at 15℃ for 16h; Lane 2: whole cell sample induced at 37℃ for 4h; Lane NC 1 Lane 4: cell lysis supernatant sample induced at 37℃ for 4 h; Lane NC: cell lysis supernatant sample induced at 15℃ for 16 h; Lane 4: cell lysis supernatant sample induced at 37℃ for 4 h; Lane NC 2 Lane 5: Uninduced cell lysis precipitate sample; Lane 5: Cell lysis precipitate sample induced at 15℃ for 16h; Lane 6: Cell lysis precipitate sample induced at 37℃ for 4h.

[0017] Figure 3 The results of SDDS-PAGE gel electrophoresis analysis of the purified HSP70-P113 fusion protein are shown in Figure 1. Lane M: Protein marker; Lane 1: purified HSP70-P113 fusion protein.

[0018] Figure 4 The results of immunogenicity analysis of HSP70-P113 fusion protein. Lane M: Protein marker; A: primary antibody is mouse anti-HSP70-P113 polyclonal antibody; B: primary antibody is mouse anti-Movi polyclonal antibody; C: primary antibody is normal control mouse serum (blank control). DETAILED DESCRIPTION

[0019] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0020] Example 1 HSP70-P113 Fusion gene design and synthesis

[0021] Analysis of heat shock protein genes of Mycoplasma ovipneumoniae FJ-01CL isolate using Protean software in DNAStar7.0 software HSP70 and the adhesin gene of Mycoplasma ovipneumoniae ATCC 29419 isolate P113 The dominant antigen region in the gene sequences of the two dominant antigen regions were optimized for rare codons and then spliced, and the GGGGSGGGGS sequence linker was added in the middle to obtain HSP70-P113 Fusion gene. HSP70-P113 The fusion gene is 1392 bp in length and has a nucleotide sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2. HSP70-P113 The fusion gene synthesis was completed by Nanjing GenScript Co., Ltd.

[0022] Example 2 HSP70-P113 -pET-30a(+) recombinant plasmid construction

[0023] Through gene cloning HSP70-P113 The fusion gene was inserted into the E. coli plasmid pET-30a N and Bm I Between the sites, construct a recombinant plasmid HSP70-P113 -pET-30a(+). HSP70-P113 -pET-30a(+) plasmid map Figure 1 shown.

[0024] Example 3 HSP70-P113 - Prokaryotic expression of pET-30a(+)

[0025] Place the stored BL21(DE3) on ice for 30 min and add 100 ng of recombinant plasmid HSP70-P113-pET-30a(+), gently pipette to mix thoroughly, react on ice for 30 min; heat shock at 42℃ for 90 s; place on ice for 3 min after heat shock; then add 100 mL LB liquid culture medium; shake and culture at 37℃, 200 rpm for 60 min in a shaker; mix the bacterial solution and spread it on a 50 μg / mL kanamycin resistance plate, invert the plate and culture it at 37℃ overnight. The next day, pick 3 single clones and inoculate them into test tubes of LB liquid culture medium containing 50 μg / mL kanamycin, and culture them at 37℃ in a shaker; when the culture OD 600 When it reached 0.6-0.8, 0.5 mM IPTG was added to two of the test tubes, and the cells were cultured at 15°C for 16 h and 37°C for 4 h, respectively. The last test tube was used as a negative reference. Samples were prepared for SDS-PAGE analysis to detect protein expression.

[0026] Sample preparation for SDS-PAGE analysis:

[0027] 1) Take 450 μL of the culture medium after centrifugation, resuspend the pellet in 300 μL of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5% glycerol, pH 8.0), and sonicate for 1 min.

[0028] 2) Whole bacterial sample: Take 80 μL of lysate and mix evenly with 20 μL SDS-PAGE protein loading buffer (5X) (Biyuntian Biotechnology Co., Ltd.), heat at 100℃ for 10 min, and then centrifuge at 15000 rpm for 5 min. Keep the supernatant and discard the precipitate for loading.

