CN110257405B

CN110257405B - Mycoplasma bovis alcohol dehydrogenase gene and encoding protein and application thereof

Info

Publication number: CN110257405B
Application number: CN201910541509.XA
Authority: CN
Inventors: 陈曦; 郭爱珍; 刘东明; 陈颖钰; 赵刚; 路豆昆; 胡长敏; 陈建国; 陈焕春
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-06-20
Filing date: 2019-06-20
Publication date: 2020-10-27
Anticipated expiration: 2039-06-20
Also published as: CN110257405A

Abstract

The invention discloses a mycoplasma bovis alcohol dehydrogenase gene, which has a nucleotide sequence shown as SEQ ID NO. 1, and also discloses a protein coded by the mycoplasma bovis alcohol dehydrogenase gene, which has an amino acid sequence shown as SEQ ID NO. 2, and belongs to the field of animal infectious disease prevention and treatment and biotechnology. The recombinant protein has the activity of ethanol dehydrogenase, can adhere to bovine lung epithelial cell line (EBL), can be combined with bovine fibronectin (Fn), has good immunogenicity and reactogenicity, is a virulence related protein of mycoplasma bovis, and has good application prospect in research of mycoplasma bovis pathogenicity mechanisms, development of vaccines and diagnostic reagents.

Description

Mycoplasma bovis alcohol dehydrogenase gene and encoding protein and application thereof

Technical Field

The invention belongs to the field of animal infectious disease prevention and biotechnology, and particularly relates to a mycoplasma bovis Alcohol Dehydrogenase (ADH) gene, and a protein coded by the same and application of the gene.

Background

Mycoplasma bovis (m.bovis) belongs to the genus Mycoplasma of the class mollicutes, is one of the important pathogens currently causing respiratory diseases in cattle, and causes various clinical symptoms, mainly including bronchopneumonia, mastitis, arthritis, otitis media, genital tract inflammation, tenosynovitis, meningitis, keratoconjunctivitis, abortion, infertility, and the like. As the virulence factors are unclear and the pathogenic mechanism is unknown, so far, the vaccine which effectively aims at the mycoplasma bovis infection is still lacked, the antibiotic treatment effect is poor, the treatment course is long, and the drug resistance of pathogenic bacteria to antibiotics is also continuously increased, so that the disease still lacks of effective prevention and control means at present, and brings huge economic loss to the beef cattle industry and the dairy cow industry.

Mycoplasma bovis was originally isolated in 1961 from a case of cow's milk with mastitis in the United states, and it was demonstrated in 1976 that this pathogen causes respiratory diseases in cattle. Later, the prevalence of the disease was reported in succession in different countries, and has now become widespread worldwide, resulting in huge economic losses. About 25-33% of calf pneumonia in Europe is caused by mycoplasma bovis, and the economic loss is more, at least reaching 1.44-1.92 hundred million euros. In addition to the above-mentioned direct economic losses, mycoplasma bovis causes serious indirect economic losses, such as the cost of medication during illness, the cost of labor expending the culling and handling of sick and dead cattle, and the cost of preventing unspecified pathogens, and the trade-off of animals and animal products is a significant cause of the epidemic.

The mycoplasma bovis is separated from mastitis milk of cows for the first time in 1983 in China, mycoplasma bovis pneumonia is reported by a laboratory where the applicant is located for the first time in 2008, and then the fact that the disease begins to be epidemic in most provinces of China is discovered. Except Hubei province, the mycoplasma bovis is isolated from respiratory tract infected cattle in Henan, Jiangxi, Xinjiang, Hunan, Guangdong, Xiamen, etc.

The discovery of virulence related proteins helps to recognize the interaction relationship of mycoplasma and organisms from the protein level and to elucidate the pathogenesis of the disease; in addition, new virulence associated proteins are likely targets for novel vaccine development; meanwhile, virulence associated proteins with immunogenicity may become a novel diagnostic marker for the disease.

Disclosure of Invention

The invention aims to provide a mycoplasma bovis alcohol dehydrogenase gene and a protein coded by the same, and provides a potential candidate protein for developing a novel vaccine and a diagnostic reagent.

In order to realize the purpose of the invention, the applicant takes M.bovis HB0801 strain mycoplasma bovis separated from important laboratory ruminant pathogens in agricultural microbiology country of university of agriculture in Huazhong as a template, designs a primer, and clones and expresses a recombinant ADH protein (rADH). Further verification confirms that rADH has the activity of alcohol dehydrogenase, can catalyze the conversion of alcohol into acetaldehyde, and can reduce NAD + into NADH at the same time. In vitro studies further found that the protein also adhered bovine lung epithelial cells (EBL) and bound fibronectin (Fn), and was immunogenic and reactogenic. Because both the adhesion function and the immunogenicity are related to the virulence of bacteria, the mycoplasma bovis ADH protein is a virulence related protein and is a candidate target for developing mycoplasma bovis vaccines and diagnostic reagents.

