CN110257405A - Mycoplasma bovis alcohol dehydrogenase gene and its coding albumen and application - Google Patents
Mycoplasma bovis alcohol dehydrogenase gene and its coding albumen and application Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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Abstract
The invention discloses a kind of Mycoplasma bovis alcohol dehydrogenase genes, with the nucleotide sequence as shown in SEQ ID NO:1, the invention also discloses a kind of albumen of Mycoplasma bovis alcohol dehydrogenase gene coding, with the amino acid sequence as shown in SEQ ID NO:2, belong to zoonosis prevention and treatment and field of biotechnology.Recombinant protein of the invention has alcohol dehydrogenase activity, ox pulmonary epithelial cells system cell (EBL) can be sticked, it can be combined with ox fibronectin (Fn), also there is good immunogenicity and reactionogenicity simultaneously, it is a kind of virulence-associated protein of Mycoplasma bovis, in the research and development of the research of Mycoplasma bovis pathogenic mechanism, vaccine and diagnostic reagent with good application prospect.
Description
Technical field
The invention belongs to zoonosis prevention and treatment and field of biotechnology, and in particular to a kind of Mycoplasma bovis alcohol dehydrogenase
Albumen and the application of (alcohol dehydrogenase, ADH) gene and its coding.
Background technique
Mycoplasma bovis (Mycoplasma bovis, M.bovis) belongs to Mollicutes Mycoplasma, is currently ox to be caused to exhale
One of the important pathogen for inhaling tract disease, and leads to various clinical illness, mainly include Bronchopneumonia, mazoitis, arthritis, in
Otitis, genital tract inflammation, tenosynovitis, meningitis, keratoconjunctivitis sicca, miscarriage with it is infertile etc..Since its virulence factor is unclear, pathogenesis
It is unknown, up to the present, still lack the vaccine effectively for Mycoplasma bovis infection, antibiotic treatment effect is poor, and the course for the treatment of is long, and
Pathogen Antibiotic Resistance is also continuously increased, this disease is caused still to lack effective preventions at present, to beef raising and
Dairy brings huge economic loss.
Mycoplasma bovis is initially 1961 isolated in the cow's milk juice that mazoitis is suffered from from an example by the U.S., is confirmed within 1976
The pathogen leads to ox respiratory disease.Later, country variant reported the prevalence of the disease successively, at present in world wide
It is inside widely current, leads to huge economic loss.There are about 25~33% calf pneumonia caused by Mycoplasma bovis in Europe, made
At economic loss it is more, at least up to 1.44~1.92 hundred million Euros.Mycoplasma bovis is in addition to causing above-mentioned direct economic loss
Except, also result in serious indirect economic loss, such as drug therapy expense during one's sickness, to illness and dead ox slaughter and
Handle the expense of a large amount of labours paid, and the spending of the nonspecific pathogen of prevention, the trade of animal and animal product
Easily come and go is the major reason of disease prevalence.
China is separated to Mycoplasma bovis in 1983 from bovine mastitis milk for the first time, and 2008 for the first time where the applicant
Mycoplasma bovis pneumonia is reported in laboratory, is hereafter found, all come into vogue in major part province the disease in China.In addition to Hubei Province, Henan,
The ground such as Jiangxi, Xinjiang, Hunan, Guangdong, Xiamen are in succession from there is suffering from ox body for respiratory symptom to be separated to Mycoplasma bovis.
The excavation of virulence-associated protein facilitate the interaction relationship that mycoplasma and body are recognized from protein level and
Illustrate the pathogenesis of the disease；In addition, the target that new virulence-associated protein is researched and developed possible as new generation vaccine；Meanwhile having
There is the virulence-associated protein of immunogenicity to be likely to become the new diagnostic mark of the disease.
Summary of the invention
The purpose of the present invention is to provide a kind of Mycoplasma bovis alcohol dehydrogenase gene and its albumen of coding, new to research and develop
Type vaccine and diagnostic reagent provide potential candidate albumen.
