CN111235082B

CN111235082B - Application of protein encoded by mycoplasma bovis gene in adhering host cell

Info

Publication number: CN111235082B
Application number: CN202010077232.2A
Authority: CN
Inventors: 郭爱珍; 朱习芳; 董亚旗; 李茜茜; 陈颖钰; 胡长敏; 陈焕春
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-02-21
Filing date: 2019-02-21
Publication date: 2021-05-14
Anticipated expiration: 2039-02-21
Also published as: CN109837226B; CN111235082A; CN109837226A

Abstract

本发明属于动物传染病防治领域，涉及黏附能力降低的牛支原体基因缺失的突变株。蛋白基因Mbov_0503从牛支原体HB0801基因组中克隆获得。根据大肠杆菌对密码子的偏爱性，对Mbov_0503基因进行了修饰，将牛支原体色氨酸密码子UGA突变成大肠杆菌中编码色氨酸的密码子UGG，获得重组蛋白Mbov0503。本发明克隆的蛋白基因的序列如SEQ ID NO:13所示，编码的蛋白质序列如SEQ ID NO:14所示。本发明的突变株从突变体库筛选的黏附缺陷株。该突变株对宿主EBL细胞的黏附能力，对MDBK细胞的跨膜传播能力及对细胞间紧密连接的破坏能力较野生株显著下降。可在牛支原体致病和防控中应用。The invention belongs to the field of animal infectious disease prevention and treatment, and relates to a mutant strain of Mycoplasma bovis gene deletion with reduced adhesion ability. The protein gene Mbov_0503 was cloned from the genome of Mycoplasma bovis HB0801. According to the codon preference of Escherichia coli, the Mbov_0503 gene was modified, and the tryptophan codon UGA of Mycoplasma bovis was mutated into the codon UGG encoding tryptophan in Escherichia coli to obtain the recombinant protein Mbov0503. The sequence of the cloned protein gene of the present invention is shown in SEQ ID NO: 13, and the encoded protein sequence is shown in SEQ ID NO: 14. The mutant strain of the present invention is an adhesion-deficient strain screened from a mutant library. The ability of the mutant strain to adhere to host EBL cells, to transmit across the membrane of MDBK cells and to destroy the tight junction between cells was significantly lower than that of the wild strain. It can be used in the pathogenesis and prevention and control of Mycoplasma bovis.

Description

Application of protein encoded by mycoplasma bovis gene in adhering host cell

Technical Field

The invention is a divisional application with application number of 2019101283763 and application date of 2019, 2 and 21.

The invention belongs to the technical field of prevention and treatment of animal infectious diseases, and particularly relates to a mycoplasma bovis Mbov _0503 gene deletion mutant strain T4.4 and a function-unknown protein Mbov0503 encoded by the gene. The mutation site is located behind the 586830 site of Mycoplasma bovis genome and behind the 313 site of the Mbov _0503 gene. Compared with wild strains, the mutant strain has obvious defect of adhesion capability to host cells, and obviously reduces the capability of damaging intercellular tight junctions and transmembrane transmission capability. The recombinant protein rMbov0503 can adhere to host cell and cell membrane proteins. The mutant strain and the protein are expected to have application prospects in the fields of mycoplasma bovis pathogenetic mechanism and immune prevention and control.

Background

Mycoplasma bovis (m.bovis) mainly causes bovine bronchopneumonia, and also causes various diseases such as mastitis, arthritis, abortion and the like, thereby seriously harming the cattle industry in the world. In europe, about one third of calf pneumonia is caused by mycoplasma bovis every year with losses of up to 5.76 billion euros; losses due to respiratory diseases and mastitis in cattle caused by mycoplasma bovis amount to $ 1.08 million per year in the united states (Rosengarten et al, 1999). Mycoplasma bovis pneumonia is reported for the first time by agriculture university in China 2008, and then the mycoplasma bovis pneumonia is found in most provinces and cities in China, the morbidity is more than 80%, the average fatality rate is 10%, and the highest fatality rate can reach 60% (Shilian et al, 2008). Meanwhile, the prevalence of bovine mastitis is very common (Zhang Hui et al, 2015), which causes great economic loss to cattle raising industry in China.

Adhesion is the first step of mycoplasma infection and pathogenesis, and is an important target for researching mycoplasma disease prevention and control measures. The research on the adhesion mechanism of mycoplasma bovis is still in the early stage, and the main adhesion protein and apical adhesion organelle similar to mycoplasma pneumoniae of human are not found so far. However, some adhesion-related proteins such as α -enolase (Song et al, 2012), VSPs (Sachse et al, 2000) and NADH Oxidase (Zhao et al, 2017) have been reported, but functional studies of these proteins are mostly based on recombinant proteins and have not been known to play a role in the actual adhesion process at mycoplasma bovis level. In order to understand the adhesion gene of Mycoplasma bovis, the applicant identified adhesion-deficient mutants using a library of Mycoplasma bovis mutants, cell adhesion and transfer chamber (transwell) transmembrane propagation tests, and the like. The invention relates to a cell adhesion ability deficient mutant, wherein a mutant gene is Mbov _ 0503; the gene codes a protein Mbov0503 with unknown function, and the protein is proved to be an adhesion related protein by the invention.

Disclosure of Invention

The invention aims to provide application of a protein coded by a mycoplasma bovis gene in adhering host cells, wherein the application is based on that an applicant obtains a mutant strain T4.4 of a mycoplasma bovis deleted gene Mbov-0503 with reduced adhesion capability. The protein Mbov0503 coded by the mycoplasma bovis deleted gene Mbov-0503 can be independently adhered to host cells, and the mutant strain T4.4 obtained by the invention has a remarkable adhesion defect phenotype compared with wild strains, and the damage capability to intercellular tight junction and transmembrane transmission capability are reduced compared with wild strains. Based on the fact that adhesion, damage to intercellular tight junction, transmembrane propagation capacity and the like are virulence related factors of mycoplasma infection and pathogenicity, the mutant strain is expected to have potential application prospects in the fields of mycoplasma bovis pathogenicity mechanism and immune control.

In order to realize the purpose of the invention, an adhesion-deficient strain T4.4 is screened from a mycoplasma bovis gene deletion mutant library by a ruminant pathogen division laboratory in the national emphasis laboratory of agricultural microbiology of university of Huazhong agriculture of the applicant. Further verification and determination show that the mutant strain has obviously reduced disruption capacity and transmembrane transmission capacity to intercellular tight junction compared with the wild strain. The mutant strain deletion gene is an unknown gene Mbov _0503, the N end of the mutant strain deletion gene is provided with a transmembrane structural domain, and the protein Mbov0503 coded by the gene has the capability of adhering host cells through further verification. The invention provides a theoretical basis for clarifying the pathogenic mechanism of mycoplasma bovis and provides a potential target for developing a specific prevention and control preparation.

In order to achieve the purpose, the invention adopts the following technical scheme:

the applicant obtains a Mycoplasma bovis local isolate HB0801 separated from a cattle lung tissue of Hubei province corresponding to a certain cattle farm in city in 6 months in 2008, is named as Mycoplasma bovis HB0801 and Mycoplasma bovis HB0801, and is delivered to China center for type culture collection of university of Wuhan, China at 2 months and 1 day 2010, with the collection number of CCTCC NO: and M2010040. Further, a random mutant library of Mycoplasma bovis HB0801 was constructed using the pMT85 transposon vector. On the basis, firstly, a cell model is utilized to screen mutant strains with adhesion defects from a mutant library to obtain mutant strains T4.4 and Mycoplasma bovis T4.4 with the most obvious adhesion defect phenotype, the mutant strains are delivered to the China center for type culture Collection of university of Wuhan and Wuhan in 2018, 8 and 31 months, and the preservation number is CCTCC NO: and M2018581. The mutant strain was identified as an Mbov _ 0503-deficient strain by identifying the mutant gene. Further, the gene was cloned using the genome of Mycoplasma bovis HB0801 (accession number CP002058 on GenBank) as a template. In the cloning process, gene modification is carried out according to the codon preference of escherichia coli, namely, the tryptophan codon UGA of the mycoplasma bovis is mutated into the tryptophan codon UGG in the escherichia coli, and mutation of 5 codons is carried out in total so as to ensure the expression of the tryptophan codon UGG in the escherichia coli.

