CN104151406A - Bovine brucellosis outer membrane protein Omp22, coding gene as well as cloning method thereof, and application - Google Patents

Abstract

The invention discloses a bovine brucellosis outer membrane protein Omp22 gene, and a preparation method and application of the gene, relates to a gene and in particular relates to an outer membrane protein Omp22 gene expressed on the outer membrane of bovine brucellosis and a preparation method and a use of the gene. The invention provides a bovine brucellosis outer membrane protein Omp22 and a coding gene thereof. The coding gene has the nucleotide sequence used for coding the amino acid sequence of the outer membrane protein Omp22. The invention also provides a method for preparing the Omp22 gene by use of the recombinant technology. The method comprises the steps of operably connecting the purified nucleotide sequence for coding the active peptide with the Omp22 to an expression regulation sequence to form an expression vector of the Omp22, transferring the expression vector in a host cell to form a recombinant cell of the outer membrane protein Omp22, and culturing the recombinant cell. The invention further provides application of the bovine brucellosis outer membrane protein Omp22 and the coding sequence of the Omp22.

Description

Brucella bovis outer membrane protein Omp22, coding gene and its cloning method and application

technical field

The invention relates to a gene, in particular to an outer membrane protein (outer membrane protein, Omp) expressed on the outer membrane of Bovine brucellosis, a gene, a gene cloning method and an application thereof.

Background technique

Brucellosis, referred to as brucellosis, also known as Mediterranean relaxation fever, Malta fever, wave fever or undulating fever, is a zoonotic systemic infectious disease caused by Brucella. It belongs to the second category of infectious diseases in my country. The prevalence of brucellosis is wide and lasts for a long time, which not only seriously threatens human health and animal husbandry production, but also causes huge economic losses and serious public health problems. There are as many as one million cattle, sheep, and pigs infected every year in China, and the economic losses caused can reach more than one billion yuan. Brucellosis is a long-term problem that has plagued the development of China's animal husbandry and people's health since the founding of the People's Republic of China. All countries in the world list it as a key disease for prevention and control, and the International Organization for Animal Health stipulates that this disease must be reported by all countries. With the integration of my country and the World Trade Organization and the improvement of people's living standards, the control and elimination of brucellosis in animal husbandry is becoming more and more important. In the field of dairy industry, the infection incidents of production personnel lead to the waste and recycling of products in batches, causing serious economic losses to manufacturers, and the harm of brucellosis has attracted special attention.

In order to effectively curb the impact of brucellosis on the dairy industry and ensure public health safety, it is extremely important to strengthen the comprehensive prevention and control of brucellosis in dairy cattle and disease surveillance. Regular quarantine is an important measure for the prevention and purification of brucellosis in dairy farms. The detection method of brucellosis is an important factor affecting the quarantine effect of brucellosis. At present, the method commonly used in China is: the initial screening of the tiger red plate agglutination test, and then the final qualitative analysis by the test tube agglutination test, while the primary screening methods used in the world mainly include: indirect enzyme-linked immunosorbent assay (I-ELISA) and fluorescence Polarization test. The tiger red plate agglutination test has low specificity. Due to the influence of test temperature, relatively short agglutination time and human subjective factors, it is not easy to judge the results accurately, and it can only be used as a preliminary screening; while the test tube agglutination test is the legal standard for brucellosis in my country. However, this method is cumbersome and time-consuming, and is not suitable for on-site use, and is also affected by human subjective factors. The OIE standard stipulates the use of ELISA and fluorescence polarization as the initial diagnostic method for international trade of brucellosis. ELISA and fluorescence polarization methods have good sensitivity and specificity for detection and monitoring, short reaction time, avoid the interference of human factors, and are easy to standardize and quality control, but these two methods can only be used for primary screening. Due to its more prominent specificity, sensitivity and high efficiency, the fluorescence polarization test requires a higher level of operator skills. The current brucellosis monitoring and prevention work expects a simpler and more efficient monitoring tool and treatment method, so to explore new protective antigens and immune mechanisms for animal brucellosis, induce high levels of humoral and cellular immunity, and improve The detection of brucellosis and the level of infection control, and reducing the economic loss of animal husbandry have become urgent issues in animal husbandry.