In order to achieve the object of the present invention, applicant is with the experiment of place Hua Zhong Agriculture University agromicrobiology state key
HB0801 plants of Mycoplasma bovis of M.bovis of room ruminant cause of disease locellus separation are template, and design primer is cloned and expressed
It recombinates ADH albumen (rADH).It further verifies and determines that rADH has alcohol dehydrogenase activity, second can be converted into catalysis ethanol
Aldehyde, while NAD+ can be reduced to NADH.In vitro study is it has furthermore been found that the albumen can also stick ox pulmonary epithelial cells (EBL)
And it is combined with fibronectin (Fn), and there is immunogenicity and reactionogenicity.And carefully due to adhesive function and immunogenicity
Bacterium power is related, and therefore, Mycoplasma bovis ADH albumen is a kind of virulence-associated protein, is that mycoplasma bovis vaccine and diagnostic reagent are ground
The candidate targets of hair.
Technical solution of the present invention is specific as follows:
Isolated one in the lung tissue for the ox that applicant falls ill in June, 2008 from Hubei Province Yingcheng City vaccary
Strain Mycoplasma bovis local separation strains HB0801, applicant are named as Mycoplasma bovis HB0801 (Mycoplasma bovis
HB0801), China typical culture collection center (Wuhan) preservation, deposit number CCTCC were delivered on 2 1st, 2010
NO:M2010040.The Mycoplasma bovis separation strains disclose in the patent document of CN 109750054A.
The present invention is first using Mycoplasma bovis HB0801 (genome is in GenBank accession number for CP002058) genome as mould
Plate clones the gene.Escherichia coli are different from the expression system of mycoplasma, and the expression system of Escherichia coli will be compiled in mycoplasma
The codon UGA of code tryptophan is as its terminator codon, therefore, if the mycoplasma gene order containing UGA is inserted into greatly
In enterobacteria expression system, obtained gene expression product is exactly the product being truncated.It therefore will using Overlap extension PCR method
UGA base rite-directed mutagenesis in mycoplasma is at the UGG base for equally expressing tryptophan in Escherichia coli, ADH protein gene
(Mbov_0338) 3 UGA base-pairs are shared in and need codon mutation, just can guarantee obtained in Escherichia coli it is total length expressed.
The nucleotide sequence of M. bovis genes Mbov_0338 after artificial mutation is sequence table SEQ ID NO:1
1-1062 bit base shown in sequence, the length is 1062bp；Wherein at 57 of the sequence, 165 and 279 generations etc.
Position gene mutation.
The ADH protein sequence of ox Zhi Yuanbai Mbov_0338 gene coding is as shown in sequence table SEQ ID NO:2, altogether
Encode 353 amino acid.
The nucleotide sequence of Mycoplasma bovis Mbov_0338 gene of the invention is passed through into building plasmid vector pET-30a-
Mbov_0338 conversion bacillus coli DH 5 alpha obtains recombinant escherichia coli strain, and the Escherichia coli of the recombination are named as by applicant
Escherichia coli pET-30a-Mbov_0338 (Escherichia coli pET-30a-Mbov_0338), on April 2nd, 2019
China typical culture collection center (CCTCC) preservation positioned at Wuhan City, Hubei Province Wuhan University is delivered, deposit number is
CCTCC NO:M2019226.The bacterial strain is under IPTG induction, the recombinant protein rADH of expression Mbov_0338 gene coding.
By verifying, the rADH that the present invention purifies has alcohol dehydrogenase activity, NAD+ can be reduced to NADH, in addition,
The albumen also has immunogenicity and reactionogenicity, and can stick host EBL epithelial cell and combine fibronectin (Fn).
Particular content detailed in Example.
The invention has the following advantages that
1, Mycoplasma bovis ADH recombinant protein is encoded by M. bovis genes Mbov_0338, confirms there is ethyl alcohol by inventor
Dehydrogenase activity.
2, Mycoplasma bovis ADH recombinant protein has immunogenicity and reactionogenicity, can be used for preparing vaccine prevention Niu Zhiyuan
Body-sensing dye, it can also be used to which the detection of Mycoplasma bovis antibody can also prepare polyclonal or monoclonal antibody by immune animal, use
In the detection of Mycoplasma bovis antigen.
3, Mycoplasma bovis ADH recombinant protein can be specifically bound with ox pulmonary epithelial cells (EBL) and fibronectin (Fn), be
One kind sticking GAP-associated protein GAP, and generated antibody can be with Mycoplasma bovis competitiveness in conjunction with receptor, to reduce Mycoplasma bovis
Stick, reduces the pathogenic of Mycoplasma bovis.