The nucleotide sequence of the mycoplasma bovis gene Mbov _0503 subjected to artificial mutation is shown in a sequence table SEQ ID NO:13, between 1 and 1509bp in length; wherein allelic mutations occur at positions 354, 549, 810, 1023 and 1050 of the sequence;

the protein sequence of the Mycoplasma bovis Mbov0503 protein gene code is shown in a sequence table SEQ ID NO:14, and a total of 502 amino acid residues.

The nucleotide sequence of the mycoplasma bovis Mbov _0503 gene is transformed into Escherichia coli DH5 alpha by constructing a plasmid vector pET-30a-Mbov _0503 to obtain a recombinant Escherichia coli strain, the applicant names the recombinant Escherichia coli strain as Escherichia coli pET-30a-Mbov _0503 and Escherichia coli pET-30a-Mbov _0503, and the recombinant Escherichia coli strain is delivered to China university Wuhan university culture collection for preservation in 2018, 9 and 12 months, wherein the preservation number is CCTCC NO: m2018614. The strain expresses recombinant protein rMbov0503 coded by the Mbov _0503 gene under IPTG induction.

Through verification, the purified recombinant protein rMbov0503 has an adhesion function on both host cells and cell membranes.

The mycoplasma bovis mutant strain disclosed by the invention has obviously reduced adhesion capacity to fetal bovine lung cells (EBL), transmembrane transmission capacity to bovine kidney cells (MDBK) and intercellular tight junction destruction capacity, and the related detection method comprises the following steps:

and (3) detecting the adhesion capability: the adhesive capacity of mycoplasma bovis HB0801 and T4.4 is detected by a colony counting method, a chemical staining method and an ELISA method. Respectively culturing HB0801 and T4.4 strains to late log stage, counting, simultaneously spreading EBL cells on a 24-hole cell culture plate for overnight culture to form a monolayer cell layer, adding the treated mycoplasma bovis into adherent cells at an infection ratio of 1000, reacting for 30min, washing the non-adhered mycoplasma bovis, and using precooled ddH₂O cells were split and mycoplasma adhered to EBL cells were released for colony counting. Extracting EBL cell membrane protein, coating the membrane protein on an ELISA plate, incubating the Mbov0503 protein and the cell membrane protein, detecting the adhesion of target protein to the membrane protein by using an ELISA method, dyeing the mycoplasma bovis/Mbov 0503 protein and the EBL cytoskeleton by using chemical dye, and observing and determining that the mycoplasma bovis mutant strain/Mbov 0503 protein has the adhesion capacity to the EBL cells by using a fluorescence microscope.

Transmembrane transmission and intercellular tight junction disruption: the transmembrane propagation ability and the ability to disrupt intercellular tight junctions of Mycoplasma bovis HB0801 and T4.4 were examined by colony counting and chemical staining using the Transwell model. Respectively culturing HB0801 and T4.4 strains to the late logarithmic phase and counting, simultaneously paving MDBK cells (stored by the experiment) in a Transwell chamber, culturing for 3 days to form tight connection, adding the treated mycoplasma bovis into the chamber with the infection ratio of 1000, simultaneously setting an HB0801 infected group as a positive control and an uninfected group as a blank control, taking a proper amount of Transwell lower chamber culture medium at different reaction times to count bacterial colonies, determining that the penetrating capacity of the mycoplasma bovis mutant strain to the MDBK is remarkably reduced, staining a Transwell chamber membrane, and observing by using a fluorescence microscope to determine that the destroying capacity of the mycoplasma bovis mutant strain to the tight connection is reduced.

The invention has the following advantages:

1. the T4.4 strain is a mutant strain which is obtained by screening mycoplasma bovis gene deletion mutant libraries by the inventor and has remarkably reduced adhesion capacity.

2. In the T4.4 strain of the invention, the gene mutation site is located behind 586830 sites of the mycoplasma bovis genome and behind 313 sites of the Mbov _0503 gene.

3. The Mycoplasma bovis Mbov _0503 gene is an adhesion related gene obtained by screening a Mycoplasma bovis gene deletion mutant library by the inventor, and is artificially modified according to the codon preference of escherichia coli. And successfully expresses the recombinant protein, and proves that the recombinant protein rMbov0503 has the adhesion capability.

4 the T4.4 strain of the present invention has been confirmed by the inventors to have significantly reduced ability to disrupt intercellular tight junctions and transmembrane transmission compared to the wild-type strain.

The more detailed scheme of the invention is described in the detailed description.

Drawings

FIG. 1: the invention relates to a quantitative detection analysis chart of mycoplasma bovis adhesion-deficient strains. The mutant strain T4.4 with the most pronounced adhesion deficiency is shown in boxes.

FIG. 2: plasmid map of the empty plasmid pET-30a in the examples of the present invention. pET-30a is a commercial plasmid, purchased from Novagen.

FIG. 3: the map of the recombinant plasmid pET-30a-Mbov _0508 prepared by the invention. The recombinant plasmid pET-30a-Mbov _0503 is formed by connecting and recombining the pET-30a plasmid and the full length (except a transmembrane region) of the Mbov _0503 gene after mutation by restriction enzyme digestion.

FIG. 4: the protein glue map of the purified mycoplasma bovis rMbov 0503. Description of reference numerals: lane M: protein marker; 1: purified mycoplasma bovis rMbov0503 recombinant proteins.

FIG. 5: the adhesion capability of the mycoplasma bovis Mbov0503 recombinant protein to EBL cells and membrane proteins is detected. Description of reference numerals: a: adhesion of Mbov0503 to EBL cells, B: adhesion of negative serum pre-hatched Mbov0503 to EBL cells, C: adhesion of multiple resistant incubated Mbov0503 to EBL cells, D: and (5) negative control. E: adhesion of Mbov0503 to EBL cell membrane proteins; f: the adhesion inhibition of Mbov0503 to EBL cell membrane proteins (p <0.05, p <0.01, ns represents differences from the BSA group, significant differences, very significant differences, no differences, respectively).

FIG. 6: the invention discloses a mycoplasma bovis growth curve detection map.

FIG. 7: the mycoplasma bovis T4.4 disclosed by the invention is used for detecting the adhesion capacity of EBL cells. Description of reference numerals: a: confocal detection of adhesion of mycoplasma bovis to EBL cells, green fluorescence is that mycoplasma bovis monoclonal antibodies and enzyme-labeled secondary antibodies Alexa-488 mark mycoplasma bovis wild strains and T4.4, red fluorescence is that rhodamine phalloidin marks EBL cytoskeleton, blue fluorescence is that DAPI marks EBL cell nucleus, and PBS is used as negative control; b: adhesion of mycoplasma bovis to EBL cells at different temperatures.