The technical scheme of the invention is as follows:

the applicant obtains a Mycoplasma bovis local isolate HB0801 separated from lung tissues of cattle of Hubei province, which are attacked by a certain cattle farm in the city of Hubei province in 6 months in 2008, the applicant names the Mycoplasma bovis HB0801(Mycoplasma bovisHB0801), and the Mycoplasma bovis local isolate is delivered to the China center for type culture Collection (Wuhan) for preservation in 2 months and 1 day in 2010, and the preservation number is CCTCCNO: and M2010040. This isolated strain of Mycoplasma bovis is disclosed in the patent document CN 109750054A.

The invention firstly uses mycoplasma bovis HB0801 (genome is in GenBank accession number CP002058) as a template to clone the gene. Coli, unlike mycoplasma expression systems, which use the codon UGA encoding tryptophan in mycoplasma as its stop codon, and thus, if the mycoplasma gene sequence containing UGA is inserted into the e.coli expression system, the resulting gene expression product is a truncated product. Therefore, UGA base in mycoplasma is subjected to site-directed mutagenesis by adopting an overlap extension PCR method to obtain UGG base for expressing tryptophan in escherichia coli, and 3 UGA base pairs in total in an ADH protein gene (Mbov _0338) need codon mutation to ensure that full-length expression can be obtained in escherichia coli.

The nucleotide sequence of the mycoplasma bovis gene Mbov _0338 subjected to artificial mutation is a sequence shown by 1-1062 bases of a sequence table SEQ ID NO. 1, and the length of the nucleotide sequence is 1062 bp; wherein allelic mutations occur at positions 57, 165 and 279 of the sequence.

The ADH protein sequence coded by the bovine mycoplasma albicans Mbov _0338 gene is shown in a sequence table SEQ ID NO. 2, and 353 amino acids are coded in total.

The nucleotide sequence of the mycoplasma bovis Mbov _0338 gene is transformed into Escherichia coli DH5 alpha by constructing a plasmid vector pET-30a-Mbov _0338 to obtain a recombinant Escherichia coli strain, and the recombinant Escherichia coli is named as Escherichia coli pET-30a-Mbov _0338(Escherichia coli pET-30a-Mbov _0338) by an applicant and is delivered to a China type culture collection (CCTCC) for preservation in Wuhan university, Wuhan, Hubei province in 4 and 2 months in 2019, wherein the preservation number is CCTCC NO: m2019226. The strain expresses recombinant protein rADH coded by Mbov _0338 gene under IPTG induction.

The rADH purified by the invention has the activity of alcohol dehydrogenase, can reduce NAD + into NADH, and in addition, the protein has immunogenicity and reactogenicity, and can adhere to host EBL epithelial cells and bind fibronectin (Fn).

The details are shown in the examples.

The invention has the following advantages:

1. mycoplasma bovis ADH recombinant protein is encoded by Mycoplasma bovis gene Mbov _0338 and confirmed by the inventors to have ethanol dehydrogenase activity.

2. The mycoplasma bovis ADH recombinant protein has immunogenicity and reactogenicity, can be used for preparing vaccines to prevent mycoplasma bovis infection, can also be used for detecting mycoplasma bovis antibodies, and can also be used for preparing polyclonal or monoclonal antibodies through immune animals to be used for detecting mycoplasma bovis antigens.

3. The mycoplasma bovis ADH recombinant protein can be specifically combined with bovine lung epithelial cells (EBL) and fibronectin (Fn) and is an adhesion-related protein, and the generated antibody can be combined with the mycoplasma bovis competitively and the receptor, so that the adhesion of the mycoplasma bovis is reduced, and the pathogenicity of the mycoplasma bovis is reduced.

Description of sequence listing:

SEQ ID NO:1 is the nucleotide sequence of Mycoplasma bovis gene Mbov _0338 after base mutation (original gene source: Mycoplasma bovis HB0801 strain, Accession number of original gene GenBank Accession: CP002058), the position of Mbov _0338 gene in the genome: 399768-400829, forward direction. The nucleotide sequence of the modified Mycoplasma bovis Mbov _0338 gene is represented by bases 1-1062, wherein codon mutations occur at positions 57, 165 and 279 of the sequence.

SEQ ID NO:2 is the amino acid sequence of Mycoplasma bovis ADH protein, which codes for 353 amino acids in total.

SEQ ID NO: 3 is the sequence of primer 0338a1 for amplifying an Mbov _0338 gene fragment.

SEQ ID NO: 4 is the sequence of primer 0338a2 for amplifying an Mbov _0338 gene fragment.

SEQ ID NO: 5 is the sequence of primer 0338b1 for amplifying an Mbov _0338 gene fragment.

SEQ ID NO: 6 is the sequence of primer 0338b2 for amplifying an Mbov _0338 gene fragment.