Researchers have found that brucellosis is difficult to control because Brucella bacteria can evade the host's immune recognition. As pathogenic bacteria evolve, they require the necessary virulence factors to invade and proliferate in the host. The outer membrane protein is the main immunogen and protective antigen of Brucella. For more accurate vaccine immunization, control and treatment of brucellosis, it is of great significance to search for protein-based diagnostic and immunogens. Foreign research work has carried out immunogenicity and protective research on some Brucella outer membrane proteins.

Currently, Brucella outer membrane proteins are divided into 3 groups according to their molecular mass. The first group has outer membrane proteins 10k, 18k, 19k, among which Omp10, Omp16 and Omp19 are a kind of lipoprotein. Group 2 includes 36k-38k outer membrane proteins. Group 3 includes 31k~34k outer membrane proteins and 25k~27k outer membrane proteins, Omp25 and Omp31 are closely related to the virulence and immunogenicity of Brucella. The mutant strains of Omp22 gene have enhanced sensitivity to negative serum reaction and difficulty growing in the stable phase, which may be related to the weakened virulence of Omp22 gene mutant strains. Studies have found that Omp22 has strong immunogenicity and can induce high-titer specific antibodies in immunized experimental animals , can play an important role in the body's protective immune response. However, there are very few researches on the application of Omp22 sequence of the Brucella outer membrane protein, and there is no report about the outer membrane protein Omp22 of Brucella in cattle, which greatly limits the monitoring and prevention of diseases in dairy cows as economic animals in the dairy industry.

Invention content:

The object of the present invention is to provide a kind of Brucella bovis outer membrane protein Omp22 and its coding gene and the cloning method of the coding gene, cloning recombinant bacterial strain and application thereof. The invention has good application prospects in the in-depth study of the pathogenic mechanism of the bovine brucellosis, disease prevention and further development into medical products and animal husbandry diagnostic products, and lays a foundation for monitoring the health status of animals.

The present invention can produce the cloning vector and transformed bacterial strain of the coding gene of Brucella bovine outer membrane protein Omp22, which is Bovine brucellosis recombination strain (Bovine brucellosis recombination strain) PGEM-7Zf-omp22, which has a replication such as sequence <400> 4 nucleotide sequence capabilities.

In order to achieve the above object, the solution of the present invention is:

1. a kind of brucella bovis outer membrane protein Omp22 is characterized in that, by 1) the protein that the aminoacid sequence shown in SEQID NO.1 forms, or 2) the aminoacid sequence shown in SEQIDNO.1 is substituted , A protein derived from 1) that has one or more amino acids deleted or added and has the same activity.

A gene encoding the above-mentioned Brucella bovis outer membrane protein Omp22.

The coding gene of the Omp22 has the following a) or b):

a) the nucleotide sequence is as shown in SEQ ID NO.2; or

B) obtain the nucleotide sequence of the Brucella bovis outer membrane protein Omp22 described in coding claim 1 through substitution, deletion or addition of one or several nucleotides by the nucleotide sequence shown in SEQ ID NO.2.

A primer pair for amplifying the sequence described in claim 2 or 3, characterized in that, the primer pair sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4.

A transformed cell line containing the vector according to claim 5.

A cloning method of the brucella bovis outer membrane protein Omp22 gene described in claim 2 or 3, is characterized in that comprising the steps:

1) Genomic DNA is extracted from Brucella bovis liquid culture as a template,

2) design the PCR primers of Brucella bovis outer membrane protein Omp22 gene as follows:

3) Omp22 gene PCR amplification reaction,

4) purification of Omp22 gene PCR amplification product,

5) digestion and connection of Omp22 gene PCR amplification product and cloning vector,

6) Transformation of host cells,

7) Screening and identification of transformed cell lines.

The application of the gene according to claim 2 or 3, characterized in that the Omp22 gene clone can be used for the monitoring of brucellosis and as a target for therapeutic drug research.

In particular, it can be used for the preparation of diagnostic reagents containing any one of claims 1-3.