Sequence table explanation:
SEQ ID NO:1 is the nucleotide sequence (protogene of the M. bovis genes Mbov_0338 after base mutation
Source: Mycoplasma bovis HB0801 bacterial strain, the accession number GenBank Accession:CP002058 of protogene),
Position of the Mbov_0338 gene in genome: 399768-400829, it is positive.Mycoplasma bovis Mbov_0338 after the modification
The nucleotide sequence of gene is as shown in 1-1062 bit base, wherein at 57 of the sequence, 165 and 279 generation codons
Mutation.
SEQ ID NO:2 is the amino acid sequence of Mycoplasma bovis ADH albumen, encodes 353 amino acid altogether.
SEQ ID NO:3 is the sequence for expanding the primer 0338a1 of Mbov_0338 genetic fragment.
SEQ ID NO:4 is the sequence for expanding the primer 0338a2 of Mbov_0338 genetic fragment.
SEQ ID NO:5 is the sequence for expanding the primer 0338b1 of Mbov_0338 genetic fragment.
SEQ ID NO:6 is the sequence for expanding the primer 0338b2 of Mbov_0338 genetic fragment.
SEQ ID NO:7 is the sequence for expanding the primer 0338c1 of Mbov_0338 genetic fragment.
SEQ ID NO:8 is the sequence for expanding the primer 0338c2 of Mbov_0338 genetic fragment.
SEQ ID NO:9 is the sequence for expanding the primer 0338d1 of Mbov_0338 genetic fragment.
SEQ ID NO:10 is the sequence for expanding the primer 0338d2 of Mbov_0338 genetic fragment.
Detailed description of the invention
Fig. 1: pET-30a plasmid map.PET-30a is commercialization plasmid, is purchased from Novagen company.
Fig. 2: the map of recombinant plasmid pET-30a-Mbov_0338.Recombinant plasmid pET-30ab-Mbov_0338 be by
Mbov_0338 full length gene after pET-30a plasmid and mutation connects after digestion with restriction enzyme to be recombinated.
Fig. 3: the rADH protein SDS-PAGE figure of purifying.Swimming lane M: high molecule mass protein standards；Swimming lane 1: purifying
RADH albumen.
Fig. 4: Mycoplasma bovis rADH albumen alcohol dehydrogenase activity measurement result.
Fig. 5: ELISA detects immune rabbit anteserum potency.
The reactionogenicity of Fig. 6: Western blot analysis rADH albumen.Swimming lane M: high molecule mass protein standards；Swimming
Road 1-8: Mycoplasma bovis whole bacterial protein and Mycoplasma bovis positive cow's serum act on；Swimming lane 9: Mycoplasma bovis whole bacterial protein and negative ox
Serum effect.
The immunogenicity of Fig. 7: Western blot analysis rADH albumen.Swimming lane M: high molecule mass protein standards；Swimming
Road 1: Mycoplasma bovis whole bacterial protein.
Fig. 8: indirect immunofluorescence detection rADH sticks with ox pulmonary epithelial cells (EBL's).A figure: 100 μ g rADH with
EBL cell (1 × 105/ hole) after albumen is incubated for, detected with the fluorescence secondary antibody that rabbit-anti rADH hyper-immune serum and Alexa488 are marked glutinous
Attached albumen；B figure: the control group that EBL cell and PBS are incubated for；Cell membrane and nucleus use DiI and DAPI to dye respectively.Laser
Observe the different colours fluorescence of each processing group cell under Laser Scanning Confocal Microscope, Bar=20 μm.
Fig. 9: anti-rADH rabbit anteserum inhibits Mycoplasma bovis to stick EBL cell detection.1×108CFU Mycoplasma bovis in advance with
After rabbit anteserum or rabbit-anti rADH hyper-immune serum (1:20 dilutes in PBS) are incubated for before 200 μ L are immune, then with EBL cell (1 ×
105/ hole) effect, finally calculate the Mycoplasma bovis quantity (hole CFU/) being attached on EBL cell.Numerical value, which represents, in figure comes from 3
Average value ± the SEM, * * of independent experiment indicate p < 0.01.
Figure 10: rADH albumen and fibronectin (Fn) binding ability qualification figure.A figure: spot hybridization detects rADH and Fn
Binding ability；B figure: the combination of ELISA detection Fn and the coated rADH of microwell plate.
