CN111393513A

CN111393513A - Mycoplasma bovis secretory adhesive protein MbovP581

Info

Publication number: CN111393513A
Application number: CN202010225626.8A
Authority: CN
Inventors: 郭爱珍; 穆罕默德·祖贝尔; 赵刚; 张慧; 陈颖钰; 胡长敏; 陈曦; 陈建国
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2020-03-26
Filing date: 2020-03-26
Publication date: 2020-07-10

Abstract

The invention belongs to the technical field of animal infectious disease prevention and treatment, and particularly relates to mycoplasma bovis secretory adhesive protein MbovP58, wherein the nucleotide sequence of a protein gene is shown as SEQ ID NO. 1, and the coded amino acid sequence is shown as SEQ ID NO. 2, the Mbov _0581 gene is mutated according to the codon preference of escherichia coli, the mutated sequence is shown as SEQ ID NO. 3, an immune transfer method is used for verifying that the protein is secretory protein and can react with infected bovine serum, an immunofluorescence test and a flow cytometry are used for detecting the adhesion of recombinant protein rMbovP581 expressed by the escherichia coli to bovine lung epithelial cells (EB L), and the MbovP581 is found to have good adhesion capacity to EB L cells.

Description

Mycoplasma bovis secretory adhesive protein MbovP581

Technical Field

The invention belongs to the technical field of prevention and treatment of animal infectious diseases, and particularly relates to mycoplasma bovis secretory mucin MbovP 581.

Background

Mycoplasma bovis (m.bovis) belongs to Mollicutes, Mycoplasma order, Mycoplasma family, Mycoplasma genus, can cause important diseases such as pneumonia, mastitis, arthritis and the like of beef cattle and dairy cows, and can also cause various symptoms such as genital tract inflammation, conjunctivitis, otitis media and the like, the average morbidity is 40%, and the mortality is 10%. Mycoplasma bovis was first isolated in 1961 from the milk of cows with mastitis in the united states and was shown in 1976 to be an important pathogen causing pneumonia. In 2008, respiratory diseases are introduced to beef cattle from other places in Hubei province, the beef cattle are attacked about 2 weeks after the cattle are introduced, the beef cattle are manifested as fever, cough, runny nose, and serious attack to calves and weak cattle, the disease is determined as infectious mycoplasma bovis pneumonia in China central laboratory of agricultural microbiology of university of Huazhong agriculture at first, and then the disease is found to be prevalent in China. Mycoplasma bovis pneumonia of cattle on shelf is often associated with transportation stress of cattle in offsite breeding, while newborn calves can be infected by sucking breast milk of cows suffering from mycoplasma bovis mastitis, causing mycoplasma bovis pneumonia, arthritis, and otitis (gouo heit, 2011). After mycoplasma bovis pneumonia occurs, the immunity of cattle is further reduced, and other conditional pathogenic bacteria such as pasteurella multocida A, mannheimia haemolytica, respiratory syncytial virus and other secondary infections are caused, so that the clinical symptoms are synergistically aggravated, and the disease is collectively called Bovine respiratory disease syndrome (BRD).

The disease is prevalent worldwide. In france, the isolation rate of mycoplasma bovis in a cattle farm with pneumonia was 30%; in britannia, the positive rate of mycoplasma bovis antibodies in a cattle farm with pneumonia is 20% -25%; in Ireland, the isolation rate of Mycoplasma bovis in a cattle farm with pneumonia is 13% -23%. New zealand reported for the first time in 2017 that cows were infected with mycoplasma bovis, after which months and more farms were affected and tens of thousands of cows were killed. The government is implementing eradication programs through mass killing, trying to become the first world to eradicate mycoplasma bovis.

At present, specific prevention and control means for preventing and controlling bovine mycoplasma disease are lacked, and specific molecular targets are needed for creating specific prevention and control measures. The determination of virulence related factors and immunogenic proteins of mycoplasma bovis is a prerequisite for the discovery of specific targets of mycoplasma bovis. Secreted proteins are often associated with pathogen virulence and immune response and are therefore preferred targets for finding specific molecular targets. However, since most genes in the mycoplasma bovis genome are unknown genes and the research of mycoplasma secretory proteins is just started, the related research is slow. The applicant identifies that the MbovP581 protein exists in a mycoplasma bovis secretory protein group through two-dimensional electrophoresis and mass spectrometry and has immunogenicity. It was further demonstrated that the secreted protein is a cell adhesion protein of mycoplasma bovis. Since adhesion is the first step in the development of infection, this protein is a promising molecular target.