SEQ ID NO: 7 is the sequence of primer 0338c1 for amplifying an Mbov _0338 gene fragment.

SEQ ID NO: FIG. 8 shows the sequence of primer 0338c2 for amplifying an Mbov _0338 gene fragment.

SEQ ID NO: 9 is the sequence of primer 0338d1 for amplifying an Mbov _0338 gene fragment.

SEQ ID NO: 10 is the sequence of primer 0338d2 for amplifying an Mbov _0338 gene fragment.

Drawings

FIG. 1: plasmid map of pET-30 a. pET-30a is a commercial plasmid, purchased from Novagen.

FIG. 2: map of recombinant plasmid pET-30a-Mbov _ 0338. The recombinant plasmid pET-30ab-Mbov _0338 is formed by connecting and recombining a pET-30a plasmid and a mutant Mbov _0338 gene after the full length is digested by restriction enzymes.

FIG. 3: SDS-PAGE of purified rADH protein. Lane M: high molecular mass protein standard; lane 1: purified rADH protein.

FIG. 4: and (3) determining the activity of the mycoplasma bovis rADH protein ethanol dehydrogenase.

FIG. 5: ELISA was used to detect the serum titer of the immunized rabbits.

FIG. 6: western blot analysis of the reactogenicity of rADH protein. Lane M: high molecular mass protein standard; lanes 1-8: the mycoplasma bovis holomycelial protein and mycoplasma bovis positive bovine serum act; lane 9: the mycoplasma bovis holomycelial protein acts with negative bovine serum.

FIG. 7: western blot analysis of the immunogenicity of rADH protein. Lane M: high molecular mass protein standard; lane 1: mycoplasma bovis holothurian protein.

FIG. 8: indirect immunofluorescence detects the adhesion of rADH to bovine lung epithelial cells (EBL). A, picture A: 100. mu.g of rADH and EBL cells (1X 10)⁵/well) protein incubation, detecting the adhered protein with rabbit anti-rADH hyperimmune serum and Alexa 488-labeled fluorescent secondary antibody; and B, drawing: a control group of EBL cells incubated with PBS; cell membranes and nuclei were stained with DiI and DAPI, respectively. The cells of each treatment group were observed under a confocal laser microscope for fluorescence of different colors, Bar being 20 μm.

FIG. 9: and (5) detecting the anti-rADH rabbit serum inhibition bovine mycoplasma adhesion EBL cells. 1X 10⁸CFU M bovis was incubated with 200. mu.L preimmune rabbit serum or rabbit anti-rADH hyperimmune serum (1:20 dilution in PBS) and then with EBL cells (1X 10)⁵/well) and finally the number of mycoplasma bovis adhering to EBL cells (CFU/well). Values in the figure represent mean ± SEM from 3 independent experiments, representing p<0.01。

FIG. 10: the binding ability of rADH protein to fibronectin (Fn) was identified. A, picture A: detecting the binding capacity of rADH and Fn by a dot hybridization method; and B, drawing: ELISA was used to detect the binding of Fn to microplate-coated rADH.

Detailed Description

Example 1: expression and purification of Mycoplasma bovis ADH recombinant protein (rADH)

1.1 Mycoplasma bovis Mbov _0338 gene cloning expression

Since codon preference of E.coli is given, in the present invention, codon UGA encoding tryptophan in Mycoplasma bovis Mbov _0338 gene is used as a terminator in E.coli, and thus, when the Mycoplasma bovis gene is expressed using E.coli, mutation of codon UGA to codon UGG capable of expressing tryptophan in E.coli is required to be performed on Mycoplasma bovis Mbov _0338 gene. The method comprises the following specific steps: using the Mbov _0338 gene in Mycoplasma bovis HB0801 (genome GenBank accession number: WP _038582875.1) as a template, 4 pairs of primers (numbered: 0338a1/0338a2, 0338b1/0338b2, 0338c1/0338c2, 0338d1/0338d2) designed as follows were used to amplify 4 fragments of the mutated Mbov _0338 gene, respectively, and 0338a1/0338d2 primer pairs were used to amplify the 4 fragments after mutation as a template, thereby obtaining the entire sequence of the mutated Mbov _0338 gene, the sequence length being 1062bp (see the sequence represented by bases 1-1062 in SEQ ID NO:1, and the coding region thereof also being a sequence corresponding to bases 1-1062).

The primer sequences for amplifying the Mbov _0338 gene are as follows:

1. the primer 0338a1/0338a2, the position of the amplified fragment in the genome is 1-67 bases, and the length of the PCR amplified product is 67 bp.

(1) Forward primer 0338a1: 5'-TTAGGTACCATGAAACAAATTCCAGCA-3', (corresponding to the sequence shown in SEQ ID NO: 3 of the sequence Listing).

(2) Reverse primer 0338a 2: 5' -CCTTAACACTCCATTTTTTAGGTTCTGTT-3' (corresponding to the sequence shown in SEQ ID NO: 4 in the sequence listing; the underlined part is a mutation site, i.e., the mutation from T to C).