Specific embodiment
Embodiment 1: the expression and purifying of Mycoplasma bovis ADH recombinant protein (rADH)
1.1 Mycoplasma bovis Mbov_0338 gene cloning and expressions
Since Escherichia coli are to the preferences of codon, coding colors in Mycoplasma bovis Mbov_0338 gene in the present invention
The codon UGA of propylhomoserin is used as terminator in Escherichia coli, therefore, when with Bacillus coli expression M. bovis genes, needs
Mycoplasma Mbov_0338 gene is mutated, codon UGA is sported can be in the password of expression in escherichia coli tryptophan
Sub- UGG.It comprises the concrete steps that: in Mycoplasma bovis HB0801 (genome GenBank accession number is WP_038582875.1)
Mbov_0338 gene is template, and using the 4 pairs of primers designed as follows, (number is respectively as follows: 0338a1/0338a2,0338b1/
0338b2,0338c1/0338c2,0338d1/0338d2) amplify respectively mutation after Mbov_0338 gene 4 segments,
Then, the Mbov_ using 4 segments after being mutated as template, using 0338a1/0338d2 primer pair amplifies, after being mutated
The complete sequence of 0338 gene, sequence length are 1062bp (see sequence shown in 1-1062 bit base in sequence table SEQ ID NO:1
Column, code area is also the corresponding sequence of 1-1062 bit base).
The primer sequence for expanding Mbov_0338 gene is as follows:
1, primer 0338a1/0338a2, position of the amplified fragments in genome are pcr amplification product at 1-67 base
Length is 67bp.
(1) forward primer 0338a1:5 '-TTAGGTACCATGAAACAAATTCCAGCA-3 ', (corresponding sequence table SEQ ID
Sequence shown in NO:3).
(2) reverse primer 0338a2:5 '-CCTTAACACTCCATTTTTTAGGTTCTGTT-3 ', (corresponding sequence table SEQ
Sequence shown in ID NO:4；Underscore part is mutational site, i.e., sports C by T).
2, primer 0338b1/0338b2, amplified fragments are PCR at 45-177 base in the position in Mbov_0338 gene
Amplified production length is 133bp.
(1) forward primer 0338b1:5 '-ACCTAAAAAATGG(wherein: underscore part is AGTGTTAAGGAAG-3 '
Mutational site sports G by A；Sequence shown in corresponding sequence table SEQ ID NO:5).
(2) reverse primer 0338b2:5 '-AGGTTCTACTAAC(underscore part is mutation to CAGTCATAATTTGCT-3 '
Site sports C by T；Sequence shown in corresponding sequence table SEQ ID NO:6).
3, primer 0338c1/0338c2 amplified fragments are PCR at 150-292 base in the position in Mbov_0338 gene
Amplified production length is 143bp.
(1) forward primer 0338c1:5 '-AGCAAATTATGACTGG(underscore part is mutation to TTAGTAGAACCT-3 '
Site sports G by A；Sequence shown in corresponding sequence table SEQ ID NO:7).
(2) reverse primer 0338c2:5 '-AAGCATCATGTAAC(underscore part is mutation position to CATGCTAAAG-3 '
Point, i.e., sport C by T；Sequence shown in corresponding SEQ ID NO:8).
4, primer 0338d1/0338d2 amplified fragments the position in Mbov_0338 gene be 261-1062 base at,
Pcr amplification product length is 802bp.
(1) forward primer 0338d1:5 '-TAGAGTTGCTTTAGCATGG(underscore part is mutation to TTACATGAT-3 '
Site sports G by A；Sequence shown in corresponding sequence table SEQ ID NO:9).
(2) (underscore part is reverse primer 0338d2:5 '-CCGGAATTCTTATTTTCTAAAGTCAATAACAG-3 '
Mutational site sports C by T；Sequence shown in corresponding sequence table SEQ ID NO:10).
5, primer 0338a1/0338d2 amplified fragments are PCR at 1-1062 base in the position in Mbov_0338 gene
Amplified production length is 1062bp.
(1) forward primer 0338a1:5 '-TTAGGTACC(straight underscore part is Kpn to ATGAAACAAATTCCAGCA-3 '
I restriction enzyme site, wave part are protectiveness base；Sequence shown in corresponding sequence table SEQ ID NO:3).