Disclosure of Invention

The invention aims to provide a mycoplasma bovis secretory adhesive protein MbovP581, and the protein gene is expected to be used as a specific molecular target of mycoplasma bovis and can be used for developing and controlling bovine mycoplasma disease specificity.

The technical scheme of the invention is as follows:

the applicant finds that the protein can adhere to bovine lung epithelial cells by an immunofluorescence method.

The applicant obtains a Mycoplasma bovis local isolate HB0801 separated from the lung tissue of a cattle of Hubei province, which is attacked by a certain cattle farm in the city of Hubei province in 6 months in 2008, and the applicant names the Mycoplasma bovis HB0801 and Mycoplasma bovisHB0801, and sends the Mycoplasma bovis HB0801 to the China center for type culture collection of the university of Wuhan, in 2 months and 1 day 2010, wherein the collection number is CCTCC NO: and M2010040.

A trichloroacetic acid (TCA) acetone precipitation method is used for extracting a mycoplasma bovis HB0801 strain secretory protein group, a lysis buffer solution of two-dimensional electrophoresis is used for dissolving the secretory protein group, and the protein is proved to be a secretory protein by a conventional Western blot method.

The Mbov _0581 gene is cloned by taking HB0801 genome as a template, the gene is modified according to the codon preference of escherichia coli, and recombinant rMbov P581 is expressed and purified, the protein is incubated with bovine lung epithelial cells (EB L), and the rMbov P581 protein is detected and confirmed to be specifically adhered to EB L cells by an immunofluorescence method, so that the protein is confirmed to be an adhesive protein of mycoplasma bovis.

The invention has the following advantages:

the invention discovers that the mycoplasma bovis secretory protein MbovP581 is an adhesion protein for the first time and is expected to be used as a molecular target for development and utilization.

The detailed technical scheme is described in the detailed description.

Drawings

FIG. 1: pET-30a plasmid map.

FIG. 2: the recombinant plasmid pET-30a-Mbov _0581 map constructed by the invention.

FIG. 3: is an electrophoretogram of the purified Mycoplasma bovis recombinant MbovP581 (or written rMbovP581) protein. Description of reference numerals: rP represents purified rMbovp581 protein. The molecular weight of the protein is 80 kDa.

FIG. 4: is a western blot map for verifying the protein secretion of the M.bovis Mbovp581 protein. Description of reference numerals: m: a protein Marker; WP: mycoplasma bovis HB0801 strain holothurin; and c: mycoplasma bovis HB0801 strain secretes proteome.

FIG. 5: is a western blot map of the reaction of the mycoplasma bovis rMbovp581 of the present invention and bovine serum artificially infected with mycoplasma bovis. Description of reference numerals: m: a protein Marker; 1: rMbovP581 protein and bovine serum after mycoplasma bovis infection reaction group; 2: rMbovP581 protein and bovine serum negative reaction group.

FIG. 6 is a map of M.bovis rMbovP581 adhered EB L cells of the present invention, wherein the reference numerals indicate panel A in FIG. 6, nuclei stained with DAPI in the adhered group, panel B in FIG. 6, F-actin stained red with rhodamine in the adhered group, panel C in FIG. 6, FITC-labeled rMbovP581 protein adhered to EB L cells, panel D in FIG. 6, fusion map of rMbovP581 adhered group, panel E in FIG. 6, fusion map of control group, and panel F in FIG. 6, flow cytometry to detect adhesion of rMbovP581 protein to L cells (p < 0.001).

Detailed Description

Description of sequence listing:

the sequence table SEQ ID NO 1 is the nucleotide sequence of the Mycoplasma bovis Mbov _0581 gene of the invention, and the sequence length is 2103 bp.

The sequence table SEQ ID NO 2 is the amino acid sequence of the M.bovis Mbovp P581 protein of the present invention, and encodes 700 amino acids.

The sequence table SEQ ID NO 3 is the nucleotide sequence of the cloned Mycoplasma bovis Mbov _0581 gene of the invention through artificial mutation according to the codon preference of escherichia coli, and the sequence length is 2103 bp.

Example 1: mycoplasma bovis Mbov _0581 gene cloning and expression purification

1. Mycoplasma bovis Mbov _0581 Gene cloning and expression and Mbov P0581 purification

The nucleotide sequence of the Mbov _0581 gene in Mycoplasma bovis HB0801 (genome GenBank accession number is CP002058) is shown in SEQ ID NO:1, and the sequence size is 2103 bp. The original sequence is taken as a template, and the codon UGA of the gene is mutated into the codon UGG which can express tryptophan in escherichia coli according to the preference of the escherichia coli codon, the mutated sequence is shown as SEQ ID NO. 3, and the sequence size is 2103 bp. The sequence shown in SEQ ID NO. 3 was sent to a commercial gene cloning company for synthesis. The original gene sequence and the protein sequence coded by the mutated gene sequence are the same (the sequence is shown in SEQ ID NO:2), and 700 amino acids are coded.