FIG. 8: the mycoplasma bovis provided by the invention is used for detecting the transmembrane propagation capacity and the capability of destroying intercellular tight junctions. A: tranwell transmembrane propagation count results; b: the ability of Mycoplasma bovis to disrupt tight junctions of cells was measured confocal, using anti-ZO-1 antibody from murine origin as the primary antibody, the tight junction protein ZO-1 was labeled with the enzyme-labeled secondary antibody IgG-Alexa 488 (green), and the MDBK nuclei were stained with DAPI (blue). The HB0801 infected group was a positive control, and the NC group was a blank control.

Detailed Description

Description of sequence listing:

sequence listing SEQ ID NO:1 is the sequence of forward primer 0503a1 amplifying the Mbov _0503 gene fragment.

Sequence listing SEQ ID NO: 2 is the sequence of reverse primer 0503a2 amplifying the Mbov _0503 gene fragment.

Sequence listing SEQ ID NO: 3 is the sequence of forward primer 0503b1 amplifying the Mbov _0503 gene fragment.

Sequence listing SEQ ID NO: reference numeral 4 denotes the sequence of the reverse primer 0503b2 for amplifying the Mbov _0503 gene fragment.

Sequence listing SEQ ID NO: reference numeral 5 denotes the sequence of forward primer 0503c1 for amplifying the Mbov _0503 gene fragment.

Sequence listing SEQ ID NO: reference numeral 6 denotes the sequence of reverse primer 0503c2 for amplifying the Mbov _0503 gene fragment.

Sequence listing SEQ ID NO: reference numeral 7 denotes the sequence of forward primer 0503d1 for amplifying the Mbov _0503 gene fragment.

Sequence listing SEQ ID NO: reference numeral 8 denotes the sequence of primer 0503d2 for amplifying the Mbov _0503 gene fragment.

Sequence listing SEQ ID NO: reference numeral 9 denotes the sequence of primer 0503e1 for amplifying the Mbov _0503 gene fragment.

Sequence listing SEQ ID NO: reference numeral 10 denotes the sequence of the reverse primer 0503e2 for amplifying the Mbov _0503 gene fragment.

Sequence listing SEQ ID NO: reference numeral 11 denotes the sequence of forward primer 0503f1 for amplifying the Mbov _0503 gene fragment.

Sequence listing SEQ ID NO: reference numeral 12 denotes the sequence of reverse primer 0503f2 for amplifying the Mbov _0503 gene fragment.

Sequence listing SEQ ID NO:13 is the nucleotide sequence of the artificial mutant mycoplasma bovis Mbov _0503 gene of the invention, and the sequence length is 1509 bp. Wherein: allelic mutations occur at positions 354, 549, 810, 1023 and 1050 of the sequence.

Sequence listing SEQ ID NO:14 is a protein sequence encoded by the Mycoplasma bovis Mbov0503 gene of the present invention.

Example 1: screening and identification of mycoplasma bovis adhesion-deficient mutants

1. Primary screening of mycoplasma bovis adhesion-defective mutants

(1) Culturing and counting the mycoplasma bovis mutant strain: the mycoplasma bovis mutant library is constructed by the focus laboratory ruminant pathogen of agricultural microbiology country of Huazhong agriculture university of China where the applicant is located, is stored at-80 ℃, and mycoplasma bovis mutants with different ORFs inserted into transposons of 197 strains of transposons are preliminarily screened in the test, wherein the ratio of 1: 1000 (wt.) inoculation liquid medium (PPLO powder 10.5g, sodium pyruvate powder 0.5g, yeast 2.5g, add 440mL ddH₂Fixing O volume, sterilizing with high temperature steam at 121 deg.C for 18min, adding 10% horse serum 50mL, 10 × MEM 5mL, 40 ten thousand units/mL penicillin solution 1mL, phenol red growth indicator 500 μ L), recovering, standing at 37 deg.C and 5% CO₂Culturing for 36h in an incubator, namely after logarithmic phaseAnd then the ratio of 1: inoculating 1000 of the extract into the above liquid culture medium, culturing to late log stage, counting CFU, diluting the cultured bacterial solution by 10 times, spreading 10 μ L of the diluted bacterial solution on PPLO solid culture medium (500mL PPLO liquid culture medium with agar 7.5g), inverting at 37 deg.C, and adding 5% CO₂After 3-7 days of culture in the incubator, counting colonies under a stereoscopic microscope, wherein the colony count formula is as follows: CFU/mL-colony count × dilution × 100, and the counted bacterial suspension was used as the infectious bacterial suspension for each test. Mycoplasma pellets were collected by centrifugation at 12000rpm before the assay and resuspended in an equal volume of cell culture medium.

(2) Culture and enumeration of RAW264.7 cells: mouse peritoneal macrophage cell line RAW264.7(ATCC TIB-71) in DMEM complete medium containing 10% FBS (Hyclone) 5% CO₂Culturing in a 37 ℃ cell culture box, hanging adherent cells gently by using a spatula when the cells grow to 80% full monolayer cells, blowing and beating the cells by using a DMEM complete culture medium with proper volume to prepare cell suspension, and counting the cell suspension by using a blood counting plate, wherein the counting method is briefly as follows: taking a proper amount of the cell suspension which is evenly resuspended and slowly dripping the cell suspension into a blood counting plate along the edge of the cover plate to ensure that the suspension is filled under the cover plate, counting the cells in 5 lattices at the periphery and in the middle of the blood counting plate under a high power microscope, wherein the cell number/mL is (the cell number of 5 lattices/20) multiplied by the dilution multiple multiplied by 10⁶。

(3) Primary screening of mycoplasma bovis mutant with altered cell adhesion ability to mouse macrophage (RAW264.7, stored in this laboratory): RAW264.7 cells were plated at 1X 10 per well⁵Inoculating in 24-well plates at 37 ℃ with 5% CO₂Culturing overnight under the conditions to allow cell growth adherent to the wall, and adding Phosphate Buffer Solution (PBS) (formula: KCl 0.2g, NaCl 8g, Na) at infection ratio of 1000 after microscopic examination of good cell state₂HPO₄ 1.44g，KH₂PO₄0.24g, 1000mL distilled water, pH 7.6), while setting Mycoplasma bovis wild strain HB0801 as positive control and cell complete medium as negative control, at 37 deg.C and 5% CO₂Adhering for 1h in an incubator, and then usingThe unsticked Mycoplasma bovis was washed 3-4 times with sterile internally opened PBS, and then with precooled internally opened ddH₂And O, treating the cells to enable the cells to expand and crack to release the adhered strains, repeatedly blowing by using a liquid transfer gun to enable the adhered mycoplasma bovis to be uniformly distributed, and then counting the CFU. 19 adhesion-deficient mutants were initially selected.

2. Rescreening bovine mycoplasma mutant strains with altered adhesive capacity by fetal bovine lung cells

To further determine the adhesion ability of the primary mutant strain to the host cells, 19 M.bovis mutant strains obtained by primary screening were rescreened with fetal bovine lung cells (EBL), and EBL cells were plated in 24-well plates at 1X 10/well⁵Cell, 5% CO at 37 ℃₂Culturing for 12h under the condition to ensure that the cells grow in an adherent way, and then culturing according to an infection ratio of 1: 1000, respectively adding a mycoplasma bovis wild strain and the mutant strain prepared by the invention, acting for 1h at 37 ℃, culturing and counting colonies of the mycoplasma bovis adhered to the EBL cells, wherein the counting result shows that: the T4.4 mutant strain had the most significant decrease in adhesion (fig. 1).