(2) reverse primer 0338d2:5 '-CCGGAATTC(the straight underscore part TTATTTTCTAAAGTCAATAACAG-3 '
For I restriction enzyme site of EcoR, wave part is protectiveness base；Sequence shown in corresponding sequence table SEQ ID NO:10).
The PCR reaction system of above-mentioned 4 segments is as follows:
2.5 μ L, pfu enzyme (Thermo) of template DNA, 1.5 μ L, pfu buffer with MgSO4(Thermo) 5 μ L, 10
× dNTP mix (Thermo) 1 μ L, each 2 μ L of primer, 36 μ L of ultrapure water.
The pcr amplification product for recycling above 4 segments is expanded using primer 0338a1/0338d2 and is mutated as template
Mbov_0338 gene afterwards, PCR reaction system are as follows: each 2.5 μ L, pfu enzyme (Thermo) of segment 1.5 μ L, pfu
buffer with MgSO4(Thermo) 5 μ L, 10 times of 1 μ L of dNTP mix (Thermo), each 2 μ L of primer, 36 μ L of ultrapure water.On
Primer is stated to be synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
The amplified production for recycling Mbov_0338 gene, with I digestion of Kpn I and EcoR, while by pET-30a plasmid (Fig. 1)
(being purchased from Novagen company) carries out double digestion with identical enzyme.By the Mbov_0338 gene and pET-30a plasmid use after digestion
DNA ligase (T4DNA ligase) connection, obtains recombinant plasmid pET-30a-Mbov_0338 (Fig. 2).By the recombinant plasmid
It after pET-30a-Mbov_0338 converts 5 α of Escherichia coli DH, is placed in 37 DEG C of shaking tables and is cultivated 12 hours at 180r/min, extract
Plasmid through sequencing it is correct after convert e. coli bl21 bacterium, by the Escherichia coli recombinant strain cultivated in LB liquid medium to
Take 1mL bacterium solution as control before induction when OD=0.6, while it is extremely final concentration of that isopropylthiogalactoside (IPTG) is added
0.8mM, 37 DEG C of shaking table inducing expression 3h.Take 1mL bacterium solution to be further processed: sample treatment is 12000r/min centrifugation
Supernatant is discarded after 1min, and 1mL phosphate buffer i.e. PBS (formula: KCl0.2g, NaCl 8g, Na is added2HPO41.44g
KH2PO40.24g, 1000mL distilled water, pH=7.6) solution be resuspended after, then with 12000r/min centrifugation 1min discard supernatant, so
Be added afterwards 30 μ L PBS and 30 μ L sample-loading buffers [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] it is resuspended.10min is boiled in 100 DEG C of boiling water.With
Whether PAGE gel electroresis appraisal expresses.
The nucleotide sequence of albumen of the invention is transformed into large intestine by construction recombination plasmid pET-30a-Mbov_0338
Escherichia coli recombinant strain is obtained in bacillus DH5 α, which is named as Escherichia coli pET-30a- by applicant
Mbov_0338(Escherichia coli pET-30a-Mbov_0338)；Wuhan City, Hubei Province is delivered on April 2nd, 2019
China typical culture collection center (CCTCC) preservation of Wuhan University, deposit number are CCTCC NO:M2019226.
The purifying of 1.2rADH albumen
After the e. coli bl21 of recombination according to the above method inducing expression, abandoned after taking bacterium solution 8000r/min to be centrifuged 10min
Fall supernatant, with PBS centrifuge washing 2 times of 500mL, 8000r/min is centrifuged 10min/ times.Discard the PBS that 30mL is added after supernatant
It is resuspended, is added protease inhibitors (being purchased from Roche Holding Ag), it is broken using hydraulic crushing instrument.It is centrifuged after broken with 12000r/min
30min takes 30 μ L supernatants that 30 μ L sample-loading buffers are added, 10min is boiled in boiling water as supernatant group.A little precipitating is taken to be added
The PBS of 30 μ L and 30 μ L sample-loading buffers boil 10min and make precipitating group.After PAGE gel electrophoresis, rADH egg is determined
White major part is expressed in supernatant.