2. The primer 0338b1/0338b2, the position of the amplified fragment in the Mbov _0338 gene is 45-177 bases, and the length of the PCR amplified product is 133 bp.

(1) Forward primer 0338b 1: 5' -ACCTAAAAAATGGAGTGTTAAGGAAG-3' (wherein the underlined part is a mutation site, i.e., from A to G; corresponding to the sequence shown in SEQ ID NO: 5 of the sequence Listing).

(2) Reverse primer 0338b 2: 5' -AGGTTCTACTAACCAGTCATAATTTGCT-3' (the underlined part is the mutation site, i.e.from T to C; corresponding to the sequence shown in SEQ ID NO: 6 of the sequence Listing).

3. The position of the amplified fragment of the primer 0338c1/0338c2 in the Mbov _0338 gene is 150-292 bases, and the length of the PCR amplified product is 143 bp.

(1) Forward primer 0338c 1: 5' -AGCAAATTATGACTGGTTAGTAGAACCT-3' (the underlined part is the mutation site, i.e.from A to G; corresponding to the sequence shown in SEQ ID NO: 7 of the sequence Listing).

(2) Reverse primer 0338c 2: 5' -AAGCATCATGTAACCATGCTAAAG-3' (the underlined part is the mutation site, i.e.from T to C; corresponding to the sequence shown in SEQ ID NO: 8).

4. The position of the amplified fragment of the primer 0338d1/0338d2 in the Mbov _0338 gene is 261-1062 bases, and the length of the PCR amplified product is 802 bp.

(1) Forward primer 0338d 1: 5' -TAGAGTTGCTTTAGCATGGTTACATGAT-3' (the underlined part is the mutation site, i.e.from A to G; corresponding to the sequence shown in SEQ ID NO: 9 of the sequence Listing).

(2) Reverse primer 0338d2: 5'-CCGGAATTCTTATTTTCTAAAGTCAATAACAG-3' (the underlined part is the mutation site, i.e.from T to C; corresponding to the sequence shown in SEQ ID NO: 10 of the sequence Listing).

5. The position of the amplified fragment of the primer 0338a1/0338d2 in the Mbov _0338 gene is 1-1062 bases, and the length of the PCR amplified product is 1062 bp.

(1) Forward primer 0338a1: 5-TTAGGTACCATGAAACAAATTCCAGCA-3' (the straight lower line part is Kpn I cleavage site, the wavy line part is protective base; corresponding to the sequence shown in SEQ ID NO: 3 of the sequence Listing).

(2) Reverse primer 0338d2: 5-CCGGAATTCTTATTTTCTAAAGTCAATAACAG-3' (the straight-down line part is EcoRI enzyme cutting site, the wavy line part is protective base, and the corresponding sequence is shown in sequence table SEQ ID NO: 10).

The PCR reaction system for the above 4 fragments was as follows:

template DNA 2.5. mu.L, pfu enzyme (Thermo) 1.5. mu.L, pfu buffer with MgSO₄(Thermo) 5. mu.L, 10. mu.L of dNTP mix (Thermo)1. mu.L, each of primers 2. mu.L, and ultra pure water 36. mu.L.

The PCR reaction system for the mutated Mbov _0338 gene using the PCR amplification products of the above 4 fragments as templates and primers 0338a1/0338d2 was as follows: 2.5. mu.L of each fragment, 1.5. mu.L of pfu enzyme (Thermo), pfubuffer with MgSO₄(Thermo) 5. mu.L, 10-fold dNTP mix (Thermo)1. mu.L, primers 2. mu.L each, and ultrapure water 36. mu.L. The primer is synthesized by Shanghai Biotechnology engineering service Co., Ltd.

The amplification product of the Mbov _0338 gene was recovered and digested with Kpn I and EcoRI, while the pET-30a plasmid (FIG. 1) (purchased from Novagen) was double-digested with the same enzyme. The digested Mbov _0338 gene and the pET-30a plasmid were ligated with a DNA ligase (T4DNA ligase) to obtain a recombinant plasmid pET-30a-Mbov _0338 (FIG. 2). After the recombinant plasmid pET-30a-Mbov _0338 is transformed into escherichia coli DH5 alpha, the escherichia coli DH5 alpha is placed in a 37 ℃ shaking table to be cultured for 12 hours at 180r/min, the escherichia coli BL21 bacteria are transformed after the plasmid is extracted and sequenced correctly, when the escherichia coli recombinant bacteria are cultured in an LB liquid culture medium until OD is 0.6, 1mL of bacteria liquid is taken as a control before induction, isopropyl thiogalactoside (IPTG) is added until the final concentration is 0.8mM, and the shaking table at 37 ℃ is used for induction expression for 3 hours. Taking 1mL of bacterial liquid for further treatment: the sample is processed by centrifuging at 12000r/min for 1min, discarding supernatant, and adding 1mL Phosphate Buffer Solution (PBS) (formula: KCl0.2g, NaCl 8g, Na)₂HPO₄1.44g，KH₂PO₄0.24g, 1000mL of distilled water, pH 7.6), and then centrifuged at 12000r/min for 1min to discard the supernatant, 30 μ L of PBS and 30 μ L of loading buffer [ 1M Tris-HCl (pH 6.8)1mL, 200mM DDT 0.31g, 4% SDS 0.4g, 0.2% bromophenol blue 0.02g, 20% glycerol 2mL, 7mL ultrapure water ] were added to resuspend the suspension. Boiling in boiling water at 100 deg.C for 10 min. Identification by SDS-PAGE gel electrophoresisWhether or not to express.