The amplified product of the synthesized Mbov _0581 gene was digested with Xho I and BamH I, while the pET-30a plasmid (map: FIG. 1) (purchased from Merck China Co., Ltd.) was digested with both Xho I and BamH I.the digested Mbov _0581 gene and pET-30a plasmid were ligated with DNA ligase (T4 DNA ligase (purchased from NEB Co.)) to obtain a recombinant plasmid pET-30a-Mbov _0581 (map: FIG. 2). after E.coli DH 5 α was transformed with the recombinant plasmid pET-30a-Mbov _0581, it was placed on a shaker at 37 ℃ and 180 r.Culturing for 12 hr at min, extracting plasmid, sequencing with universal vector T7, determining that the insertion sequence is correct, transforming Escherichia coli B L21, culturing the recombinant Escherichia coli B L21 in L B liquid culture medium until OD is 0.6, taking 1m L bacterial liquid as control before induction, adding isopropyl thiogalactoside (IPTG) to final concentration of 0.8mM, inducing and expressing for 3 hr at 37 deg.C on shaker, taking 1m L bacterial liquid for further treatment, centrifuging at 12000r/min for 1min, discarding supernatant, adding 1m L Phosphate Buffer Solution (PBS) (formula: KCl 0.2g, NaCl 8g, Na)₂HPO₄1.44g，KH₂PO₄0.24g, 1000M L distilled water, pH 7.6) solution was resuspended and centrifuged at 12000r/min for 1min to discard the supernatant, then 30u L PBS and 30u L loading buffer (1M Tris-HCl (pH 6.8)1M L, 200mM DDT 0.31g, 4% SDS 0.4g, 0.2% bromophenol blue 0.02g, 20% glycerol 2M L, 7M L ultrapure water) were added and resuspended, boiling in 100 ℃ boiling water for 10min, 12% SDS-PAGE gel electrophoresis was used to determine whether rMbovP581 protein size is 80 kDa.

2. Purification of Mycoplasma bovis rMbovp581

After recombinant Escherichia coli B L21 is induced and expressed by 0.8mMIPTG according to the method of the step 1, taking a bacterium solution 8000r/min, centrifuging for 10min, discarding a supernatant, washing once with 500m L PBS, centrifuging for 10min at 8000r/min, washing once with 500m L PBS again, discarding a supernatant, adding 30m L PBS for resuspension, adding a protease inhibitor (purchased from Shanghai Roche pharmaceutical Co., Ltd.), crushing with a hydraulic crusher, centrifuging for 30min at 12000r/min after crushing, taking 30 mu L supernatant, adding 30 mu L sample buffer, boiling for 10min in boiling water as a supernatant group, taking a little precipitate, adding 30 mu L PBS and 30 mu L sample buffer, boiling for 10min as a precipitate group, and determining that most of rMbovP581 is expressed in the supernatant after SDS-PAGE electrophoresis.

The rMbovP581 protein is specifically purified as follows:

(1) adding 1m L Ni-NTA metal chelating His protein purification medium filler (from GE company) to the affinity chromatography column;

(2) adding 12m L ddH to the affinity chromatography column₂Washing with water;

(3) binding buffer of 12m L was added(20mM Na₃PO₄0.5M NaCl, 20mM imidazole, pH 7.4) equilibration column;

(4) adding protein expression supernatant filtered by a filter with the aperture of 0.45 mu m, and collecting the first few drops of filtered liquid, wherein the number is 1;

(5) adding 50m L binding buffer solution to balance the column, and collecting the first drops of liquid, wherein the serial number is 2;

(6) 50m L washing buffer (20mM Na) was added₃PO₄0.5M NaCl, 60mM imidazole, pH 7.4) to wash away the contaminating proteins, and collect the first few drops, numbered 3;

(7) 12m L elute buffer (20mM Na) was added₃PO₄0.5M NaCl,1M imidazole, pH 7.4) eluting the target protein, collecting the first few drops, numbered 4;

(8) adding 50 mu L loading buffer solution into each tube numbered 1 to 4, boiling for 10 min;

(9) preparing 12% SDS-PAGE polyacrylamide gel, adding the treated sample into a hole (20 mu L/hole), carrying out electrophoresis (the electrophoresis condition of a concentrated gel is 80 volts direct current voltage, and the electrophoresis condition of a separation gel is 120V direct current voltage), taking the gel down after the electrophoresis is finished, staining the gel with Coomassie brilliant blue overnight, and then decoloring to determine to obtain the purified target protein.