In particular, it includes applications as brucellosis, targets for drug research, diagnostic reagents for animal brucellosis, kits for animal brucellosis investigation, preparation of brucellosis vaccines, preparation of brucellosis Applications in DNA vaccines.

The "isolated" and "purified" DNA in the present invention means that the DNA or fragment has been separated from the sequences on both sides of it in the natural state, and it also means that the DNA or fragment has been accompanied by the natural state. The components of the nucleic acid are separated and have been separated from the proteins that accompany them in the cell.

The vectors mentioned in the present invention can be selected from various vectors known in the art, such as commercially available vectors, including plasmids, cosmids and the like. Most of the cloning vectors are high-copy vectors, usually prokaryotic bacteria. The gene to be cloned is connected to the plasmid of the cloning vector, and then introduced into the prokaryotic bacteria. The gene is not necessarily expressed, but it must be copied in large numbers. The purpose of cloning vectors is to replicate enough target plasmids, so they often have strong self-replicating elements, such as replication origin sites, etc., and there are often multiple copies in the bacteria, so a large number of plasmids will be extracted. But it does not have expression elements. In the present invention, the plasmid is preferred, and PGEM-7Zfvector is particularly preferred to prepare a cloning vector as an empty vector. -7Zf(+) and -7Zf(-)Vector is composed of vector -3Zf(+) derived and contains the origin of replication of filamentous bacteriophage f1. These two vectors can be used as standard cloning vectors, as well as templates for in vitro transcription and preparation of circular ssDNA. There are multiple cloning sites in the α-peptide coding region of β-galactosidase on the vector, followed by the multiple cloning sites also contain SP6 and T7 RNA polymerase promoters. By selecting a suitable E.coli strain (such as JM109), the recombinant clones with α-peptide insertion inactivation can be directly identified by the method of blue/white screening. The multiple cloning site region is unique and contains some restriction sites such as ApaI, AatII, SphI, XbaI, XhoI, EcoRI, KpnI, SmaI, Csp45I, ClaI, HindIII, BamHI, SacI, BstXI and NsiI. These structural features are beneficial to the mass replication and screening of the target gene of the present invention.

When producing the outer membrane protein Omp22 of the present invention, the Brucella protein Omp22 coding sequence can be operably linked to the replication initiation sequence, thereby forming a Brucella outer membrane protein Omp22 coding gene cloning vector. The term "operably linked" refers to the condition that certain parts of a linear DNA sequence can affect the activity of other parts of the same linear DNA sequence.

The "host cell" is a prokaryotic cell. Commonly used prokaryotic host cells are Escherichia coli (E.coli) or Bacillus subtilis, etc., preferably E.coli DH5α, E.coli BL21 and E.coli TOP10, etc., especially preferably E.coli DH5α.

The clone of the full-length sequence of the gene encoding the outer membrane protein Omp22 of Brucella bovis of the present invention or its fragments can usually be obtained by PCR amplification, recombination or library screening, and PCR amplification is particularly preferred. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, and Brucella can be used as a template to amplify to obtain relevant sequences.

In the cloning method of the brucella bovis outer membrane protein Omp22 encoding gene of the present invention, the preparation method of the template is simple, and only the simple steps of centrifugation, boiling and supernatant absorption are used. The primers were designed to introduce BamH I and Xho I cleavage sites into the upstream and downstream primers respectively, and the formation of sticky ends is conducive to the efficient enzyme ligation effect.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial chemical synthesis. Before this application, in the prior art, it was possible to obtain the nucleic acid sequence encoding the outer membrane protein Omp22 of the present invention by first synthesizing multiple small polynucleotide fragments and then connecting them. Then, the nucleic acid sequence can be introduced into various existing DNA molecules (such as vectors) and cells in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The cloning vector of the gene encoding the outer membrane protein Omp22 of Brucella bovis obtained by the above technical scheme provides sufficient material for the applicant of the present invention to carry out in-depth bioinformatics analysis on the clone.