The nucleotide sequence of the protein is transformed into Escherichia coli DH5 alpha by constructing a recombinant plasmid pET-30a-Mbov _0338 to obtain an Escherichia coli recombinant strain, and the applicant names the Escherichia coli recombinant strain as Escherichia coli pET-30a-Mbov _0338(Escherichia coli pET-30a-Mbov _ 0338); the culture is delivered to China Center for Type Culture Collection (CCTCC) of Wuhan university in Wuhan city, Hubei province in 2019 in 4 and 2 months, and the preservation number is CCTCC NO: m2019226.

1.2 purification of rADH protein

After the recombinant Escherichia coli BL21 is induced and expressed according to the method, taking 8000r/min of the bacterial liquid, centrifuging for 10min, discarding the supernatant, centrifuging and washing for 2 times by using 500mL PBS, and centrifuging for 10 min/time at 8000 r/min. After discarding the supernatant, 30mL of PBS was added for resuspension, protease inhibitor (from Roche) was added, and the mixture was disrupted using a hydraulic disrupter. After disruption, the cells were centrifuged at 12000r/min for 30min, and 30. mu.L of the supernatant was added to 30. mu.L of the buffer solution and boiled in boiling water for 10min to prepare a supernatant group. A small amount of the precipitate was added to 30. mu.L of PBS and 30. mu.L of the loading buffer and boiled for 10min to prepare a precipitate group. After SDS-PAGE gel electrophoresis, the rADH protein is mostly expressed in the supernatant.

The rADH protein was purified as follows:

(1) 1mL of Ni-NTA metal-chelating His protein purification media packing (from GE) was added to the affinity chromatography column;

(2) add 12mL ddH to affinity column₂Washing with water;

(3) 12mL of binding buffer (20mM Na) was added₃PO₄0.5M NaCl, 20mM imidazole, pH 7.4) equilibration column;

(4) adding the protein expression supernatant filtered by a filter with the aperture of 0.45 mu m;

(5) adding 50mL binding buffer solution to balance the column;

(6) 50mL of washing buffer (20mM Na) was added₃PO₄0.5M NaCl, 60mM imidazole, pH 7.4) to wash away the contaminating proteins;

(7) add 12mL of elute buffer (20mM Na)₃PO₄0.5M NaCl,1M imidazole, pH 7.4) washRemoving target protein, and collecting the first drops with the number of 1;

(8) adding 50 mu L of loading buffer solution into the tube with the number of 1, and boiling for 10 min;

(9) SDS-PAGE polyacrylamide gels were prepared, the treated samples were added to wells (20. mu.L/well), electrophoresed (80V DC for concentration gel electrophoresis and 120V DC for separation gel electrophoresis), and after electrophoresis was complete, the gels were removed and stained with Coomassie Brilliant blue overnight. Then decolorized to confirm the purified target protein (FIG. 3).

Example 2: ethanol dehydrogenase Activity assay of rADH protein

The detection is carried out by using an ethanol Dehydrogenase Activity assay kit (Sigma) according to the operation of a product instruction, and the method is briefly described as follows: 22.5ng of rADH was added to a 96-well plate, followed by addition of ethanol, NAD +, ethanol dehydrogenase-detecting solution and developing solution, and after thorough mixing, incubation was carried out at 37 ℃ for 3 min. Then, absorbance was measured every 5min at 37 ℃ and 450nm with an enzyme-labeled detector. The detection result proves that the rADH protein can catalyze the substrate ethanol to form acetaldehyde, simultaneously reduce NAD + into NADH, the generated quantity of the NADH is proportional to the absorbance value of the substrate, and then the absorbance at the wavelength of 450nm is increased along with the prolonging of the enzyme catalysis time (figure 4).