[0029] 3) Supernatant and inclusion body samples: Take 200 μL of lysate, centrifuge at 15000 rpm for 10 min, and separate the supernatant and precipitate. Mix 40 μL of SDS-PAGE protein loading buffer (5X) and 160 μL of supernatant evenly to use as the supernatant sample. Resuspend the precipitate with 150 μL5 SDS-PAGE protein loading buffer (5X) to use as the inclusion body sample. Heat the supernatant sample and inclusion body sample at 100℃ for 10 min, centrifuge at 15000rpm for 5 min, keep the supernatant and discard the precipitate for loading.

[0030] SDS-PAGE results showed that ( Figure 2 ), the expressed HSP70-P113 fusion protein was about 51 KD in size; when the induction temperature was 15°C, the HSP70-P113 fusion protein was expressed in both the bacterial supernatant (soluble expression) and the precipitate (inclusion body); when the induction temperature was 37°C, the HSP70-P113 fusion protein was mainly expressed in the form of inclusion bodies.

[0031] Example 3 Purification of HSP70-P113 fusion protein

[0032] Place the stored BL21(DE3) on ice for 30 min, add 100 ng of recombinant plasmid HSP70-P113-pET-30a(+), gently pipette to mix thoroughly, react on ice for 30 min; heat shock at 42℃ for 90 s; place on ice for 3 min after heat shock; then add 100 mL LB liquid culture medium; shake and culture at 37℃, 200 rpm for 60 min in a shaker; mix the bacterial solution and spread it on a 50 μg / mL kanamycin resistance plate, invert the plate and culture it at 37℃ overnight. The next day, pick a single clone and inoculate it into LB liquid culture medium containing 50 μg / mL kanamycin, and culture it at 37℃ in a shaker; when the culture OD 600 When the p-value reached 0.6-0.8, 0.5 mM IPTG was added and induced at 15°C for 16 hours; after induction, the mixture was centrifuged at 10,000 rpm and 4°C for 10 min, the supernatant was discarded, and the precipitate was resuspended in PBS buffer (0.01M, pH7.2); the mixture was centrifuged at 10,000 rpm and 4°C for 10 min, the supernatant was discarded, and the precipitate was resuspended in PBS buffer (0.01M, pH7.2); the mixture was centrifuged at 10,000 rpm and 4°C for 10 min, the supernatant was discarded, and the precipitate was resuspended in PBS buffer (0.01M, pH7.2), and ultrasonically treated in an ice bath for 20 min to break the bacteria; the mixture was centrifuged at 12,000 rpm and 4°C for 10 min, the supernatant was collected, and the target protein was purified according to the instructions of ProteinIso® Ni-NTA Resin to obtain the target protein. The results of SDDS-PAGE gel electrophoresis analysis of the purified protein are shown in Figure 3 The fusion protein showed an obvious band at the corresponding position, and a 51 KD HSP70-P113 fusion protein was obtained, which matched the expected result.

[0033] Example 4 Preparation and immunogenicity analysis of polyclonal antibodies against Mycoplasma ovipneumoniae HSP70-P113 fusion protein

[0034] Preparation of polyclonal antibody against HSP70-P113 fusion protein of Mycoplasma ovipneumoniae: The purified HSP70-P113 fusion protein was mixed with an equal volume of Freund's complete adjuvant, ultrasonically emulsified, and subcutaneously immunized into Balb / c mice for a total of 3 times. The 15th and 30th days after the first immunization were the second and third immunization periods, respectively. For the first immunization, HSP70-P113 fusion protein was mixed with an equal volume of Freund's complete adjuvant for immunization injection, and the injection dose was 50 μg fusion protein / mouse; for the second and third immunizations, HSP70-P113 fusion protein was mixed with an equal volume of Freund's incomplete adjuvant for booster immunization injection, and the injection dose was 50 μg fusion protein / mouse. On the 9th day after the third immunization, the mice were tail-cut and blood was collected, serum was separated, antibody levels were detected, and antibody titers were determined. The mouse serum before immunization was used as a negative control. When the antibody titer reached the requirement (antibody titer>1:5000), the whole blood of mice was collected by orbital bleeding, and the serum was separated. The serum was then subjected to antigen-antibody affinity purification to obtain the polyclonal antibody against the HSP70-P113 fusion protein of Mycoplasma ovipneumoniae.