The purified rMbovP581 protein was 80kDa in size and a single band (FIG. 3).

Example 2: mycoplasma bovis Mbovp581 secretory validation

Mycoplasma bovis HB0801 strain was cultured in PPP L O medium to logarithmic phase, centrifuged at 140000g for 20 minutes and the supernatant was filtered using a 0.22 μ M filter, TCA reagent was added to the supernatant to a final concentration of 10%, incubated overnight at 4 ℃, centrifuged at 65000g for 20 minutes, the supernatant was removed and the pellet was washed with precooled acetone, the washed pellet was dissolved with lysis buffer (8Murea, 4% CHAPS,2M thiourea,60mM DTT, 2% Amidosultaine-14 (ASB-14),40mM Tris-HCl pH 8.8) to obtain an extract of secreted protein group, the concentration of protein was measured using 2D Quant, PMSF was added to the protein group to prevent protein degradation, and the protein group was stored at-80 ℃.

Meanwhile, the whole mycoprotein is extracted by the following method that 10ml of mycoplasma bovis growing to the logarithmic phase is taken, centrifuged at 12000r/min for 10min, 10ml of PBS is used for washing and precipitating for 1 time, then 500u of L PBS is used for re-suspending and precipitating for one time, the mycoplasma bovis (200W and 5min) is crushed by an ultrasonic crusher, the protein concentration of the crushed sample is measured by a BCA kit (purchased from Biyuntian biotechnology limited company), and the sample is stored at-20 ℃ for later use.

The whole bacterial protein load was 1 μ g, the secreted proteome load was 50 μ g, 5 × SDS-PAGE protein loading buffer was added, the total amount was 8 μ l, heated in boiling water for 5 minutes to denature the proteins sufficiently, after cooling to room temperature, the protein samples were loaded directly to 12% SDS-PAGE gel loading wells, the top gel electrophoresis voltage was 80V and the bottom gel electrophoresis voltage was 120V, after electrophoresis was completed, proteins were transferred using nitrocellulose membrane by wet transfer, the voltage was 100V, the membrane transfer time was 1 hour, after membrane transfer was completed, 5% skim milk was used to block the membranes, and the membranes were placed at 4 ℃ for blocking overnight, TBST (10mM Tris-HCl, 150mM NaCl, 150mM Tween-20 (0.05% (V/V)), pH 7.4) was washed three times with 5 minutes each time interval, and then incubated with mouse anti-Mbovinp 581 diluted with TBST (volume ratio 1:500) at room temperature for 3 hours, washed with 5 minutes with TBST, and the molecular weight of the secreted anti-rMbovins protein was obtained after five times incubation with anti-mouse anti-Mbovins (TBST) diluted with 5 minutes, and anti-rat-S, and the molecular weight was obtained after five times of incubation with light-emitted proteins (TBST, the results of the protein was obtained with 5: 1: 5min, and the protein was obtained after 5. the protein was obtained after incubation with 5. the staining).

Example 3: mycoplasma bovis Mbovp581 natural antigenicity verification

Purified rMbovP581 protein was treated with 5 × SDS-PAGE protein loading buffer, boiled for 5 minutes, cooled to room temperature, and protein samples were directly loaded onto SDS-PAGE gel loading wells at 80V for top gel electrophoresis and 120V for bottom gel electrophoresis, and proteins were transferred using nitrocellulose membrane, which was applied to a wet transfer method at 100V for 1 hour after electrophoresis, after transfer, 5% skim milk was used to block the membrane and left at 4 ℃ overnight, TBST was washed three times with 5 minutes intervals, and then incubated with mixed serum of 5 cattle in a TBST-diluted Mycoplasma bovis HB0801 artificial infection test (laboratory retention) (volume ratio 1:500) for 3 hours at room temperature, uninfected bovine serum (laboratory retention) (volume ratio 1:500) was used as a negative control, TBST was washed five times with 5 minutes each, and then incubated with anti-bovine IgG diluted with TBST (Southern IgG) (5000: 1:5000) for 5 minutes, and the results of the artificial serum was obtained after incubation with positive bovine serum and the protein was reacted with light-induced by five times with positive test (positive test).