The specific purification step of rADH albumen is as follows:
(1) it is (public purchased from GE that 1mL Ni-NTA metal-chelating His protein purification medium filler is added into affinity column
Department)；
(2) 12mL ddH is added into affinity column2O washing；
(3) binding buffer (the 20mM Na of 12mL is added3PO4, 0.5M NaCl, 20mM imidazoles, pH=7.4) and flat
Weigh pillar；
(4) the protein expression supernatant by the filtering of 0.45 μm of aperture filter is added；
(5) 50mL binding buffer buffer is added and balances pillar；
(6) 50mL washing buffer buffer (20mM Na is added3PO4, 0.5M NaCl, 60mM imidazoles, pH=
7.4) foreign protein is washed away；
(7) 12mL elute buffer buffer (20mM Na is added3PO4, 0.5M NaCl, 1M imidazoles, pH=7.4) and it washes
De- destination protein, collects former drops, number 1；
(8) 50 μ L sample-loading buffers are added in the pipe for being 1 by number and boil 10min；
(9) SDS-PAGE polyacrylamide gel is configured, by (the 20 every hole μ L/), electrophoresis in the sample adding hole handled well
(it is 80 volts that concentration gel electrophoresis condition, which is DC voltage, and separation gel deposition condition is that DC voltage is 120 volts), electrophoresis is completed
Afterwards, gel coomassie brilliant blue staining is removed to stay overnight.Then it decolourizes, determines the destination protein (Fig. 3) purified.
The alcohol dehydrogenase activity of embodiment 2:rADH albumen is tested
Use alcohol dehydrogenase activity assay kit (Alcohol Dehydrogenase Activity Assay
Kit, Sigma) detection, it is operated by product description, is summarized as follows: 22.5ng rADH is added in 96 orifice plates, then added again
Enter ethyl alcohol, NAD+, alcohol dehydrogenase detects liquid and developing solution, after being sufficiently mixed, is incubated for 3min at 37 DEG C.Then it is examined with enzyme mark
Instrument is surveyed under 37 DEG C, 450nm wavelength, measures an absorbance every 5min.Testing result confirms that rADH albumen can be by substrate
Alcohol catalysis forms acetaldehyde, while NAD+ is reduced to NADH, and the amount that NADH is generated is proportional to substrate light absorption value, at this moment
Absorbance under 450nm wavelength extends with the enzymatic time and increases (Fig. 4).
The how anti-preparation of embodiment 3:rADH albumen
By the rADH protein immunization male Japan large ear rabbit of purifying, the amount of being immunized is about 1mg/, is calculated according to immune amount
The volume of recombination Mbov_338 albumen used, and it is complete with isometric Freund's complete adjuvant mixing and emulsifying, subcutaneous multi-point injection into
Row is immune, be immunized every two weeks later it is primary, from second it is immune when use incomplete Freund's adjuvant instead and emulsified, and in from third
Secondary immune ear edge vein exploitating blood after a week carries out indirect ELISA detection white rabbit and generates antibody level, and general immune 4-5 times i.e. reachable
To required potency, when white rabbit antibody level no longer increases, it is mostly anti-that Culling heart blood purifying can be carried out.The result shows that rADH egg
The white antibody titer that can produce is 211× 100, i.e., 2.0 × 103(Fig. 5).
The reactionogenicity of embodiment 4:rADH albumen is analyzed
The identification of rADH albumen reactionogenicity, key step are as follows: by the recombination of purifying are carried out using Western blot method
Albumen rADH (2.0 μ g/ swimming lane) carries out SDS-PAGE electrophoresis, and albumen on glue is transferred on pvdf membrane, after film washing, uses
HT-Blot Western Blot couveuse by pvdf membrane respectively with natural infection and experimental infection Mycoplasma bovis positive serum (
1:100 dilutes in TBST) incubation at room temperature 1h, after washing, film rabbit-anti ox IgG-HRP secondary antibody (1:3000 dilutes in TBST) room
Temperature is incubated for 1h, and TBST is washed three times；After film is acted on 2~5min with Bio-rad chemiluminescent substrate, in chemiluminescence detector
Upper detection signal.As the result is shown: rADH albumen can react with Mycoplasma bovis positive cow's serum, and specific reaction can be detected
Band, molecular weight are about 44kDa (Fig. 6, swimming lane 1-8), meanwhile, which cannot react with negative cow's serum, not detect
To signal (Fig. 6, swimming lane 9), therefore, rADH albumen has good reactionogenicity.