Example 2: expression of Mycoplasma bovis Mbov0503 protein

1. Cloning and expression of Mycoplasma bovis Mbov _0503 Gene

In the present invention, the codon UGA encoding tryptophan in the Mycoplasma bovis genome is used as a terminator in E.coli due to the preference of E.coli for the codon, and therefore, when the Mycoplasma bovis gene is expressed using E.coli, it is necessary to mutate the codon UGA to the codon UGG capable of expressing tryptophan in E.coli. In order to express the Mycoplasma bovis Mbov _0503 gene, the applicant carries out mutation of corresponding 5 codons on the gene by using a self-designed PCR primer, and the specific steps are as follows: the Mbov _0503 gene of Mycoplasma bovis HB0801 (genome GenBank accession number CP002058) was used as a template, 6 pairs of primers (0503 a1/0503a2, 0503b1/0503b2, 0503c1/0503c2, 0503d1/0503d2, 0503e1/0503e2, 0503f1/0503f2) designed as follows were used to amplify 6 fragments of the mutated Mbov _0503 gene, and then the mutated Mbov _0503 gene was amplified in its full nucleotide sequence of 1509bp (see the sequence shown in the sequence No. 1-1509 in SEQ ID NO:13, and the coding region thereof also had a sequence corresponding to the 1-1509 base) using the 0503a1/0503f2 primers designed as a template.

The 6 primer sequences used for amplification of the Mbov _0503 whole gene are shown below:

(1) the primer 0503a1/0503a2, the position of the amplified fragment in the Mbov _0503 gene is 586656 and 587028 bases, and the length of the PCR amplified product is 373 bp. The sequences of the specific primer pairs are as follows:

1) forward primer 0503a 1: 5'

CGCGGATCCTTCAAAATTAATTTAGAAAAGAAAAATGTAATTAG 3' (corresponding to the sequence shown in SEQ ID NO:1 of the sequence Listing).

2) Reverse primer 0503a 2: 5 'TATAGTTAGGCGTAAAGCTCCAGTATATA 3' (corresponding to the sequence shown in SEQ ID NO: 2 of the sequence Listing; the underlined part is a mutation site, i.e., from T to C)

(2) The position of the 0503b1/0503b2 amplified fragment in the Mbov _0503 gene is 587014-587220 bp, the length of the PCR amplified product is 207bp, and the sequence of the specific primer pair is as follows:

1) forward primer 0503b 1:5 'TTACGCCTAACTATAATGAAAAAAATATG 3' (corresponding to the sequence shown in SEQ ID NO: 3 of the sequence Listing).

2) Reverse primer 0503b 2: 5' CATCTTTAAAATAATACCAGCCTGGGT 3' (the underlined part is the mutation site, i.e.from T to C; corresponding to the sequence shown in SEQ ID NO: 4 of the sequence Listing).

(3) The position of the 0503c1/0503c2 amplified fragment in the Mbov _0503 gene is 587212-587479 bp, the length of the PCR amplified product is 268bp, and the sequence of the specific primer pair is as follows:

1) forward primer 0503c 1: 5' TTAAAGATGGCATTTTACACTTAATAATTGG3' (corresponding to the sequence shown in SEQ ID NO: 5 of the sequence Listing).

2) Reverse primer 0503c 2: 5' TTACTGTCTAAAAACCACTGATAATACTTTG 3' (the underlined part is the mutation site, i.e.from T to C; corresponding to the sequence shown in SEQ ID NO: 6).

(4) The position of the 0503d1/0503d2 amplified fragment in the Mbov _0503 gene is 587468-587695 bp, the length of the PCR amplified product is 228bp, and the sequence of the specific primer pair is as follows:

1) forward primer 0503d 1:5 'TTTAGACAGTAATGAGCATAACGAACAC 3' (corresponding to the sequence shown in SEQ ID NO: 7 of the sequence Listing).

2) Reverse primer 0503d 2: 5' TTATATAAATATCTAAACCAAGGGACATTT 3' (the underlined part is the mutation site, i.e.from T to C; corresponding to the sequence shown in SEQ ID NO: 8 of the sequence Listing).

(5) The position of the 0503e1/0503e2 amplified fragment in the Mbov _0503 gene is 587682-587727 basic groups, the length of the PCR amplification product is 46bp, and the sequence of the specific primer pair is as follows:

1) forward primer 0503e 1: 5' AGATATTTATATAATAATTTATGGACCGAAAATT 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 0503e 2: 5' CAGTTGAAAGTAAATTTTCGGTCCATAAATTATT 3' (corresponding to the sequence shown in SEQ ID NO: 10 of the sequence Listing).

(6) The position of the 0503f1/0503f2 amplified fragment in the Mbov _0503 gene is 587718-588164 bp, the length of the PCR amplified product is 447bp, and the sequence of the specific primer pair is as follows:

1) forward primer 0503f 1:5 'CTTTCAACTGAAGTAAACAGTGATGATT 3' (corresponding to the sequence shown in SEQ ID NO: 11 of the sequence Listing).

2) Reverse primer 0503f 2: 5 'CCGCTCGAGTTATACATTATAAAAATATTTTATTTTAATCTTTGTACA 3' (corresponding to the sequence shown in SEQ ID NO: 12 of the sequence Listing).

(7) The position of the 0503a1/0503f2 amplified fragment in the Mbov _0503 gene is 586656-588164 bases, the length of the PCR amplified product is 1509bp, and the sequence of the specific primer pair is as follows:

1) forward primer 0503a 1: 5'CGCGGATCCTTCAAAATTAATTTAGAAAAGAAAAATGTAATTAG 3' (underlined part is the BamH1 restriction site, wavy line part is a protective base; corresponding to the sequence shown in SEQ ID NO:1 of the sequence Listing).

2) Reverse primer 0503f 2: 5' CCGCTCGAGTTATACATTATAAAAATATTTTATTTTTAATCTTTGTACA 3' (the underlined part is an XhoI cleavage site and the wavy line part is a protective base; corresponding to the sequence shown in SEQ ID NO: 12 of the sequence Listing).

The PCR reaction system for the above 6 fragments was as follows:

mu.L of template DNA, 1.5. mu.L of pfu enzyme (Thermo), 5. mu.L of pfu buffer with MgSO4(Thermo), 1. mu.L of 10-fold dNTPmix (Thermo), 2. mu.L of each primer, and 36. mu.L of ultrapure water.

The PCR amplification products of the above six fragments were recovered, and the mutated Mbov _0503 gene was amplified using primer 0503a1/0503f2 as a template, and the PCR reaction system was as follows: 2.5. mu.L of each fragment, 1.5. mu.L of pfu enzyme (Thermo), pfu buffer with MgSO 2₄(Thermo) 5. mu.L, 1. mu.L of 10-fold dNTP mix (Thermo), 2. mu.L of each primer, and 23.5. mu.L of ultrapure water. The above primers were synthesized by Tianyihui Biotech, Inc.

The amplification product of the Mbov _0503 gene was recovered and digested with BamH I and XhoI, while the pET-30a plasmid (FIG. 2) (purchased from Merck China, Inc.) was digested with BamH I and XhoI in two. The digested Mbov _0503 gene and pET-30a plasmid were ligated with DNA ligase (T4 DNA ligase) to obtain a recombinant plasmid pET-30a-Mbov _0503 (FIG. 3). Transforming Escherichia coli DH5 alpha with the recombinant plasmid pET-30a-Mbov _0503, culturing for 12 hours at 37 ℃ in a shaker at 180r/min, extracting the plasmid, correctly sequencing, transforming Escherichia coli BL21, culturing the Escherichia coli in LB liquid culture medium until OD is 0.6, taking 1mL of bacterial liquid as a control before induction, simultaneously adding isopropyl thiogalactoside (IPTG) until the final concentration is 0.8mM, and performing shake induction expression for 3 hours at 37 ℃. 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: KCl 0.2g, NaCl 8g, Na)₂HPO₄1.44g，KH₂PO₄0.24g, 1000mL of distilled water, pH 7.6) solution was resuspended and centrifuged at 12000r/min for 1min to discard the supernatant, then 30uL of PBS and 30uL of loading buffer [ 1M Tris-HCl (pH 6.8)1mL, 200mM DDT 0.31g, 4% SDS 0 were added.4g, 0.2% bromophenol blue 0.02g, 20% glycerol 2mL, 7mL ultrapure water ] weight suspension. Boiling in boiling water at 100 deg.C for 10 min. SDS-PAGE gel electrophoresis was used to determine whether expression was observed.