Example 4: functional verification of M.bovis Mbovp581 adhered cells

1. Qualitative adhesion test

Bovine lung epithelial cells (EB L) were seeded in 6-well plates plated with cell slide, cultured overnight, and after the cells were attached, 500. mu. L cells of complete medium were added, the cells were fixed with 4% paraformaldehyde for 10min, then washed once with 0.5% Triton X-100 at room temperature through 5min, and then washed once with PBS, 1% BSA (bovine serum albumin) was used to seal 2h at 37 ℃ in the present adhesion test, the present adhesion test was divided into 2 wells, 10. mu.g rMbovP581 protein was added to the first well, 10. mu.2 PBS was added to the second well as a blank, the above liquid was added to the fixed and permeated EB L cell well, incubated at 37 ℃ for 1h, washed 3 times with PBS, mouse anti-rMovP protein polyclonal antibody (prepared according to the conventional method) diluted with 1% PBS containing 1% of mouse anti-rMovP protein polyclonal antibody (prepared according to the volume ratio of 1:300), after 3 times of washing with FV, mouse anti-mBvP protein polyclonal antibody was added to each well diluted with PBS, the stained with the white stained sperm binding protein polyclonal antibody was added to the stained by a blue stained gel stained with PBS, the blue stained gel stained sperm binding protein stained with PBS, the stained sperm binding protein polyclonal antibody (see FIG. 12) was added to the stained by the stained gel stained with PBS) of the blue stained sperm binding protein (see FIG. 12), the stained sperm binding protein stained with the stained sperm binding protein of the mouse anti-binding.

2. Quantitative adhesion detection

Will 10⁶EB L cells were incubated with 10. mu.g of rMovP581 protein or PBS in 1.5m L EP tubes at 37 ℃ for 1h at 1000rpm, centrifuged at room temperature for 5min, the supernatant was discarded, 1m L PBS was added to each tube to resuspend the cells, after 3 times of centrifugation and washing under the above conditions, murine anti-rMovP 581 polyclonal antibody (volume ratio 1:300) was added to the EP tubes, the EP tubes were left to stand at 37 ℃ for 30min, after 3 times of washing under the above conditions, FITC-labeled goat anti-mouse IgG (southern Biotech) (volume ratio 1:1000) was added, after 30min at 37 ℃ and adhesion of rNOX protein to L cells was detected using a flow cytometer after washing under the above conditions<0.001) (fig. 6F).

Appendix: the term in the specification states:

m. bovis MbovP581 recombinant proteins, expressed as rMbovP 581.

The gene encoding M.bovis Mbovp P581 protein is represented by Mbov _ 0581.

A native isolate of Mycoplasma bovis, designated Mycoplasma bovis HB 0801.

Sequence listing

<110> university of agriculture in Huazhong

<120> Mycoplasma bovis secretory adhesion protein Mbovp P581

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tacaaatatt gtatgaacac actaaaagct attgaaaacg gagagcaaga agtagctaaa 1080

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gaagacttaa aaagattaaa gcacaacttc aaacatatgc gttttgttgc ctataagatt 1200

tttaaatctt ttgctgatga aatttatgct aaatataatc ttgatgaagt tatgaaagat 1260

gacaatgagc ataaaaatgc taatttgtcc gaagcacaaa gagatgaaat ttatgaactt 1320

tcaaaagaca taatttcaat taaaaaagct atttttaatg aagttatgga agttgctaaa 1380

agagttgaaa ttcttcctat tttgcaaaag aaacctacaa gactttcagg tggtcaacaa 1440

caacgtgttg ctatcgcccg tgctattgtt aaaaagcctg acattttatt aatggatgaa 1500

ccgctttcaa accttgatgc taaattacgt atatcaacaa gacaatgaat tagacaaatt 1560

caaagaaatc ttggtattac aacagttttt gttactcacg accaagaaga agctatgtct 1620

attagtgata ttgttgtatg tatgtcaaca tcaaaagttc aacaactagg ctcaccatta 1680

gaactatata atagacctaa aaataaattt gtggctcgtt tcttgggtat gcctgaaatg 1740

ggactattcc caggtaaata tgaaaacgga aaacttagcg tattaggcaa accagttgat 1800

ggcattacat tttctgaata tgaccaattt acctgtaatg taggggtaag agccgaagac 1860

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tag 2103

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Lys Val Thr Asp Asn Asp Ile Ala Glu Tyr Asp Arg Leu Ser Ala Lys

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Ser Phe Lys Ile Pro Asn Gly Lys Leu Val Thr Leu Leu Gly Pro Ser

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Gly Ser Gly Lys Thr Thr Thr Leu Asn Ala Ile Ser Gly Leu Leu Arg