The immunogenicity of embodiment 5:rADH albumen
Using the immunogenicity of Western Blot method detection rADH albumen, Mycoplasma bovis whole bacterial protein is transferred to PVDF
After film, after being incubated for anti-rMbov_338 albumen rabbit anteserum, TBST wash three times, by film and goat anti-rabbit igg-HRP antibody (1:
5000) it is incubated for 1h at room temperature；TBST is washed three times；After film is acted on 2-5min with Bio-rad chemiluminescent substrate, sent out in chemistry
Signal is detected on optical detector.As the result is shown: being able to detect that apparent immunoblotting band, protein size and Mycoplasma bovis
ADH albumen theoretical molecular weight is identical, shows that rADH albumen has immunogenicity, infected animal can be caused to generate specific antibody
(Fig. 7).
Embodiment 6:rADH albumen detects the Adhering capacity of host cell
6.1 stick detection
Indirect immunofluorescence combination laser confocal scanning microscope detection rADH albumen sticks with EBL cell.By EBL
After cell cultivates 24~36h in 24 orifice plates, after being fixed with 4% paraformaldehyde, 200 μ L rADH albumen (0.5 μ g/ μ L) is added
Enter in cell culture well, with EBL cell (1 × 105/ hole) in 4 DEG C of incubation 1h, after washing, sealed at room temperature with 1%BSA-PBS
Close cell 1h；Then, cell and anti-rADH rabbit anteserum (1:300) are incubated at room temperature 1h.Sufficiently after washing, by cell and donkey
488 antibody of anti-rabbit IgG-Alexa (1:400) is incubated at room temperature 1h；Cell membrane and nucleus then use CellMask respectively
Deep Red and DAPI dyeing liquor dyes red (film) and (core) blue.Finally, being detected under laser confocal scanning microscope
Fluorescence signal.The result shows that: rADH albumen is attached on EBL cell surface, detects green fluorescence (figure in EBL cell surface
8A), and under same experimental conditions, in the control group that EBL cell is only incubated for PBS, green fluorescence (Fig. 8 B) cannot be detected,
Confirm: rADH albumen is in conjunction with EBL cell-specific.
The detection of 6.2 Adherence inhibitions
Anti- rADH albumen rabbit anteserum inhibits Mycoplasma bovis to stick EBL cell experiment.EBL cell inoculation is in 24 hole cell culture
In plate, at 37 DEG C, 5%CO220h is cultivated in incubator.Before Inhibition test, culture medium is discarded, with 1%BSA-MEM 37
DEG C closing cell 15min.It in advance will about 1 × 108The Mycoplasma bovis of CFU and 56 DEG C of heat-inactivated rabbit-anti rADH hyper-immune serums and
Rabbit anteserum is in 4 DEG C of incubation 2h before immune.Then Mycoplasma bovis is added to together with the mixture of serum containing single layer EBL cell
(about 1 × 105A/hole) cultivation plate hole in, and in 37 DEG C of oscillation incubation 30min.After sufficiently washing four times, with 0.25% pancreas egg
Cell suspension is applied on PPLO agar plate by white enzymic digestion cell monolayer using serial dilution, cultivates microscope after 72h
Lower observation calculates the colony counts of the Mycoplasma bovis sticked, calculates the hole CFU/.The result shows that: compared with preimmune serum, rabbit-anti
RADH hyper-immune serum (1:20 dilution) significantly reduces the Mycoplasma bovis quantity (p < 0.01) (Fig. 9) being attached on EBL cell.
Embodiment 7:rADH albumen Adhesion fibronectin (Fn) detection
The detection of 7.1Dot-Blot method
Nitrocellulose filter is mounted on Bio-Dot millipore filter, with continuous twice of dilution rADH of PBS (from 1 μ g to
0.0625 μ g), each dilution is by every 100 μ L rADH point to nitrocellulose filter of hole.Then film is taken out from device,
And 4h is closed with the TBS solution containing 5% skimmed milk.It is after washing three times, film is molten in 1% skimmed milk-TBS with 10 μ g/mL ox Fn
4 DEG C of overnight incubations in liquid, then use rabbit-anti ox Fn antibody (1:1000 dilution) and goat antirabbit-HRP antibody (1:4000 dilution)
It is incubated at room temperature 1h.Chemiluminescent substrate process film is finally used, signal is detected on chemiluminescence detector.The result shows that: Fn with
RADH albumen combines (Figure 10 A) in dose dependent, and as rADH protein concentration increases, the signal of reaction is more and more stronger.