The nucleotide sequence of the protein is transformed into Escherichia coli DH5 alpha by constructing a plasmid vector pET-30a-Mbov _0503 to obtain recombinant Escherichia coli, and the applicant names the recombinant Escherichia coli as Escherichia coli pET-30a-Mbov _0503 and Escherichia coli pET-30a-Mbov _0503, and the recombinant Escherichia coli is delivered to China at 9 and 12 months in 2018, Wuhan university China center for type culture Collection with the preservation number of CCTCC NO: and M2018614.

2. Expression and purification of recombinant proteins of mycoplasma bovis rMbov0503

After the recombinant Escherichia coli BL21 is induced and expressed according to the method, taking 8000r/min of bacterial liquid, centrifuging for 10min, discarding the supernatant, washing once with 500mL PBS, centrifuging for 10min at 8000r/min, and washing once with 500mL PBS again. 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, rMbov0503 protein is mostly expressed in the supernatant.

The rMbov0503 protein is specifically purified by the following steps:

(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 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 50mL binding buffer solution to balance the column, and collecting the first drops of liquid, wherein the serial number is 2;

(6) adding50mL washing buffer (20mM Na)₃PO₄0.5M NaCl, 60mM imidazole, pH 7.4) to wash away the contaminating proteins, and collect the first few drops, numbered 3;

(7) add 12mL of elute buffer (20mM Na)₃PO₄0.5M NaCl, 1M imidazole, pH 7.4) eluting the target protein, collecting the first few drops, numbered 4;

(8) adding 50 μ L of loading buffer solution into each tube numbered 1-4, 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 in the case of gel-concentrate electrophoresis and 120V DC in the case of gel-separate electrophoresis), and after electrophoresis was complete, the gels were removed and stained with Coomassie Brilliant blue overnight. Then decolorized to confirm that the purified target protein is obtained (FIG. 4)

Example 3: detection of adhesion capability of rMbov0503 of recombinant protein

1. Detection of EBL cell adhesion by recombinant protein rMbov0503

(1) Spread 1X 10⁵EBL cells in 24-well plates in 37 ℃ 5% CO₂Carrying out cell adherent culture under the condition;

(2) mu.g of Mbov0503 was interacted with EBL cells for 1h in a reaction volume of 200. mu.L to allow sufficient contact of the protein with the cells, and the following controls were set: a blank with MEM minimal medium (Hyclone) alone; rMbov0503 was incubated with polyclonal antibody against Mbov0503 for 1h at 37 ℃; rMbov0503 was incubated with mouse negative serum for 1h at 37 ℃;

(3) and (3) sealing: washing the protein which is not adhered with the internally opened PBS for 5 times, adding PBS for washing along different directions each time, adding 5% skimmed milk, sealing for 2h at room temperature, and washing for 3 times with PBS;

(4) fixing and permeabilizing: adding 1mL of commercial 4% paraformaldehyde into each well, fixing at room temperature for 30min, washing with PBS buffer solution for 3 times, adding 10% of TX-100, and performing permeabilization at room temperature for 5 min;

(5) primary antibody incubation: antiserum of murine antibody (prepared in laboratory) rMbov0503 is diluted at a ratio of 1:500, added into each well at a rate of 200. mu.L/well, reacted for 1h at room temperature, and washed 3 times with PBS;

(6) and (3) secondary antibody incubation: rMbov0503 was labeled with goat anti-mouse 488(ThermoFisher, Green fluorescent) secondary antibody, incubated for 45min at room temperature, and washed 3 times with PBS;

(7) marking EBL cytoskeleton with rhodamine phalloidin, shading at room temperature for 30min, washing with PBS, adding DAPI to mark cell nucleus, and acting at room temperature for 5 min;

(8) tabletting: carefully taking out the slide with tweezers, sucking water, inverting the slide on a glass slide on which an anti-fluorescence quencher is dropped, removing the excess anti-fluorescence quencher with filter paper, and detecting under a fluorescence confocal microscope to confirm that the recombinant protein rMbov0503 can adhere to the EBL cells (A-D in FIG. 5).

2. Adhesion detection of recombinant protein rMbov0503 to EBL cell membrane protein:

1) EBL (stored in the laboratory) cells were expanded in MEM medium (Hyclone) containing 10% FBS; extracting membrane protein according to a cell membrane protein extraction kit (Novagen), and specifically comprises the following steps;

2) washing cells with inwardly opened PBS and scraping cells with a spatula, centrifuging at 700g and 4 deg.C for 5min, collecting 0.2-10 × 10⁸A cell;

3) EBL cells were washed 2-3 times with 3mL of pre-chilled PBS;

4) adding 2mL of localization Buffer Mix to shake and lyse the cells in an ice-cold environment;

5) transferring the homogenate into a centrifuge tube with the volume of 1.5mL, centrifuging at the temperature of 4 ℃ for 10min at 700g, and collecting the supernatant;

6) transferring the supernatant obtained by centrifugation into a new 1.5mL centrifuge tube, centrifuging for 30min at 1000g and 4 ℃, collecting the precipitate as cell membrane protein, re-suspending the precipitate with 300 mu L of precooled PBS, measuring the concentration, and storing at-80 ℃.

ELISA detection of rMbov0503 adhesion to EBL cell membrane protein

(1) Coating an enzyme label plate with membrane protein: EBL cell membrane protein was coated in ELISA plates at 100. mu.L/well, 400ng, 3 replicates per group and an equal amount of Bovine Serum Albumin (BSA) was set as a negative control. After coating overnight at 4 ℃ and 3 washes with PBST, 5% skim milk diluted with PBST was added and blocked for 2h at 37 ℃.

(2) rMbov0503 reacts with membrane protein adhesion: the blocked plates were washed 3 times with PBST, patted dry, added with diluted rMbov0503 in two-fold, acted for 1h at 37 ℃ and washed 3 times with PBST (PBS buffer containing 0.05% Tween-20); then adding a mouse source anti-rMbov 0503 polyclonal antibody diluted by 1:300, reacting for 1h at 37 ℃, and washing for 3 times by using PBST; adding a goat anti-mouse IgG enzyme-labeled secondary antibody (southern Biotech) diluted at the ratio of 1:3000, and reacting for 1h at the temperature of 37 ℃; adding color development liquid TMB (biological products Co., Ltd. before Wuhan Ke) to react for 10min in dark, adding stop solution, and measuring light absorption value at OD 630. The dose dependence of rMbov0503 on the adherence of EBL cell membrane proteins was determined (fig. 5E).