Example 3: preparation of rADH protein polyclonal antibody

The method comprises the steps of immunizing male Japanese big-ear white rabbits with purified rADH protein, wherein the immunization amount is about 1 mg/rabbit, calculating the volume of the used recombinant Mbov _338 protein according to the immunization amount, mixing and completely emulsifying the recombinant Mbov _338 protein with equal volume of Freund complete adjuvant, carrying out subcutaneous multi-point injection for immunization, immunizing once every two weeks, emulsifying the recombinant Mbov _338 protein by using the Freund incomplete adjuvant when immunizing for the second time, carrying out ear marginal vein blood sampling after immunizing for the third time for one week for carrying out indirect ELISA for detecting the level of the antibody generated by the white rabbits, generally immunizing for 4-5 times to reach the required titer, and carrying out heart blood sampling and purifying the polyclonal antibody when the level of the white rabbits does not. The results show that the rADH protein can produce antibody titer of 2¹¹ X 100, i.e. 2.0X 10³(FIG. 5).

Example 4: analysis of the reactogenicity of rADH protein

The method for identifying the rADH protein reactogenicity by adopting a Western blot method mainly comprises the following steps: subjecting the purified recombinant protein rADH (2.0. mu.g/lane) to SDS-PAGE, transferring the proteins on the gel to a PVDF membrane, washing the membrane, incubating the PVDF membrane with naturally and experimentally infected bovine mycoplasma positive serum (diluted 1:100 in TBST) for 1h at room temperature by using an HT-Blot Western Blot incubator, washing the membrane, incubating the membrane with a rabbit anti-bovine IgG-HRP secondary antibody (diluted 1:3000 in TBST) for 1h at room temperature, and washing the TBST for three times; and (3) after the membrane is acted for 2-5min by a Bio-rad chemiluminescent substrate, detecting a signal on a chemiluminescence detector. The results show that: the rADH protein can react with bovine mycoplasma positive bovine serum, a specific reaction band can be detected, the molecular weight is about 44kDa (figure 6, lanes 1-8), meanwhile, the protein cannot react with negative bovine serum, and no signal is detected (figure 6, lane 9), so the rADH protein has good reactogenicity.

Example 5: immunogenicity of rADH protein

Detecting the immunogenicity of the rADH protein by using a Western Blot method, transferring mycoplasma bovis holoprotein to a PVDF membrane, incubating the PVDF membrane with anti-rMbov _338 protein rabbit serum, washing the PVDF membrane with TBST for three times, and incubating the PVDF membrane with a goat anti-rabbit IgG-HRP antibody (1:5000) at room temperature for 1 h; TBST washing three times; after the membrane was exposed to Bio-rad chemiluminescent substrate for 2-5min, the signal was detected on a chemiluminescent detector. The results show that: an obvious immunoblot band can be detected, the protein size is the same as the theoretical molecular weight of the mycoplasma bovis ADH protein, and the rADH protein is immunogenic and can cause the sick animals to produce specific antibodies (figure 7).

Example 6: detection of adhesion capability of rADH protein to host cell

6.1 adhesion detection

And detecting the adhesion of the rADH protein and the EBL cells by combining indirect immunofluorescence with a laser confocal scanning microscope. EBL cells are cultured in a 24-well plate for 24-36 h, fixed by 4% paraformaldehyde, and 200. mu.L of rADH protein (0.5. mu.g/. mu.L) is added into the cell culture well to react with EBL cells (1X 10)⁵Perwell) was incubated at 4 ℃ for 1h, washed, and then incubated with 1% BSA-PB at room temperatureS blocking the cells for 1 h; the cells were then incubated with anti-rADH rabbit serum (1:300) for 1h at room temperature. After extensive washing, cells were incubated with donkey anti-rabbit IgG-Alexa 488 antibody (1:400) for 1h at room temperature; cell membranes and cell nuclei were stained Red (membrane) and blue (nucleus) with cellmaskdiep Red and DAPI staining solutions, respectively. Finally, the fluorescence signal is detected under a confocal laser scanning microscope. The results show that: the rADH protein adhered to the surface of EBL cells and green fluorescence was detected on the surface of EBL cells (FIG. 8A), whereas under the same experimental conditions, green fluorescence could not be detected in the control group in which EBL cells were incubated with PBS only (FIG. 8B), confirming that: the rADH protein binds specifically to EBL cells.

6.2 adhesion inhibition assay

Experiment of anti rADH protein rabbit serum inhibiting bovine mycoplasma from adhering EBL cell. EBL cells were seeded in 24-well cell culture plates at 37 ℃ with 5% CO₂Culturing in an incubator for 20 h. Prior to the inhibition experiments, the medium was discarded and the cells were blocked with 1% BSA-MEM for 15min at 37 ℃. About 1X 10 is preliminarily formed⁸CFU of Mycoplasma bovis and 56 degrees C heat inactivated rabbit anti rADH high immune serum and immune rabbit serum at 4 degrees C were incubated for 2 h. The mixture of M.bovis and serum was then added to the mixture containing a monolayer of EBL cells (approximately 1X 10)⁵One/well) and incubated at 37 ℃ for 30min with shaking. After washing four times thoroughly, the monolayer cells were digested with 0.25% trypsin, the cell suspension was spread on PPLO agar plates by serial dilution, and the number of adhered colonies of Mycoplasma bovis was counted by microscopic observation after 72 hours of incubation, and CFU/well was counted. The results show that: rabbit anti-rADH hyperimmune serum (1:20 dilution) significantly reduced the number of Mycoplasma bovis (p) adhering to EBL cells compared to preimmune serum<0.01) (fig. 9).