The detection of 7.2ELISA method
RADH albumen and BSA (negative control) are coated on 96 orifice plates, every hole 500ng is incubated overnight at 4 DEG C；It washes
After washing, with the PBS solution of 5% defatted milk in 37 DEG C of closing 2h；After washing, by the Fn of various concentration (0,3.125,6.25,
12.5,25,50,75,100 μ g/mL), to be incubated at room temperature 1.5h in every 100 μ L adding hole of hole；After washing, every hole is added
Rabbit-anti Fn antibody is incubated at room temperature 1h；After washing, anti-rabbit IgG-HRP antibody (1:6000 dilutes in PBST) room temperature is added in every hole
It is incubated for 1h；It is added after developing solution and reads light absorption value under 630nm wavelength with enzyme mark detector.The result shows that: Fn is with dose-dependant
Property and saturability mode and the rADH protein binding (Figure 10 B) that is coated in microwell plate.When Fn concentration is 0~5 hole g/ μ,
The amount for the Fn being integrated on microwell plate increases with the increase of Fn concentration；When Fn concentration is higher than 5 hole g/ μ, rADH recombinates egg
It is white in conjunction with Fn quantity no longer increase with the increase of Fn concentration, in conjunction with reach saturation.On the contrary, Fn and BSA are with low-level, no
The mode of saturation combines, and shows that the combination between Fn and BSA is a kind of non-specific interaction, and the combination of Fn and rADH is
One species specific combination.
Annex: vocabulary of terms illustrates in specification:
Mycoplasma bovis ADH protein gene is indicated with Mbov_0338.
Mycoplasma bovis ADH recombinant protein is indicated with rADH.
Mycoplasma bovis local separation strains are indicated with Mycoplasma bovis HB0801.
Ox fibronectin is indicated with Fn.
Sequence table
<110>Hua Zhong Agriculture University
<120>Mycoplasma bovis alcohol dehydrogenase gene and its coding albumen and application
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<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> RNA
<213>Mycoplasma bovis (Mycoplasma bovis)
<400> 2
<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 (10)
1. a kind of Mycoplasma bovis alcohol dehydrogenase gene has the nucleotide sequence as shown in SEQ ID NO:1.
2. the albumen of the coding of Mycoplasma bovis alcohol dehydrogenase gene described in claim 1, has the ammonia as shown in SEQ ID NO:2
Base acid sequence.
3. the recombinant plasmid containing Mycoplasma bovis alcohol dehydrogenase gene described in claim 1.
4. recombinant plasmid as claimed in claim 3, it is passed through by pET-30a plasmid and full length gene described in claim 1
It crosses after digestion with restriction enzyme to connect and recombinate.
5. the recombinant bacterium containing albumen described in claim 2.
6. recombinant bacterium as claimed in claim 5 is named as Escherichia coli pET-30a-Mbov_0338 (Escherichia
Coli pET-30a-Mbov_0338), it is preserved in China typical culture collection center, deposit number is CCTCC NO:
M2019226。
7. the antibody of albumen described in anti-claim 2.
8. albumen as claimed in claim 2, which has alcohol dehydrogenase activity, has and sticks ox pulmonary epithelial cells and knot
The ability of ox fibronectin is closed, while also there is immunogenicity and reactionogenicity, is the new virulence correlation of one kind of Mycoplasma bovis
Albumen.
9. albumen as claimed in claim 2 is preparing the purposes in mycoplasma bovis vaccine.
10. albumen as claimed in claim 2 and antibody as claimed in claim 7 are in preparing Mycoplasma bovis diagnostic kit
Purposes.
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Cited By (2)
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| CN111635880A (en) * | 2020-05-06 | 2020-09-08 | 华中农业大学 | Mycoplasma bovis VspX gene mutant strain and construction method and application thereof |
| CN111856005A (en) * | 2020-06-22 | 2020-10-30 | 华中农业大学 | Application of mycoplasma bovis secretory protein MbovP280 |
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| CN111856005B (en) * | 2020-06-22 | 2021-07-30 | 华中农业大学 | Application of mycoplasma bovis secretory protein MbovP280 |
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