(3) Adhesion inhibition by rMbov0503 antisera: incubating 400ng of protein diluted by PBST and polyclonal antibody against rMbov0503 diluted in multiple proportion with 37 ℃ for 1h in advance, adding the treated rMbov0503 into a closed ELISA plate, performing reaction at the temperature of 37 ℃ for 1h, performing reaction at 100 mu L/hole for 3 times of each group, setting a group without the antibody as a positive control, and performing reaction at the temperature of 37 ℃ for 1 h; PBST wash 3 times; antiserum raised against rMbov0503 from murine origin (1:300 dilution) was then added; PBST wash 3 times; adding horseradish peroxidase-labeled goat anti-mouse IgG (southern Biotech) (1:3000 dilution) as a secondary antibody, and reacting at 37 ℃ for 1 h; after 10min of addition of color developing solution, stop solution TMB (probiological products of Wuhan's family Co., Ltd.) was added and absorbance was measured at OD 630. It was determined that antiserum to rMbov0503 inhibited the adhesion of recombinant proteins to EBL cell membrane proteins (panel F in FIG. 5).

Example 4: detection of growth curves of Mycoplasma bovis

Taking mycoplasma bovis HB0801 and T4.4, and mixing the components in a ratio of 1: 1000, inoculating PPLO liquid medium, standing, and culturing at 37 deg.C and 5% CO₂CFU counts were taken after 36h incubation in the incubator, after log phase. Diluting the counted bovine mycoplasma with PPLO medium to 10⁵CFU/mL, as 1: inoculating 10% of the culture medium into PPLO, standing at 37 deg.C, and adding 5% CO₂Continuously culturing for 72h in an incubator every timeAnd taking appropriate bacteria liquid for colony counting after 12 hours. The mycoplasma bovis count results were plotted against the sampling time points to obtain growth curves. The growth of the mutant strain was observed to be consistent with that of the wild strain in comparison of the growth of the different strains at each stage (FIG. 6).

Example 5: verification of adhesion capability of mycoplasma bovis on EBL cells

1. Determination of adhesive capacity of mycoplasma bovis to EBL cells at different temperatures

EBL cells were plated in 24-well plates at 1X 10 per well⁵，37℃，5％CO₂Culturing for 12h under the condition to make cells grow adherent, adding Mycoplasma bovis wild strain and mutant strain at infection ratio of 1000, reacting at different temperatures (4 deg.C, 37 deg.C, 42 deg.C) for 1h, washing unadhered Mycoplasma bovis with PBS, and precooling ddH₂And O, collecting bacterial liquid and counting bacterial colonies after the cells absorb water and are broken.

2. Visual detection of adhesion capability of mycoplasma bovis and EBL cells

(1) EBL cells were cultured at 1X 10⁵The cells were seeded in 24-well cell plates at 37 ℃ with 5% CO₂Culturing for 12h under the condition to ensure that the cells grow adherent;

(2) washing EBL cells with internally opened PBS for 2-3 times, adding 100 μ L of CFDA-SE chemical dye-labeled bacterial solution (containing about 1 × 10) at an infection ratio of 1000⁸CFU) were added to the corresponding wells and incubated at 37 ℃ for 1h, respectively, in a total volume of 200 μ L of liquid per well, to allow sufficient exposure of the cells to mycoplasma. Adding the same amount of cell culture medium into the blank group;

(3) washing the cells with an internally opened PBS buffer stored at normal temperature for 5 times, slowly adding the washed cells along different directions each time, and removing mycoplasma bovis which is not adhered to the EBL cells;

(4) fixing the cells with 1mL of 4% paraformaldehyde at room temperature for 30min, washing with PBS, adding 200 μ L of rhodamine phalloidin, staining EBL cytoskeleton, and keeping away from light at room temperature for 30 min. After PBS wash, add 200. mu.L of DAPI to stain nuclei for 10 min;

(5) and fishing out the slide from the 24-hole plate, absorbing water of the slide by using water absorption paper, inversely placing the slide on a glass slide on which the anti-fluorescence quenching agent is dropped, and observing under a fluorescence confocal microscope.

Colony counting and fluorescence microscopy showed that the T4.4 mutant strain had significantly reduced ability to adhere to EBL cells compared to the wild-type strain (fig. 7).

Adhesion is the primary condition for pathogen colonization and induction of disease (Rszin and Jacobs, 1992), not only facilitates its breakthrough through the host cell barrier structure, sustained proliferation, invasion into deep tissues, but also stimulates the host immune response to cause disease, and thus adhesion is also considered to be a virulence factor for mycoplasma infection. In mutant strains, T4.4 decreased most significantly in its ability to adhere to host cells, suggesting an important role in interacting with host cells.

Example 6: detection of transmembrane transmission ability and MDBK intercellular tight junction disruption ability of mycoplasma bovis

1. Detection of cell transmembrane transmission capability of mycoplasma bovis

(1) Detection of tight junctions between MDBK cells: at 1 × 10⁵Cells/well MDBK cells were seeded in 24-well Transwell chambers. 37 ℃ and 5% CO₂Culturing for 3 days under the condition, performing permeability detection after changing liquid, adding 100 μ L of 0.4% trypan blue into each well, collecting 100 μ L of lower chamber liquid in 1min, 5min, 10min, and 30min, placing in 96-well plate, and detecting at OD₅₈₀Measuring the absorbance value under the conditions, and measuring the standard: OD after 30min₅₈₀< 0.1 indicates that tight junctions are formed between cells at this time, and can be used for infection experiments.

(2) Detection of transmembrane transmission ability of mycoplasma bovis: after MDBK cells form tight connection, the cells in the upper chamber are washed for 2 times by using internally opened PBS, 600 mu L of DMEM complete culture medium is added into the lower chamber of each hole, the upper chamber is covered by the volume, mycoplasma bovis strains at the end of logarithm are taken, the counted and washed mycoplasma bovis strains are added into the upper chamber according to the MOI of 1000, and 100 mu L of culture medium in the lower chamber is collected for colony counting after 6h, 12h and 24h after reaction respectively. The detection result shows that the T4.4 mutant strain has obviously reduced transmembrane transmission capability to MDBK compared with the wild strain (figure 8).

2. Detection of MDBK cell-cell tight junction disruption ability by mycoplasma bovis

(1) Detection of tight junctions between MDBK cells: at 1 × 10⁵Cells/well MDBK cells were seeded in 24-well Transwell chambers. 37 ℃ and 5% CO₂Culturing for 3d under the condition to form tight connection of the cells.

(2) The treated Mycoplasma bovis was added to a Transwell chamber at an infection rate of 1000 ℃ and 5% CO at 37 ℃₂Infection was carried out for 24h under the conditions.

(3) Sealing the infected Transwell chamber membrane with 5% skimmed milk at room temperature for 3h, and washing with PBS 3 times after sealing;

(4) adding 4% paraformaldehyde, fixing at room temperature for 30min, washing, permeabilizing with TX-100, and washing with PBS for 3 times;

(5) using Anti-ZO-1 labeled tight junction protein ZO-1, acting at room temperature for 1h, adding goat Anti-mouse enzyme-labeled secondary antibody 488, acting for 1h, and washing with PBS 3 times;

(6) staining cell nuclei with DAPI for 5min at room temperature, and washing with PBS for 3 times;

(7) the Transwell chamber membrane was torn off, placed upside down on a glass slide on which an anti-fluorescence quencher was dropped, a cover glass was added, and after removing the excess anti-fluorescence quencher with filter paper, observation was performed under a confocal fluorescence microscope.

(8) And (4) judging a result: the NC blank group is tightly connected and complete, and is rarely damaged; the T4.4 mutant infected group showed only a small percentage of intercellular junction breaks; the HB0801 infected group showed a large number of tight junctions disrupted, compared to the T4.4 mutant with a severe degree of disruption (fig. 8).