The beneficial effects of the present invention are:

1. The coding gene of Brucella bovis outer membrane protein Omp22 can be produced in batches by adopting the method of the present invention, and the preparation method is simple at the same time, and the template extraction does not need to consume chemical reagents; simultaneously, the present invention can efficiently replicate the target gene, and has the advantages of environmental protection and economy .

2. the coding gene clone of the product Brucella bovis recombinant bacterial strain outer membrane protein Omp22 obtained by the technical scheme of the present invention has good application prospects in the diagnosis monitoring and prevention control of brucellosis, including in brucellosis as brucellosis The application of mycosis, the target of drug research, the diagnostic reagent of animal brucellosis, the kit of animal brucellosis epidemic investigation, the application in the preparation of Brucella vaccine and the preparation of Brucella DNA vaccine.

3. The coding gene sequence information and the cloning vector of the product Brucella bovis outer membrane protein Omp22 obtained by the technical scheme of the present invention are to enrich and deepen the understanding of the mechanism and protein function of the interaction between Brucella and the host in the pathogenic process Research and Applied Development of the Expression of the Outer Membrane Protein Omp22 Research provides sustainable feedstock production methods and is therefore of great societal and commercial value.

4. Using bioinformatics tools and network systems to analyze the physical and chemical properties of the outer membrane protein Omp22 of Brucella bovis, such as the size of the isoelectric point, the size of the molecular weight, the strength of the absorbance and the length of the amino acid sequence. Through the analysis of our above bioinformatics tools, the 56th-62nd amino acid is a region with high hydrophilicity and flexibility. These analysis and prediction results will play a guiding role in the research and analysis of the outer membrane protein Omp22 of Brucella bovis. This will help us to adopt a reasonable strategy to express the outer membrane protein OMP22 in the next step, increase the expression of OMP22 outer membrane protein, and hope to obtain an active recombinant protein, which will provide a material basis for the diagnosis, drug and vaccine research of brucellosis.

Description of drawings

Figure 1. Omp22 gene amplification results (M.DL2000DNA Marker; 1, 2.PCR product)

Figure 2. Restriction digestion identification of Omp22 gene recombinant plasmid (M.DL2000DNA Marker; 1. BamHI single digestion product; 2. BamHI and Xho I double digestion product of empty vector PGEM-7Zf. 3, 4. BamHI and Xho I double digestion product Digest recombinant plasmid PGEM-7Zf-Omp22 product)

Figure 3. Molecular flexibility analysis of Brucella Omp22 protein

Figure 4. Hydrophilicity analysis of Brucella Omp22 protein

Figure 5. B cell linear epitope analysis of outer membrane protein Omp22

Figure 6. Epitope analysis of outer membrane protein Omp22

Figure 7. The 3D structure of the outer membrane protein Omp22 and the spatial position of the domain (1. N-glycosylation site; 2. Protein kinase C phosphorylation site; 3. Casein kinase ∏ phosphorylation site)

Figure 8. Protein network system predicted to interact with the outer membrane protein Omp22

Detailed ways

The clone of embodiment 1 Brucella bovis Omp22 gene

Strains and plasmids

Brucella bovis 544A was isolated from Horqin District; Escherichia coli DH5α was preserved in our laboratory. The cloning vector PGEM-7Zf vector was purchased from PROMEGA company

Main reagents and equipment

Tool enzymes such as restriction endonuclease BamH I, Xho I, Taq polymerase, T4DNA ligase, and DNA standard molecular mass markers were purchased from Dalian Bao Biological Company; plasmid extraction kits were purchased from Qiagen Company, DNA gel recovery kits were purchased from From Shanghai Sangon Bioengineering Technology Service Co., Ltd.; gel imaging system was purchased from (France); PCR instrument (USA); low temperature and constant temperature circulator (Ningbo Tianheng); other reagents were of domestic analytical grade.

1. Cultivation of the original Omp22 bovine strain and preparation of genomic DNA

Streak culture 3% TSB solid medium of Brucella bovis isolated from the Horqin area, grow for 48 hours, pick a single colony in 3% TSB liquid medium, shake at 200r/min for 48 hours, take 2ml of the bacterial solution and place it in two sterile In a 1.5ml centrifuge tube, centrifuge at 12000r/min, add sterilized distilled water to wash 4 times, boil for 10min, centrifuge to absorb the supernatant, which is the DNA template, store in a -20°C refrigerator for later use.