Example 7: rADH protein adhesion fibronectin (Fn) assay

7.1Dot-Blot detection

The nitrocellulose membrane was mounted on a Bio-Dot microporous filter and rADH (from 1. mu.g to 0.0625. mu.g) was serially diluted two-fold with PBS, each dilution being spotted onto the nitrocellulose membrane at 100. mu.L of rADH per well. The membrane was then removed from the apparatus and blocked with a 5% skim milk in TBS for 4 h. After three washes, the membranes were incubated with 10. mu.g/mL bovine Fn in 1% skim milk-TBS solution overnight at 4 ℃ and then incubated with rabbit anti-bovine Fn antibody (1:1000 dilution) and goat anti-rabbit-HRP antibody (1:4000 dilution) for 1h at room temperature. Finally, the membrane is treated with a chemiluminescent substrate and the signal is detected on a chemiluminescent detector. The results show that: fn bound to the rADH protein dose-dependently (FIG. 10A), and the signal of the reaction was stronger with increasing concentration of rADH protein.

7.2 detection by ELISA

rADH protein and BSA (negative control) were coated in 96-well plates, 500ng per well, incubated overnight at 4 ℃; after washing, blocking with 5% skim milk in PBS for 2h at 37 ℃; after washing, different concentrations of Fn (0, 3.125, 6.25, 12.5, 25, 50, 75, 100 μ g/mL) were added to the wells at 100 μ L per well and incubated at room temperature for 1.5 h; after washing, adding rabbit anti-Fn antibody into each hole, and incubating for 1h at room temperature; after washing, anti-rabbit IgG-HRP antibody (diluted 1:6000 in PBST) was added to each well and incubated for 1h at room temperature; after adding the color developing solution, reading the light absorption value by using an enzyme-labeled detector at the wavelength of 630 nm. The results show that: fn bound to the rath protein coated in microwell plates in a dose-dependent and saturating manner (fig. 10B). When the concentration of Fn is 0-5 mug/hole, the amount of Fn bound to the microporous plate is increased along with the increase of the concentration of Fn; when the Fn concentration is higher than 5 mug/hole, the amount of Fn bound by the rADH recombinant protein does not increase with the increase of the Fn concentration, and the binding reaches saturation. In contrast, Fn binds to BSA at low levels and unsaturation, indicating that the binding between Fn and BSA is a non-specific interaction, while Fn binds to rath specifically.

Appendix: the term in the specification states:

the Mycoplasma bovis ADH protein gene is represented by Mbov _ 0338.

The Mycoplasma bovis ADH recombinant protein is denoted by rADH.

The mycoplasma bovis native isolate is represented by mycoplasma bovis HB 0801.

Bovine fibronectin is denoted as Fn.

Sequence listing

<110> university of agriculture in Huazhong

<120> mycoplasma bovis alcohol dehydrogenase gene, and encoding protein and application thereof

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<170>SIPOSequenceListing 1.0

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<211>1062

<212>DNA

<213> Mycoplasma bovis (Mycoplasma bovis)

<400>1

atgaaacaaa ttccagcaaa aatgaaagct tttgttgtaa cagaacctaa aaaatggagt 60

gttaaggaag ttgatgttcc taaaccaaaa tataaagaag ttttaattga aatggaaact 120

tcaggtatct gtcatacaga cttgcatgca gcaaattatg actggttagt agaacctaaa 180

tacccactta ttccaggcca tgaaggaatt ggaaaggtag ttgcattagg ggaaggatgc 240

acacgtttga aaattggtga tagagttgct ttagcatggt tacatgatgc ttgtggctac 300

tgtgaatttt gtctaacagg tagagaaaca ctttgtccaa atcaaaatat gtcggcttac 360

actaaagatg gatcatatgc tgaatatgca attggtcatg aagattatgt aggattggtt 420

cctgaaaaat tagatattgt aactggtgcg ccaattgttt gcgcaggtgt tacaacttat 480

aaatcattaa aacaaaccaa agcaaaagct ggtaactttg tagctgttat cggtgtcggt 540

ggcttaggtc aaatggctat tcaatatgca aaagctatgg gactaagacc tattggtgtt 600

gacttgcaag atgaaaaatg tgaattagct cttaaatcag gcgcagaata tgcatttaac 660

tcagcaaaag atcctaaatt tattgaaaaa attattgaag taactggcgg aggtgtacat 720

gctgtagtta atacatctgt tcacccaagt gctgctgaac aaggtatgga tatgcttcgt 780

cgcggcggcc gtcaagtatt agttggttta ccagcaaaag ataaacacgg aaaagatgac 840

tttaaagtct caattttctg gtcagtatta ttagaacgtg agcttgctgg ctcaattgtt 900

ggaactagac aagacctagc agaagcttta gaatatgctg ctgaaggaaa agttaaatca 960

gaagttacta aggttgtcaa attagaagaa gttgcagata tttttgaaaa acttcaaaaa 1020

ggcgagttct taggacgtgc tgttattgac tttagaaaat aa 1062

<210>2

<211>353

<212>PRT

<213> Mycoplasma bovis (Mycoplasma bovis)