Tight junctions can be formed among MDBK cells, the tight junctions can protect host cells to a certain extent, and once the tight junctions among the cells are damaged, the propagation of pathogens among the host cells is accelerated, so that the disease damage is caused to the host. The disruption capability and transmembrane transmission capability of the T4.4 mutant strain on MDBK intercellular tight junction are reduced compared with those of a wild strain, which suggests that the mutant strain is possibly weakened in the diffusion capability among host cells, and further the pathogenicity on the host is weakened.

In conclusion, the protein Mbov0503 encoded by the Mbov _0503 gene is a main adhesion protein of mycoplasma bovis; the Mbov0503 gene mutant exhibits a defect in adhesion to a host cell as compared with a wild-type strain; the disruption capability and transmembrane propagation capability of intercellular tight junction are weakened, and the characteristics result in that the mutant strain has potential application prospect in pathogenesis and prevention and control.

The noun terms describe:

in this specification:

the Mycoplasma bovis Mbov0503 recombinant protein is represented by Mbov 0503.

The M.bovis Mbov0503 protein gene is represented by Mbov _ 0503.

The mutant strain of M.bovis Mbov0503 gene is represented by M.bovis T4.4.

A local isolate (or wild-type strain) of Mycoplasma bovis is designated Mycoplasma bovis HB 0801.

Sequence listing

<110> university of agriculture in Huazhong

<120> Mycoplasma bovis gene-deleted mutant strain having reduced adhesion ability

<141> 2019-02-16

<160> 14

<170> SIPOSequenceListing 1.0

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<211> 44

<212> DNA

<213> Mycoplasma bovis (Mycoplasma bovis)

<220>

<221> primer_bind

<222> (1)..(44)

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<210> 2

<211> 29

<212> DNA

<213> Mycoplasma bovis (Mycoplasma bovis)

<220>

<221> primer_bind

<222> (1)..(29)

<400> 2

tatagttagg cgtaaagctc cagtatata 29

<210> 3

<211> 29

<212> DNA

<213> Mycoplasma bovis (Mycoplasma bovis)

<220>

<221> primer_bind

<222> (1)..(29)

<400> 3

ttacgcctaa ctataatgaa aaaaatatg 29

<210> 4

<211> 27

<212> DNA

<213> Mycoplasma bovis (Mycoplasma bovis)

<220>

<221> primer_bind

<222> (1)..(27)

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catctttaaa ataataccag cctgggt 27

<210> 5

<211> 31

<212> DNA

<213> Mycoplasma bovis (Mycoplasma bovis)

<220>

<221> primer_bind

<222> (1)..(31)

<400> 5

ttaaagatgg cattttacac ttaataattg g 31

<210> 6

<211> 31

<212> DNA

<213> Mycoplasma bovis (Mycoplasma bovis)

<220>

<221> primer_bind

<222> (1)..(31)

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ttactgtcta aaaaccactg ataatacttt g 31

<210> 7

<211> 28

<212> DNA

<213> Mycoplasma bovis (Mycoplasma bovis)

<220>

<221> primer_bind

<222> (1)..(28)

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<211> 30

<212> DNA

<213> Mycoplasma bovis (Mycoplasma bovis)

<220>

<221> primer_bind

<222> (1)..(30)

<400> 8

ttatataaat atctaaacca agggacattt 30

<210> 9

<211> 34

<212> DNA

<213> Mycoplasma bovis (Mycoplasma bovis)

<220>

<221> primer_bind

<222> (1)..(34)

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<210> 10

<211> 34

<212> DNA

<213> Mycoplasma bovis (Mycoplasma bovis)

<220>

<221> primer_bind

<222> (1)..(34)

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cagttgaaag taaattttcg gtccataaat tatt 34

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<212> DNA

<213> Mycoplasma bovis (Mycoplasma bovis)

<220>

<221> primer_bind

<222> (1)..(28)

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<211> 49

<212> DNA

<213> Mycoplasma bovis (Mycoplasma bovis)

<220>

<221> primer_bind

<222> (1)..(49)

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<212> DNA

<213> Mycoplasma bovis (Mycoplasma bovis)

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<220>

<221> mutation

<222> (1023)..(1023)

<220>

<221> mutation

<222> (810)..(810)

<220>

<221> mutation

<222> (549)..(549)

<220>

<221> mutation

<222> (354)..(354)