2. Full-length cloning of Omp22 gene

(1) Primer design and synthesis

Referring to the coding region sequences of the genes of B. bovis strains AM712379.1 and AM712380.1 collected in GenBank, combined with the reading frame and multiple cloning sites of the cloning vector PGEM-7Zf, the outer membrane protein Omp22 gene of B. bovis was designed using Primer5.0 The PCR primers are as follows:

Upstream 5`-GA GGGATC CGGAATGTTCAAGCGTTCTATCACC-3;

Downstream 5`G CTCGAG CTAGAATTTGTAGTTCAG-3.

The primer sequence was synthesized by Shanghai Sangon Biotechnology Co., Ltd., and the BamH I and Xho I restriction sites were introduced into the upstream and downstream primers respectively.

(2) PCR amplification

The PCR amplification system is 50 ul; dNTP mixture 6 ul, TaKaRa Ex Taq 0.5 ul (1.25 U), Brucella DNA template 1 ul, Ex Taq Buffer 5 ul, upstream and downstream primers 1 ul, and finally MilliQ H2O to the total volume. PCR amplification conditions: pre-denaturation at 95°C for 10 minutes, denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, extension at 72°C for 2 minutes, 30 cycles, and finally a total extension at 72°C for 10 minutes.

(3) Recovery of PCR amplified fragments

5ul of the PCR product was mixed with loading buffer, electrophoresed in 1.0g/L agarose gel (containing EB), and photographed by a gel image analyzer. Electrophoresis analysis of the product in 1% agarose gel found that there is a band at about 639bp, the size is consistent with the expected size, see Figure 1.

(4) Construction and identification of recombinant plasmids

After the PCR product was digested by BamHI and Xho I, it was directional inserted into the cloning vector PGEM-7Zf which was also digested by the same double enzyme, and the recombinant plasmid obtained was named PGEM-7Zf-Omp22. The recombinant clone PGEM-7Zf-Omp22 plasmid was cut with BamHI and Xho I, respectively, and electrophoresed in 1% agarose gel at the same time, as shown in Figure 2, the target gene fragments of the expected size were obtained, indicating that the Omp22 gene was cloned successfully .

(5) Construction of recombinant strains

i. Preparation of Competent Cells and Transformation of Recombinant Plasmids

Transfer Escherichia coli DH5α culture solution to an ice-cold 1.5ml EP tube, 4°C, 4000rpm×5min; discard the supernatant, add 300μl of ice-cold 0.1mol/L CaCl ₂ to the precipitate, resuspend the bacteria, and ice-bath for 20min; 4 ℃, 4000rpm×5min, discard the supernatant, put the tube upside down on the filter paper for 1min, and make the liquid flow clean; add 100μl of ice-cold 0.1mol/L CaCl ₂ to the precipitate, resuspend the bacteria; ice-bath for 10min; add 5μl of plasmid to each tube, Gently rotate the test tube, mix the mixture, and place it on the ice bath for 20 minutes. Place the test tube in a water bath at 42°C for 90 seconds without shaking the test tube. Quickly transfer the test tube to the ice bath and cool for 1 to 2 minutes; after taking out the test tube, each tube Add 800 μl of LB medium, place on a shaker at 37°C (100-150 r/min), and shake gently for 45 minutes; spread 200 μl of the bacterial solution on the surface of the agar plate containing Amp with a sterile elbow glass spreader; place it at room temperature for 20 minutes, Let the liquid be absorbed (sweat); invert the plate, culture at 37°C, 12 hours visible colony growth;

ii. Screening and identification of recombinant clones: blue and white spot experiment

experimental reagent

X-gal: 2% mother solution (prepared with dimethylformamide, wrapped with aluminum foil or black paper to prevent damage by light, stored at -20°C for future use), working concentration 20ul/20ml plate;