[0035] Immunogenicity analysis of HSP70-P113 fusion protein: After the purified HSP70-P113 fusion protein was analyzed by SDS-PAGE, it was transferred to a nitrocellulose membrane by semi-dry method. Mycoplasma ovipneumoniae HSP70-P113 fusion protein polyclonal antibody, Mycoplasma ovipneumoniae polyclonal antibody and negative control mouse serum were used as primary antibodies, Goat anti-mouse IgG (H+L), HRP conjugate, ELISA and WB were used as secondary antibodies (using a concentration of 1:5000) for Western-blot test. The results of Western-blot analysis showed that ( Figure 4 ), HSP70-P113 fusion protein polyclonal antibody can react specifically with HSP70-P113 fusion protein antigen, and a band of 51KD appears; Mycoplasma ovipneumoniae polyclonal antibody reacts specifically with HSP70-P113 fusion protein antigen, and a band of 51KD appears; HSP70-P113 fusion protein and negative control mouse serum have no specific bands. The above results show that HSP70-P113 fusion protein has immunogenicity.

[0036] Example 5 Configuration and use of a kit for detecting antibodies to Mycoplasma ovipneumoniae

[0037] Coating ELISA plate: dilute the purified HSP70-P113 fusion protein to 5 μg / mL with coating buffer, add to 96-well ELISA plate, 100 μL / well, and set up 100 μL / well coating buffer as a control, and coat at 4°C overnight; wash the plate 3 times with PBST and pat dry; add blocking solution to block the ELISA plate, 200 μL / well, incubate at 37°C for 45 minutes, wash the plate 3 times with PBST, and pat dry.

[0038] The negative standard serum is a mouse serum that is negative for Mycoplasma ovipneumoniae antibodies.

[0039] The positive standard serum is a mouse serum that is positive for Mycoplasma ovipneumoniae antibodies.

[0040] The coating buffer was a carbonate buffer at pH 9.6 consisting of Na 2 CO 3 1.59 g, NaHCO 3 2.93 g, add double distilled water to make up to 1000 mL.

[0041] The main component of the blocking solution is 3% bovine serum albumin.

[0042] The enzyme-labeled secondary antibody was peroxidase-labeled donkey anti-sheep IgG (H+L).

[0043] The main component of the diluent is 0.5% bovine serum albumin.

[0044] The substrate solution was TMB colorimetric solution (Guangzhou Jiebei Biotechnology Co., Ltd.).

[0045] The stop solution is prepared by adding 22.2 mL of concentrated sulfuric acid to 177.8 mL of distilled water.

[0046] When in use, take 100μL of the diluted sample to be tested and add it to the test plate; set up 3 wells for positive and negative controls, 100μL in each well; gently shake the sample in the well (do not overflow), incubate at 37℃ for 45min; shake off the solution in the well, wash the plate with PBST 5 times, and then pat dry on clean absorbent paper; add 100μL of 1:10000 diluted peroxidase-labeled donkey anti-sheep IgG (H+L) to each well, incubate at 37℃ for 45min; shake off the solution in the well, wash the plate with PBST 5 times, and then pat dry on clean absorbent paper; add 100μL of TMB colorimetric solution to each well, incubate at 37℃ for 10min; add 50μL of stop solution to each well, measure the result within 10 minutes (lightly shake on the oscillator before detection), and measure OD with an enzyme reader 450nm .