<400> 13

ttc aaa att aat tta gaa aag aaa aat gta att aga tta agc tcc aat 48

Phe Lys Ile Asn Leu Glu Lys Lys Asn Val Ile Arg Leu Ser Ser Asn

1 5 10 15

act cta agt ggt cta agt agc ttt gat gat gat aaa gtg gga atc aaa 96

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

20 25 30

aat aac caa aca gtt gat ata gga gaa ttt tta agt ttt ttt gca cct 144

Asn Asn Gln Thr Val Asp Ile Gly Glu Phe Leu Ser Phe Phe Ala Pro

35 40 45

aaa agc agg gat aaa ata tcc aaa tac ata ttt aaa gag aaa att ggc 192

Lys Ser Arg Asp Lys Ile Ser Lys Tyr Ile Phe Lys Glu Lys Ile Gly

50 55 60

agt aga tat aat tta ttt tgt tct ctt aat ctt gag aaa tat gca aat 240

Ser Arg Tyr Asn Leu Phe Cys Ser Leu Asn Leu Glu Lys Tyr Ala Asn

65 70 75 80

tca tcc ata aat aag tca tat aaa tat ttt gat atc aac ttg gtt aaa 288

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

85 90 95

aaa caa ttt tta gtt aag tta tat cct tca gct gat aaa aag ttt ata 336

Lys Gln Phe Leu Val Lys Leu Tyr Pro Ser Ala Asp Lys Lys Phe Ile

100 105 110

tat tgc aat ata tac tgg agc ttt acg cct aac tat aat gaa aaa aat 384

Tyr Cys Asn Ile Tyr Trp Ser Phe Thr Pro Asn Tyr Asn Glu Lys Asn

115 120 125

atg ttt aat gca ctt tca agt gat aat gac tta tta aaa ttg cct aat 432

Met Phe Asn Ala Leu Ser Ser Asp Asn Asp Leu Leu Lys Leu Pro Asn

130 135 140

aaa ttt tac tta tca tat gca tta aca ata aat caa ttt tat ttg caa 480

Lys Phe Tyr Leu Ser Tyr Ala Leu Thr Ile Asn Gln Phe Tyr Leu Gln

145 150 155 160

aat ggg ctc agt aat agt gat att aat cat aca atc agc atg atg aat 528

Asn Gly Leu Ser Asn Ser Asp Ile Asn His Thr Ile Ser Met Met Asn

165 170 175

tta cta aag cac cca ggc tgg tat tat ttt aaa gat ggc att tta cac 576

Leu Leu Lys His Pro Gly Trp Tyr Tyr Phe Lys Asp Gly Ile Leu His

180 185 190

tta ata att ggt gta aat tca cat tta tta ctt aaa aaa ttt act aaa 624

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

195 200 205

gat gct ata aaa atg gtg aat aaa gta aat tcc tta aac tca ttt aac 672

Asp Ala Ile Lys Met Val Asn Lys Val Asn Ser Leu Asn Ser Phe Asn

210 215 220

ccg ttt ttt aat aac tgt tct ctt tta tca ttc aaa atg ccc aca gat 720

Pro Phe Phe Asn Asn Cys Ser Leu Leu Ser Phe Lys Met Pro Thr Asp

225 230 235 240

aaa agt caa gta tat gaa tat gac att aag gca aaa tac ctg ctt cat 768

Lys Ser Gln Val Tyr Glu Tyr Asp Ile Lys Ala Lys Tyr Leu Leu His

245 250 255

cat tta gat aat aac att tta aat aca aag tat tat cag tgg ttt tta 816

His Leu Asp Asn Asn Ile Leu Asn Thr Lys Tyr Tyr Gln Trp Phe Leu

260 265 270

gac agt aat gag cat aac gaa cac ttt agc gaa ttt aaa act aaa tta 864

Asp Ser Asn Glu His Asn Glu His Phe Ser Glu Phe Lys Thr Lys Leu

275 280 285

gag tcc ttt gaa aac aaa aat aat ctt ctt gaa tat gct aaa gac cct 912

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

290 295 300

gtt tat gta ctt aat tat aaa gac gat tct cag gcc aat ata aga ata 960

Val Tyr Val Leu Asn Tyr Lys Asp Asp Ser Gln Ala Asn Ile Arg Ile

305 310 315 320

cta aaa agt cgt gtt tta ggc ttt agc aat aat gat gtg aac ttt ttt 1008

Leu Lys Ser Arg Val Leu Gly Phe Ser Asn Asn Asp Val Asn Phe Phe

325 330 335

aaa aat gtc cct tgg ttt aga tat tta tat aat aat tta tgg acc gaa 1056

Lys Asn Val Pro Trp Phe Arg Tyr Leu Tyr Asn Asn Leu Trp Thr Glu

340 345 350

aat tta ctt tca act gaa gta aac agt gat gat ttt gta att gtc gaa 1104

Asn Leu Leu Ser Thr Glu Val Asn Ser Asp Asp Phe Val Ile Val Glu

355 360 365

act aat gat att aac ttt gaa aaa ctt tct cca ctt tta agt gat aaa 1152

Thr Asn Asp Ile Asn Phe Glu Lys Leu Ser Pro Leu Leu Ser Asp Lys

370 375 380

act att ttg atg ttt aaa tat cac tat cgc gac ttt aat tat gaa ttg 1200

Thr Ile Leu Met Phe Lys Tyr His Tyr Arg Asp Phe Asn Tyr Glu Leu

385 390 395 400

gtt tca aaa ctt tta aat cat att gga aat gat aaa aat tta aaa aag 1248

Val Ser Lys Leu Leu Asn His Ile Gly Asn Asp Lys Asn Leu Lys Lys

405 410 415

ctt tca att ggt tta tat ata gat aac ctt gat gaa aaa ctt ttt aat 1296

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

420 425 430

ttt agc gac ttt gcc aat att aat acc ttt gtg ttt tca aaa aat atc 1344

Phe Ser Asp Phe Ala Asn Ile Asn Thr Phe Val Phe Ser Lys Asn Ile

435 440 445

tgt gaa aac ata cta aac aat gct gaa act tac tta aaa tta caa gtg 1392

Cys Glu Asn Ile Leu Asn Asn Ala Glu Thr Tyr Leu Lys Leu Gln Val

450 455 460

ttt gta caa aaa ata ata tct atg aaa aaa tac tta att att tat gaa 1440

Phe Val Gln Lys Ile Ile Ser Met Lys Lys Tyr Leu Ile Ile Tyr Glu

465 470 475 480

aat att cca aaa aat tta gaa aat att gtt gta caa aga tta aaa ata 1488

Asn Ile Pro Lys Asn Leu Glu Asn Ile Val Val Gln Arg Leu Lys Ile

485 490 495

aaa tat ttt tat aat gta taa 1509

Lys Tyr Phe Tyr Asn Val

500

<210> 14

<211> 502

<212> PRT

<213> Mycoplasma bovis (Mycoplasma bovis)

<400> 14

Phe Lys Ile Asn Leu Glu Lys Lys Asn Val Ile Arg Leu Ser Ser Asn

1 5 10 15

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

20 25 30

Asn Asn Gln Thr Val Asp Ile Gly Glu Phe Leu Ser Phe Phe Ala Pro

35 40 45

Lys Ser Arg Asp Lys Ile Ser Lys Tyr Ile Phe Lys Glu Lys Ile Gly

50 55 60

Ser Arg Tyr Asn Leu Phe Cys Ser Leu Asn Leu Glu Lys Tyr Ala Asn

65 70 75 80

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

85 90 95

Lys Gln Phe Leu Val Lys Leu Tyr Pro Ser Ala Asp Lys Lys Phe Ile

100 105 110

Tyr Cys Asn Ile Tyr Trp Ser Phe Thr Pro Asn Tyr Asn Glu Lys Asn

115 120 125

Met Phe Asn Ala Leu Ser Ser Asp Asn Asp Leu Leu Lys Leu Pro Asn

130 135 140

Lys Phe Tyr Leu Ser Tyr Ala Leu Thr Ile Asn Gln Phe Tyr Leu Gln

145 150 155 160

Asn Gly Leu Ser Asn Ser Asp Ile Asn His Thr Ile Ser Met Met Asn

165 170 175

Leu Leu Lys His Pro Gly Trp Tyr Tyr Phe Lys Asp Gly Ile Leu His

180 185 190

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

195 200 205

Asp Ala Ile Lys Met Val Asn Lys Val Asn Ser Leu Asn Ser Phe Asn

210 215 220

Pro Phe Phe Asn Asn Cys Ser Leu Leu Ser Phe Lys Met Pro Thr Asp

225 230 235 240

Lys Ser Gln Val Tyr Glu Tyr Asp Ile Lys Ala Lys Tyr Leu Leu His

245 250 255

His Leu Asp Asn Asn Ile Leu Asn Thr Lys Tyr Tyr Gln Trp Phe Leu

260 265 270

Asp Ser Asn Glu His Asn Glu His Phe Ser Glu Phe Lys Thr Lys Leu

275 280 285

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

290 295 300

Val Tyr Val Leu Asn Tyr Lys Asp Asp Ser Gln Ala Asn Ile Arg Ile

305 310 315 320

Leu Lys Ser Arg Val Leu Gly Phe Ser Asn Asn Asp Val Asn Phe Phe

325 330 335

Lys Asn Val Pro Trp Phe Arg Tyr Leu Tyr Asn Asn Leu Trp Thr Glu

340 345 350

Asn Leu Leu Ser Thr Glu Val Asn Ser Asp Asp Phe Val Ile Val Glu

355 360 365

Thr Asn Asp Ile Asn Phe Glu Lys Leu Ser Pro Leu Leu Ser Asp Lys

370 375 380

Thr Ile Leu Met Phe Lys Tyr His Tyr Arg Asp Phe Asn Tyr Glu Leu

385 390 395 400

Val Ser Lys Leu Leu Asn His Ile Gly Asn Asp Lys Asn Leu Lys Lys

405 410 415

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

420 425 430

Phe Ser Asp Phe Ala Asn Ile Asn Thr Phe Val Phe Ser Lys Asn Ile

435 440 445

Cys Glu Asn Ile Leu Asn Asn Ala Glu Thr Tyr Leu Lys Leu Gln Val

450 455 460

Phe Val Gln Lys Ile Ile Ser Met Lys Lys Tyr Leu Ile Ile Tyr Glu

465 470 475 480

Asn Ile Pro Lys Asn Leu Glu Asn Ile Val Val Gln Arg Leu Lys Ile

485 490 495

Lys Tyr Phe Tyr Asn Val

500

Claims

1. Application of a protein encoded by Mycoplasma bovis ( Mycoplasma bovis ) gene Mbov_0503 in adhering host cells, the nucleotide sequence of the gene is shown in SEQ ID NO: 13.