Ampicillin (Amp): Prepare a 100 mg/ml stock solution with sterile water and store in a -20°C refrigerator. Working concentration 100ug/ml;

IPTG: mother liquor 100mmol/L, stored in -20°C refrigerator. Working concentration 40ul/20ml plate;

LB liquid medium: 1% peptone, 0.5% yeast extract, 1% NaCl, adjust the pH to 7.2 with NaOH, and sterilize at 121°C for 20 minutes for later use. For solid LB medium, add 1.5% to 2% agar to LB liquid medium, and sterilize it for later use;

LB solid medium containing Amp: After autoclaving the prepared LB solid medium, cool it to about 60°C, add Amp stock solution to make the final concentration 100ug/ml, shake well and plate;

Escherichia coli DH5a.

Experimental procedure

(1) Take 4 ul of the ligation product and add it to 100 ul of competent cells, blow gently with a gun to evenly, and place in ice bath for 30 min.

(2) Place the tube in a water bath for heat shock at 42°C for 90 sec, and immediately place it on ice for 5 min.

(3) Add 1ml of LB medium preheated at 37°C and mix well.

(4) Place the tube on a constant temperature shaker at 37° C. for shaking and culturing for 1 h.

(5) Place the tube in a centrifuge for 5 minutes at 3000 rpm.

(6) Discard 1ml of the supernatant, and use the remaining about 100ul of the supernatant to blow evenly with a gun, and use it to coat the plate.

(7) On an LB plate containing ampicillin, add 20 μl of 20 mg/ml X-gal and 40 μl of 100 mmol/L IPTG.

(8) Put the glass spatula into the culture plate after being overfire sterilized, and evenly coat the plate after it cools down. After overfire sterilizing the glass spatula, place it in alcohol for later use, and place the plate at room temperature for 30 minutes for later use.

(9) Aspirate the bacterial solution containing the recombinant obtained above onto the prepared flat plate containing X-gal, and spread it evenly with a glass spatula. Place the plate in a biochemical incubator and place it upside down for 30 minutes, then invert it and incubate overnight at 37°C to select white strains.

Embodiment 2 Brucella bovis strain Omp22 gene sequence determination and sequence analysis and bioinformatics analysis

The standard sequence used in the determination of the Omp22 gene sequence of Brucella bovis strain:

Show through sequencing, the outer membrane protein Omp22 gene length of recombinant bacterial strain of Brucella bovis of the present invention is 639bp; As shown in sequence SEQ ID No.4; Sequence SEQ ID No.4 is

ATGTTCAAGCGTTCTATCACCGCAGCCGCGCTCGGCGCTGCCGTCATGGCCTTTGCAGGCTCGGCTTTCGCAGCCGACATGATGGGAGGGACCGACTACACCTATAACGACCCTGTCGCCGCCGGTCCGCATGACTGGTCCGGCAATTATGTCGGCGCGCAGGTTGGTGGTTCGTCTTCCAAATTCCCAAGCCCGTTTGCCAGCCGTACCGGCGCCCTCGGCGGCATTGTCGTCGGCAAGAACATGCAGAACGGCAATATCGTTTTCGGCGCGGAGCTGGAAGGCAACTTCGCCGAAGCCGAACATCGCATCGGCCATGGCGGCACGCTACAGCAATCTTGGAATGGCAATGCCAAGGGCAAGGTCGGCTATGCCTTCGACAAGACCCTCGTTTACGGCACCGCCGGTTATGGCGTGACCCGCTTCAAGGCTAAGGACAACACCACTTCCGCTCCCGGCTGGGAAGGCGGCGTACTGATTGGTGCAGGTGTGGAACAGGCCTTGAGCGGCCCTCTCTCCGTCAAGGCCGAATATGACTTCCAGCGTTTCAACGATGTCAAATCGCAAGTGAATGGCATCGAACAGCGTAACAACCTGAAGAACCATTCGATCAAGGCCGGCCTGAACTACAAATTCTAG

According to the nucleotide sequence of the gene, the bioinformatics software (danman and Vector NTI) was used to deduce 212 amino acid residues encoded by the Omp22 gene, with a molecular weight of about 22kDa, as shown in SEQ ID No.1. SEQ ID No.1 is:

MFKRSITAAA LGAAVMAFAGSAFAADMMGGTDYTYNDPVAAGPHDWSGNYVGAQVGGSSSKFPSPFASRT GALGGIVVGK NMQNGNIVFG AELEGNFAEA EHRIGHGGTL QQSWNGNAKGKVGYAFDKTL VYGTAGYGVT RFKAKDNTTS APGWEGGVLI GAGVEQALSG PLSVKAEYDFQRFNDVKSQV NGIEQRNNLK NHSIKAGLNY KF

(1) Gene sequence analysis: After sequencing, the DNA sequence of the Omp22 gene of Brucella bovis with a length of 639 bp was successfully obtained, and the online analysis tool BLAST ORF Finder in NCBI (http://blast.ncbi.nlmg.nih.goV/ Blast.cgi) and http://blast.ncbi.nlmg.nih.goV/gorf/orfig.cgi) for comparison and ORF search found that the length of the CDS sequence in this sequence is 639bp, such as SEQ No.4; according to the The nucleotide sequence of gene, application bioinformatics software (danman and Vector NTI) deduced 212 amino acid residues encoded by Omp22 gene, molecular weight is about 22kDa; On nucleotide level, it and standard Brucella (Brucellamicroti The 11-645 sequence in the full coding sequence (GenBank Accession No.AM712379) of CCM 4915) outer membrane protein Omp22 has 88% identity (seeing table 1), and table 2 is Brucella outer membrane protein of the present invention and Homology comparison (BLAST) of the amino acid sequence (GenPept Accession No. AM712380) of the standard Brucella (Brucella microti CCM4916) outer membrane protein Omp22. At the amino acid level, it has extremely high identity and similarity with the amino acid residues of the standard Brucella outer membrane protein Omp22 (see Table 2). Among them, the same amino acid is marked with a single amino acid character between the two sequences, and the similar amino acid is marked with "+".

Table 1. the nucleotide BLAST result of the coding gene of the outer membrane protein Omp22 of the Brucella bovis recombinant bacterial strain outer membrane protein Omp22 of the present invention and the outer membrane protein Omp22 coding gene of standard bacterial strain

Table 2. The BLAST result of the amino acid sequence of the outer membrane protein Omp22 of the Brucella bovis recombinant strain outer membrane protein Omp22 of the present invention and the outer membrane protein Omp22 aminoacid sequence of standard bacterial strain

Through BLAST, it was found that the nucleotide sequence encoded by the outer membrane protein Omp22 that we isolated from cow milk had higher homology with the outer membrane protein OMP22 of the standard strain. The results showed that the protein was an outer membrane protein.

(2) Chemical characteristics of Omp22 protein molecule

Using bioinformatics software (danman and Vector NTI) and databases (prosite and PDB) to analyze the main chemical characteristics of the Brucella outer membrane protein Omp22 molecule, see Table 3, Figure 3 and Figure 4

Table 3 Main chemical properties of Brucella bovis outer membrane protein Omp22 protein molecule

Using the correlation between physical and chemical properties and B cell epitopes, the B cell epitopes of Brucella bovis OMP22 can be analyzed and predicted by phenomenological theory. Most of the B cell epitopes are located in the outer membrane protein of Brucella bovis. The amino acid segment with high hydrophilicity and flexibility scores of Omp22, the 56th-62nd amino acid is a region with high hydrophilicity and flexibility through the analysis of our above bioinformatics tools. These analysis and prediction results will play a guiding role in the research and analysis of the outer membrane protein Omp22 of Brucella bovis.

The display of protein spatial structure by Vector NTI suite software provides theoretical support for our in-depth understanding of the relationship between the structure and function of the outer membrane protein Omp22 of Brucella bovis and the pathogenic mechanism of bacteria. Through the modification of the OMP22 domain of Brucella bovis at the gene level, or the mutation of the signal peptide cleavage site, such as the potential N-glycosylation site deletion mutation, it may be possible for the outer membrane protein of Brucella bovis The changes in the immune response between Omp22 and host cells can provide a direction for in-depth study of the function of the outer membrane protein Omp22. At the same time, according to the predicted protein network system interacting with the outer membrane protein Omp22 in the prosite database, this provides a certain theoretical support for the study of protein interaction.