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Ile Thr Ser Gly Asn Val Tyr Phe Asn Gly Lys Asp Val Thr Asn Leu

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Ser Pro Gln Gln Arg Lys Leu Gly Phe Val Phe Gln Asn Tyr Ala Leu

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tat ccg cac atg tca gtt ttt gac aat atc gca ttt ccg ctg aag aac 432

Tyr Pro His Met Ser Val Phe Asp Asn Ile Ala Phe Pro Leu Lys Asn

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Asp Ile Lys Trp Gln Asn Lys Ala Ile Asp Lys Lys Asn Ser Ala Ile

145 150 155 160

att aag atc aag aac ctg tat ctg aag agc ctg ggg gca agc gat gaa 528

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

165 170 175

gaa att aat aaa att aac gat ctt tgg aac atc tac acc aat ata gac 576

Glu Ile Asn Lys Ile Asn Asp Leu Trp Asn Ile Tyr Thr Asn Ile Asp

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Lys Glu Val Asp Tyr Glu Leu Asn Asn Phe Val Val Lys Asn Asn Lys

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Ile Ile Glu Lys Ala Lys Thr Asp Tyr Lys Leu Ala Lys Ile His Tyr

210 215 220

gaa ggt gaa ctg gca acc att agc aaa acc att ctg aaa gca ttc aac 720

Glu Gly Glu Leu Ala Thr Ile Ser Lys Thr Ile Leu Lys Ala Phe Asn

225 230 235 240

tcc ctg aaa aag agc tac aaa ctg aaa aag gat aac ata aag aag cac 768

Ser Leu Lys Lys Ser Tyr Lys Leu Lys Lys Asp Asn Ile Lys Lys His

245 250 255

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

260 265 270

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Pro Lys Cys Glu Phe Leu Ala Asn Leu Asp Thr Gln Leu Leu Ser Tyr

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Lys Lys Val Ala Asn Val Asp Glu Ala Thr Glu Lys Tyr His Asn Leu

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Gln Glu Val Ile Ser Asp Ile Ser Thr Leu Asp Trp Lys Val Tyr Ser

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Glu Lys Asp Gln Tyr Glu Leu Leu Lys Val Glu Asn Lys Leu Leu Lys

325 330 335

gca agc ctg tac tac aaa tac tgt atg aat acc ctg aaa gca att gaa 1056

Ala Ser Leu Tyr Tyr Lys Tyr Cys Met Asn Thr Leu Lys Ala Ile Glu

340 345 350

aat ggt gaa cag gaa gtg gcc aaa gca aaa gaa gca tta aaa gaa gca 1104

Asn Gly Glu Gln Glu Val Ala Lys Ala Lys Glu Ala Leu Lys Glu Ala

355 360 365

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Lys Ile Ala Asn Lys Glu Leu Lys Ala Asn Asn Lys Glu Asp Leu Lys