[0047] The condition for the result judgment test to be successful is the sample OD 450nm OD value and negative control 450nmIf the ratio is greater than or equal to 2.1, it is judged as positive; otherwise, it is judged as negative.

[0048] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention. SEQUENCE LISTING <110> Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences <120> An immunogenic Mycoplasma ovipneumoniae HSP70-P113 fusion protein <130> <160> 2 <170> PatentIn version 3.3 <210> 1 <211> 1392 <212> DNA <213> Artificial sequence <400> 1 catatgcacc accaccacca ccacactgct gaagataacc aaacagaagt aaccattagc 60 gttgttcagg gcgagcgtca gctggcggcg gacaacaaaa tgctgggtcg ttttaacctg 120 agcggtattg aaccggcgcc gcgtggtctg ccgcagattg aggtgagctt cagcatcgac 180 gttaacggta ttaccaccgt gagcgcgaaa gataagaaaa ccggcaagga acagaccatc 240 accattaaaa acaccagcac cctgagcgag gaagagatca accgtatgat tcaagaagcg 300 gaagagaacc gtgaggcgga cgcgatcaag aaagataaga ttgaaaccac cgttcgtgcg 360 gagggtctga tcaaccaact ggagaagagc attaccgacc agggcgagaa gatcgatccg 420 aagcaaaag aactgctga gaacagatc cagagctga aggatctggt gaggaagg 480 aaaattgacg aactgaagac caactggat cagattgagc aagcggcgca ggcgtttgcg 540 caggcgagcg cgcagcaagc gacaacgcg agcgaaccg acaggcagg taggacaccg 600 atcgacgcgg agattaaca aaatggtggc ggtggcagcg gtggcggtgg cagcgaatttc 660 agcgatgcga agatcagcta ccagatcctg gagattctgc cggacgatgc gaaccaaac 720 tttaggtta aattccaagc gagccagaaa ctggcgaacg gcgacatcgc gagagcgat 780 atttatgaac aggtggttag cttcgttaaa gaagcacca tcctgattgc ggagttcaac 840 tttagcctgc agagattac cagccgtctg aaccagcaag tgcaaacct gatcagcgcg 900 cgtaccgcga acttgcgga ccagacagc gcgaccagca acccgaccga tccgagcacc 960 attcgtccgg tggactcca acacgatctg aaaaggcga agacagcaa agaatttggc 1020 gagaagagatca gcagctactt cccgagcctg aacaacctgc tggcgagcct gaaaaacagc 1080 caggagaca agctgccgaa cagcatggatt accatttca actttgactt cgcgcgtgat 1140 aagagcaccg gccagtttgt tagcctgcag aaccaagtgc cgagcttctt tctggaagcg 1200 aacctgacca gcagcgcgcg tcaaatgctg gacagcagca gcagcttcag caccgtggtt 1260 aacgcgatca agctggagaa aaacgacaag agcagctact ttctgaacta tagcgatttc 1320 tttgataacc tgagcctgaa gaatctgacc aagaccgact ttaagagcga catgggtgac 1380 taatgaaagc tt 1392 <210> 2 <211> 459 <212> PRT <213> Artificial Sequence <400> 2 Met His His His His His His Thr Ala Glu Asp Asn Gln Thr Glu Val 1 5 10 15 Thr Ile Ser Val Val Gln Gly Glu Arg Gln Leu Ala Ala Asp Asn Lys 20 25 30 Met Leu Gly Arg Phe Asn Leu Ser Gly Ile Glu Pro Ala Pro Arg Gly 35 40 45 Leu Pro Gln Ile Glu Val Ser Phe Ser Ile Asp Val Asn Gly Ile Thr 50 55 60 Thr Val