(3) Analysis of immunological characteristics

(i) Through B cell epitope online analysis tool

The protein was predicted to contain 12 potential linear epitopes of B cell antigens. Among them, the longest B cell linear epitope is composed of amino acids 26-71. It consists of 44 amino acids in total and can be used as a potential antigen target, as shown in Fig. 5 and Fig. 6 .

(ii) The protein contains 9 potential antigenic epitopes predicted by the epitope online analysis tool. Among them, the longest epitope is composed of 21 amino acids, located between amino acids 157-177, and can be preferentially considered as an antigen target.

(iii) Formyltransferase domain analysis

Analysis results: The Swiss-model outer membrane protein Omp22 is matched with the protein three-dimensional structure in the protein structure database, and the simulated three-dimensional structure diagram is input back. The file is opened in the vector NTI suite software package to open the protein structure file, and the structure of the outer membrane protein Domains are marked on Figure 7. The proteins predicted to interact with the outer membrane protein Omp22 according to the protein interaction network are shown in Figure 8.

Claims (10)

1. a bovine brucellosis (Bovine brucellosis) outer membrane protein Omp22, is characterized in that, by a) the protein that the amino acid sequence shown in SEQ ID NO.1 forms, or b) shown in SEQ ID NO.1 A protein derived from a) with equivalent activity by substitution, deletion or addition of one or several amino acids in the amino acid sequence. the 2. the brucella bovis outer membrane protein Omp22 gene of encoding claim 1. the 3. brucella bovis outer membrane protein Omp22 gene according to claim 2, it is following a) or b): a) The nucleotide sequence is as shown in SEQ ID NO.2; or B) obtain the nucleotide sequence of the Brucella bovis outer membrane protein Omp22 described in coding claim 1 through substitution, deletion or addition of one or several nucleotides by the nucleotide sequence shown in SEQ ID NO.2. the 4. A primer pair for amplifying the gene described in claim 2 or 3, characterized in that, said primer pair sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively: Upstream 5`-GAGGGATCCGGAATGTTCAAGCGTTCTATCACC-3; Downstream 5`-GCTCGAGCTAGAATTTGTAGTTCAG-3 The primer pair introduces BamH I and Xho I restriction sites into the upstream and downstream primers respectively. the 5. The cloning vector containing the gene of claim 2 or 3, characterized in that the empty vector connected with the gene is a plasmid or a cosmid vector. the 6. A transformed cell line containing the cloning vector of claim 5. the 7. a method for cloning of the brucella bovis outer membrane protein Omp22 gene described in claim 2 or 3, is characterized in that, comprises the steps: 1) thermal cracking method extracts genomic DNA from Brucella bovis liquid culture as template, 2) Design the PCR primer pair sequence of Brucella bovis outer membrane protein Omp22 gene as shown in SEQ ID NO.3 and SEQ ID NO.4, 3) PCR amplification of Omp22 gene 4) Purification of Omp22 gene PCR amplification product 5) Omp22 gene PCR amplification product and the cloning vector are operably connected to the empty vector to form the cloning vector, 6) Transformation of host cells, 7) Screening and identification of transformed cell lines. the 8. The preparation method of Omp22 gene as claimed in claim 7, is characterized in that, described empty vector is plasmid or cosmid vector, and described prokaryotic cell is Escherichia coli DH5α. the 9. The preparation method of the Omp22 gene preparation method as claimed in claim 7, characterized in that, in step 2), the host cell is a prokaryotic cell. the 10. the application of Omp22 albumen or coding gene described in any one of claim 1～3, it is characterized in that, comprise in the monitoring as brucellosis and the target of therapeutic drug research, animal brucellosis diagnostic reagent, The reagent kit for animal brucellosis epidemic investigation, the application in the preparation of Brucella vaccine and the preparation of Brucella DNA vaccine. the