370 375 380

cgg ctg aaa cat aat ttt aaa cac atg aga ttc gtt gca tac aaa att 1200

Arg Leu Lys His Asn Phe Lys His Met Arg Phe Val Ala Tyr Lys Ile

385390 395 400

ttt aag agc ttc gca gat gaa atc tac gca aaa tac aat tta gac gaa 1248

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

405 410 415

gtg atg aaa gac gat aat gaa cat aaa aac gca aac ctg agc gaa gcg 1296

Val Met Lys Asp Asp Asn Glu His Lys Asn Ala Asn Leu Ser Glu Ala

420 425 430

cag cgc gat gaa att tac gaa ctg agc aaa gac ata ata agc att aaa 1344

Gln Arg Asp Glu Ile Tyr Glu Leu Ser Lys Asp Ile Ile Ser Ile Lys

435 440 445

aag gca atc ttc aac gaa gtg atg gaa gtg gca aaa cgg gtg gaa atc 1392

Lys Ala Ile Phe Asn Glu Val Met Glu Val Ala Lys Arg Val Glu Ile

450 455 460

ctg ccg ata tta cag aaa aag cca acc cgg ctg agc ggc gga caa caa 1440

Leu Pro Ile Leu Gln Lys Lys Pro Thr Arg Leu Ser Gly Gly Gln Gln

465 470 475 480

caa aga gtg gcg att gcg aga gca att gtg aaa aag cca gat att ctg 1488

Gln Arg Val Ala Ile Ala Arg Ala Ile Val Lys Lys Pro Asp Ile Leu

485 490 495

ctg atg gat gaa ccg ctg agc aat ctg gat gca aaa ctgaga atc agc 1536

Leu Met Asp Glu Pro Leu Ser Asn Leu Asp Ala Lys Leu Arg Ile Ser

500 505 510

acc cgt cag tgg ata aga cag atc cag cgt aat ctg ggg ata acc acc 1584

Thr Arg Gln Trp Ile Arg Gln Ile Gln Arg Asn Leu Gly Ile Thr Thr

515 520 525

gtt ttc gtg acc cat gac caa gaa gaa gca atg agc att tca gac att 1632

Val Phe Val Thr His Asp Gln Glu Glu Ala Met Ser Ile Ser Asp Ile

530 535 540

gtt gtt tgc atg agc acc agc aaa gtt caa cag ctg ggg agc ccg tta 1680

Val Val Cys Met Ser Thr Ser Lys Val Gln Gln Leu Gly Ser Pro Leu

545 550 555 560

gag ctg tat aat cgc cca aaa aat aaa ttt gtt gca cgt ttt ctg ggt 1728

Glu Leu Tyr Asn Arg Pro Lys Asn Lys Phe Val Ala Arg Phe Leu Gly

565 570 575

atg ccg gaa atg ggt ctg ttt ccg ggt aaa tat gaa aat ggt aaa ctg 1776

Met Pro Glu Met Gly Leu Phe Pro Gly Lys Tyr Glu Asn Gly Lys Leu

580 585 590

agc gtt ctg ggt aaa ccg gtt gat ggt att acc ttt agc gaa tat gat 1824

Ser Val Leu Gly Lys Pro Val Asp Gly Ile Thr Phe Ser Glu Tyr Asp

595 600 605

cag ttt acc tgt aat gtt ggt gtt cgt gca gaa gat ttt gat att gtt 1872

Gln Phe Thr Cys Asn Val Gly Val Arg Ala Glu Asp Phe Asp Ile Val

610 615 620

aaa agc agc gca gat gca aat tat ttt ggt ctg gtt aaa gca gtt gaa 1920

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

625 630 635 640

aat ttt ggt aaa gaa agc aaa ctg att gtt gaa gtt ccg gaa gca ggt 1968

Asn Phe Gly Lys Glu Ser Lys Leu Ile Val Glu Val Pro Glu Ala Gly

645 650 655

gat gtt aat ttt ctg gtt agc aat gat tat gtt tat aat gtt cgt gat 2016

Asp Val Asn Phe Leu Val Ser Asn Asp Tyr Val Tyr Asn Val Arg Asp

660 665 670

aaa att tat ttt aat ctg ccg acc aat cgt ctg cat att ttt gat aaa 2064

Lys Ile Tyr Phe Asn Leu Pro Thr Asn Arg Leu His Ile Phe Asp Lys

675 680 685

gca aat gat gaa cgt gtt gaa tat gaa att aaa aaa taa 2103

Ala Asn Asp Glu Arg Val Glu Tyr Glu Ile Lys Lys

690 695 700

<210>3

<211>700

<212>PRT

<213> Mycoplasma bovis (Mycoplasma bovis)