<400>2

Met Lys Gln Ile Pro Ala Lys Met Lys Ala Phe Val Val Thr Glu Pro

1 5 10 15

Lys Lys Trp Ser Val Lys Glu Val Asp Val Pro Lys Pro Lys Tyr Lys

20 25 30

Glu Val Leu Ile Glu Met Glu Thr Ser Gly Ile Cys His Thr Asp Leu

35 40 45

His Ala Ala Asn Tyr Asp Trp Leu Val Glu Pro Lys Tyr Pro Leu Ile

50 55 60

Pro Gly His Glu Gly Ile Gly Lys Val Val Ala Leu Gly Glu Gly Cys

65 70 75 80

Thr Arg Leu Lys Ile GlyAsp Arg Val Ala Leu Ala Trp Leu His Asp

85 90 95

Ala Cys Gly Tyr Cys Glu Phe Cys Leu Thr Gly Arg Glu Thr Leu Cys

100 105 110

Pro Asn Gln Asn Met Ser Ala Tyr Thr Lys Asp Gly Ser Tyr Ala Glu

115 120 125

Tyr Ala Ile Gly His Glu Asp Tyr Val Gly Leu Val Pro Glu Lys Leu

130 135 140

Asp Ile Val Thr Gly Ala Pro Ile Val Cys Ala Gly Val Thr Thr Tyr

145 150 155 160

Lys Ser Leu Lys Gln Thr Lys Ala Lys Ala Gly Asn Phe Val Ala Val

165 170 175

Ile Gly Val Gly Gly Leu Gly Gln Met Ala Ile Gln Tyr Ala Lys Ala

180 185 190

Met Gly Leu Arg Pro Ile Gly Val Asp Leu Gln Asp Glu Lys Cys Glu

195 200 205

Leu Ala Leu Lys Ser Gly Ala Glu Tyr Ala Phe Asn Ser Ala Lys Asp

210 215 220

Pro Lys Phe Ile Glu Lys Ile Ile Glu Val Thr Gly Gly Gly Val His

225 230 235 240

Ala Val Val Asn Thr Ser Val HisPro Ser Ala Ala Glu Gln Gly Met

245 250 255

Asp Met Leu Arg Arg Gly Gly Arg Gln Val Leu Val Gly Leu Pro Ala

260 265 270

Lys Asp Lys His Gly Lys Asp Asp Phe Lys Val Ser Ile Phe Trp Ser

275 280 285

Val Leu Leu Glu Arg Glu Leu Ala Gly Ser Ile Val Gly Thr Arg Gln

290 295 300

Asp Leu Ala Glu Ala Leu Glu Tyr Ala Ala Glu Gly Lys Val Lys Ser

305 310 315 320

Glu Val Thr Lys Val Val Lys Leu Glu Glu Val Ala Asp Ile Phe Glu

325 330 335

Lys Leu Gln Lys Gly Glu Phe Leu Gly Arg Ala Val Ile Asp Phe Arg

340 345 350

Lys

<210>3

<211>27

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>3

ttaggtacca tgaaacaaat tccagca 27

<210>4

<211>29

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>4

ccttaacact ccatttttta ggttctgtt 29

<210>5

<211>26

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>5

acctaaaaaa tggagtgtta aggaag 26

<210>6

<211>28

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>6

aggttctact aaccagtcat aatttgct 28

<210>7

<211>28

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>7

agcaaattat gactggttag tagaacct 28

<210>8

<211>24

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>8

aagcatcatg taaccatgct aaag 24

<210>9

<211>28

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>9

tagagttgct ttagcatggt tacatgat 28

<210>10

<211>32

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>10

ccggaattct tattttctaa agtcaataac ag 32

Claims

1. The protein coded by the mycoplasma bovis alcohol dehydrogenase gene or the antibody thereof has the activity of alcohol dehydrogenase, has the capacity of adhering to bovine lung epithelial cells and combining bovine fibronectin, and also has immunogenicity and reactogenicity, the generated antibody can be combined with a receptor in a competitive way with the mycoplasma bovis, so that the adhesion of the mycoplasma bovis is reduced, and the pathogenicity of the mycoplasma bovis is reduced, wherein the amino acid sequence of the protein is shown in SEQ ID NO. 2.