Ser Ala Lys Asp Lys Lys Thr Gly Lys Glu Gln Thr Ile Thr 65 70 75 80 Ile Lys Asn Thr Ser Thr Leu Ser Glu Glu Glu Ile Asn Arg Met Ile 85 90 95 Gln Glu Ala Glu Glu Asn Arg Glu Ala Asp Ala Ile Lys Lys Asp Lys 100 105 110 Ile Glu Thr Thr Val Arg Ala Glu Gly Leu Ile Asn Gln Leu Glu Lys 115 120 125 Ser Ile Thr Asp Gln Gly Glu Lys Ile Asp Pro Lys Gln Lys Glu Leu 130 135 140 Leu Glu Lys Gln Ile Gln Glu Leu Lys Asp Leu Val Lys Glu Glu Lys 145 150 155 160 Ile Asp Glu Leu Lys Thr Lys Leu Asp Gln Ile Glu Gln Ala Ala Gln 165 170 175 Ala Phe Ala Gln Ala Ser Ala Gln Gln Ala Asn Asn Ala Ser Glu Thr 180 185 190 Asp Ser Gln Asp Ser Asn Thr Ile Asp Ala Glu Ile Lys Gln Asn Gly 195 200 205 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Phe Ser Asp Ala Lys Ile 210 215 220 Ser Tyr Gln Ile Leu Glu Ile Leu Pro Asp Asp Ala Asn Gln Asn Phe 225 230 235 240 Lys Val Lys Phe Gln Ala Ser Gln Lys Leu Ala Asn Gly Asp Ile Ala 245 250 255 Lys Ser Asp Ile Tyr Glu Gln Val Val Ser Phe Val Lys Glu Ser Thr 260 265 270 Ile Leu Ile Ala Glu Phe Asn Phe Ser Leu Gln Lys Ile Thr Ser Arg 275 280 285 Leu Asn Gln Gln Val Gln Asn Leu Ile Ser Ala Arg Thr Ala Asn Phe 290 295 300 Ala Asp Gln Asn Ser Ala Thr Ser Asn Pro Thr Asp Pro Ser Thr Ile 305 310 315 320 Arg Pro Val Asp Phe Gln His Asp Leu Asn Lys Ala Lys Asp Ser Lys 325 330 335 Glu Phe Gly Glu Lys Ile Ser Ser Tyr Phe Pro Ser Leu Asn Asn Leu 340 345 350 Leu Ala Ser Leu Lys Asn Ser Gln Glu Asn Lys Leu Pro Asn Ser Met 355 360 365 Asp Thr Ile Phe Asn Phe Asp Phe Ala Arg Asp Lys Ser Thr Gly Gln 370 375 380 Phe Val Ser Leu Gln Asn Gln Val Pro Ser Phe Phe Leu Glu Ala Asn 385 390 395 400 Leu Thr Ser Ser Ala Arg Gln Met Leu Asp Ser Ser Ser Ser Phe Ser 405 410 415 Thr Val Val Asn Ala Ile Lys Leu Glu Lys Asn Asp Lys Ser Ser Tyr 420 425 430 Phe Leu Asn Tyr Ser Asp Phe Phe Asp Asn Leu Ser Leu Lys Asn Leu 435 440 445 Thr Lys Thr Asp Phe Lys Ser Asp Met Gly Asp 450 455

Claims

1. An application of an immunogenic Mycoplasma ovipneumoniae HSP70-P113 fusion protein in the preparation of a Mycoplasma ovipneumoniae subunit vaccine, characterized in that: The amino acid sequence of the Mycoplasma ovipneumoniae HSP70-P113 fusion protein is the sequence shown in SEQ ID NO.2, and the nucleotide sequence of the fusion gene encoding the Mycoplasma ovipneumoniae HSP70-P113 fusion protein is the sequence shown in SEQ ID NO.

1.

2. A kit for detecting antibodies to Mycoplasma ovipneumoniae, characterized in that: The kit comprises the Mycoplasma ovipneumoniae HSP70-P113 fusion protein described in claim 1.

Images