<400>3

Met Ile Trp Leu Lys Lys Leu Ile Asp Asn Val Lys Leu Ser Thr Ala

1 5 10 15

Lys Val Thr Asp Asn Asp Ile Ala Glu Tyr Asp Arg Leu Ser Ala Lys

20 25 30

Ile Leu Asn Ser Asp Lys Asn Ala Glu Glu Ile Pro Ala Ile Glu Leu

35 40 45

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

50 55 60

Ser Phe Lys Ile Pro Asn Gly Lys Leu Val Thr Leu Leu Gly Pro Ser

65 70 75 80

Gly Ser Gly Lys Thr Thr Thr Leu Asn Ala Ile Ser Gly Leu Leu Arg

85 90 95

Ile Thr Ser Gly Asn Val Tyr Phe Asn Gly Lys Asp Val Thr Asn Leu

100 105 110

Ser Pro Gln Gln Arg Lys Leu Gly Phe Val Phe Gln Asn Tyr Ala Leu

115 120 125

Tyr Pro His Met Ser Val Phe Asp Asn Ile Ala Phe Pro Leu Lys Asn

130 135 140

Asp Ile Lys Trp Gln Asn Lys Ala Ile Asp Lys Lys Asn Ser Ala Ile

145 150 155 160

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

165 170 175

Glu Ile Asn Lys Ile Asn Asp Leu Trp Asn Ile Tyr Thr Asn Ile Asp

180 185 190

Lys Glu Val Asp Tyr Glu Leu Asn Asn Phe Val Val Lys Asn Asn Lys

195 200 205

Ile Ile Glu Lys Ala Lys Thr Asp Tyr Lys Leu Ala Lys Ile His Tyr

210 215 220

Glu Gly Glu Leu Ala Thr Ile Ser Lys Thr Ile Leu Lys Ala Phe Asn

225 230 235 240

Ser Leu Lys Lys Ser Tyr Lys Leu Lys Lys Asp Asn Ile Lys Lys His

245 250 255

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

260 265 270

Pro Lys Cys Glu Phe Leu Ala Asn Leu Asp Thr Gln Leu Leu Ser Tyr

275 280 285

Lys Lys Val Ala Asn Val Asp Glu Ala Thr Glu Lys Tyr His Asn Leu

290 295 300

Gln Glu Val Ile Ser Asp Ile Ser Thr Leu Asp Trp Lys Val Tyr Ser

305 310 315 320

Glu Lys Asp Gln Tyr Glu Leu Leu Lys Val Glu Asn Lys Leu Leu Lys

325 330 335

Ala Ser Leu Tyr Tyr Lys Tyr Cys Met Asn Thr Leu Lys Ala Ile Glu

340 345 350

Asn Gly Glu Gln Glu Val Ala Lys Ala Lys Glu Ala Leu Lys Glu Ala

355 360 365

Lys Ile Ala Asn Lys Glu Leu Lys Ala Asn Asn Lys Glu Asp Leu Lys

370 375 380

Arg Leu Lys His Asn Phe Lys His Met Arg Phe Val Ala Tyr Lys Ile

385 390 395 400

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

405 410 415

Val Met Lys Asp Asp Asn Glu His Lys Asn Ala Asn Leu Ser Glu Ala

420 425 430

Gln Arg Asp Glu Ile Tyr Glu Leu Ser Lys Asp Ile Ile Ser Ile Lys

435 440 445

Lys Ala Ile Phe Asn Glu Val Met Glu Val Ala Lys Arg Val Glu Ile

450 455 460

Leu Pro Ile Leu Gln Lys Lys Pro Thr Arg Leu Ser Gly Gly Gln Gln

465 470 475 480

Gln Arg Val Ala Ile Ala Arg Ala Ile Val Lys Lys Pro Asp Ile Leu

485 490 495

Leu Met Asp Glu Pro Leu Ser Asn Leu Asp Ala Lys Leu Arg Ile Ser

500 505 510

Thr Arg Gln Trp Ile Arg Gln Ile Gln Arg Asn Leu Gly Ile Thr Thr

515 520 525

Val Phe Val Thr His Asp Gln Glu Glu Ala Met Ser Ile Ser Asp Ile

530 535 540

Val Val Cys Met Ser Thr Ser Lys Val Gln Gln Leu Gly Ser Pro Leu

545 550 555 560

Glu Leu Tyr Asn Arg Pro Lys Asn Lys Phe Val Ala Arg Phe Leu Gly

565 570 575

Met Pro Glu Met Gly Leu Phe Pro Gly Lys Tyr Glu Asn Gly Lys Leu

580 585 590

Ser Val Leu Gly Lys Pro Val Asp Gly Ile Thr Phe Ser Glu Tyr Asp

595 600 605

Gln Phe Thr Cys Asn Val Gly Val Arg Ala Glu Asp Phe Asp Ile Val

610 615 620

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

625 630 635 640

Asn Phe Gly Lys Glu Ser Lys Leu Ile Val Glu Val Pro Glu Ala Gly

645 650 655

Asp Val Asn Phe Leu Val Ser Asn Asp Tyr Val Tyr Asn Val Arg Asp

660 665 670

Lys Ile Tyr Phe Asn Leu Pro Thr Asn Arg Leu His Ile Phe Asp Lys

675 680 685

Ala Asn Asp Glu Arg Val Glu Tyr Glu Ile Lys Lys

690 695 700

Claims

1. A mycoplasma bovis secretory protein gene MbovP581 with the adhesion function to bovine lung epithelial cells is characterized in that the protein sequence of the protein gene is shown as SEQ ID NO. 3.

2. The Mycoplasma bovis secretory protein gene MbovP581 as claimed in claim 1, wherein the codon UGA of the sequence shown in SEQ ID NO. 1 is mutated to a codon UGG capable of expressing tryptophan in Escherichia coli, and the nucleotide sequence of the mutated gene is shown in SEQ ID NO. 2.

3. Use of the protein gene of claim 1 or 2 for the preparation of a diagnostic reagent, a medicament and/or a vaccine for mycoplasma bovis.