CN104151406A - Bovine brucellosis outer membrane protein Omp22, coding gene as well as cloning method thereof, and application - Google Patents
Bovine brucellosis outer membrane protein Omp22, coding gene as well as cloning method thereof, and application Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/23—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Brucella (G)
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
Technical field
The present invention relates to a kind of gene, especially relate to a kind of outer membrane protein (outer membrane protein, Omp) and gene and gene clone method and application thereof of expressing on Brucella abortus (Bovine brucellosis) adventitia.
Background technology
Brucellosis (brucellosis) is called for short cloth disease, claims again Mediterranean Sea remittent fever, Malta fever, and brucellosis or Malta fever, be the infecting both domestic animals and human whole body transmissible disease being caused by brucella.Belong to the two class transmissible diseases of China.The sick Epidemic Scope of cloth is wide, and the time length is long, and not only serious threat human health and livestock industry are produced, and has also caused huge financial loss and serious public health problem simultaneously.The annual ox of China, sheep, pig infection have 1,000,000 more than, and the financial loss causing can reach tens00000000 yuan.Cloth disease is to perplex since the establishment of the nation the long-standing problem of Animal Husbandry in China development and people ' s health always, is that people cures and the important diseases of animal doctor's common defence.Countries in the world are all classified as focal disease and are prevented and treated, and this disease of International Animal Health organization prescribed is the epidemic disease that various countries must report.Along with integrating with and people's living standard raising of China and World Trade Organization, control and eliminate cloth disease and more and more seem important in livestock industry work.At dairy industry industrial circle, producers' infection event causes product discarded recovery in batches, and manufacturer is caused to serious financial loss, and the harm of brucellosis especially receives much concern.
For the effectively impact of containment brucellosis on dairy, guarantee public health security, strengthen integrated control to milk cow brucellosis and the work of disease surveillance and just seem very important.Regularly quarantine is that the control of brucellosis and the important measures of purification are carried out in diary farm.And the detection method of brucellosis is the important factor that affects brucellosis quarantine effect.The at present domestic method generally adopting is: rose bengal precipitation test screens for the first time, then utilize tube agglutination test to carry out finally qualitative, and the method for the first screening adopting in the world mainly contain: indirect enzyme-linked immunosorbent assay (I-ELISA) and fluorescence polarization test.Rose bengal precipitation test specificity is not high, and the impact of the relatively short and artificial subjective factor of the temperature that is put to the test, aggegation time, is difficult for accurate result of determination, can only serve as primary dcreening operation; And tube agglutination test is the legal detection decision method of China's brucellosis, but the method complex operation, time-consuming is unsuitable for field by using, is also subject to the impact of artificial subjective factor.OIE standard specifies to adopt ELISA and the fluorescence polarization international trade tentative diagnosis method as brucellosis.Susceptibility and specificity that ELISA and fluorescence polarization method detect monitoring are good, and the reaction times is short, has avoided the interference of human factor, easily stdn and quality control, but these two kinds of methods also can only be used for primary dcreening operation.Fluorescence polarization is tested due to its specificity of more giving prominence to, susceptibility and high efficiency, but operator's state of the art is required.Supervise prevention and cure work hope easier, the efficient monitoring tool of one and the methods for the treatment of of current cloth disease; therefore inquire into new protective antigen and the immunologic mechanism of animal brucellosis; induce high-caliber body fluid and cellular immunization; improve the detection of brucellosis and infect state of an illness level of control, the financial loss that reduces livestock industry becomes livestock industry and is badly in need of the problem solving.
Researchist finds the difficult control of brucellosis, exactly because brucella can escape host's immunity identification.Along with the evolution of pathogenic bacteria, they need necessary virulence factor to invade host, and in host internal breeding.Outer membrane protein is brucellar main immunogens and protective antigen.For more accurate vaccine immunity, to control and treatment brucellosis, Diagnosis of Primary and the immunogen of finding property of protein have great importance.External research work has been carried out the research of immunogenicity and protectiveness to some brucellar outer membrane proteins.
According to the size of molecular mass, brucella outer membrane protein is divided into 3 groups at present.First group has outer membrane protein 10k, 18k, 19k, and wherein Omp10, Omp16 and Omp19 are a kind of lipoprotein.The 2nd group comprises 36k～38k outer membrane protein.The 3rd group comprises 31k～34k outer membrane protein and 25k～27k outer membrane protein, and Omp25 and Omp31 and brucellar virulence and immunogenicity have substantial connection.The mutant strain of Omp22 gene, the susceptibility of cloudy sero-reaction strengthens, and in growth stationary phase difficulty, these are all likely relevant with the virulence attenuation of of Omp22 genovariation strain.Research finds that Omp22 immunogenicity is stronger; can induction of immunity laboratory animal producing high titre specific antibody Omp22 encoding gene and purifying protein becomes the more first-selected mark of diagnosis and the prevention of cloth disease; can be used as potential drug target simultaneously, can in body protective immune response, play important effect.But the applied research of brucella bacterial outer membrane protein Omp22 sequence is very few at present, has no report about the brucella outer membrane protein Omp22 of ox, has greatly limited the Supervise prevention and cure as the milk cow disease of dairy industry economic animal.
Summary of the invention:
The object of the present invention is to provide cloning process, clone's recombinant bacterial strain and the application thereof of a kind of Brucella abortus outer membrane protein Omp22 and encoding gene and encoding gene.The present invention in-depth research, the diseases prevention and treatment of Niu Bulushi germ mechanism of causing a disease and be further developed as pharmaceutical prod and livestock industry diagnostic products aspect have a good application prospect, and lay the foundation for the monitoring of animal health state.
The present invention can produce cloning vector and the conversion bacterial strain of the encoding gene of B.abortus outer membrane protein Omp22, it is B.abortus recombinant bacterial strain (Bovine brucellosis recombination strain) PGEM-7Zf-omp22, and it has the ability copying as the nucleotide sequence of sequence <400>4.
To achieve these goals, solution of the present invention is:
1. a Brucella abortus outer membrane protein Omp22, it is characterized in that, by 1) protein that forms of the aminoacid sequence shown in SEQ ID NO.1, or 2) the aminoacid sequence shown in SEQ ID NO.1 be substituted, lack or add one or several amino acid and have same isoreactivity by 1) derivative protein.
The gene of a kind of above-mentioned Brucella abortus outer membrane protein Omp22 that encodes.
The encoding gene of described Omp22, have lower a) or b):
A) nucleotide sequence is as shown in SEQ ID NO.2; Or
B) by nucleotide sequence shown in SEQ ID NO.2 through generation, lack or add one or several Nucleotide obtain encoding nucleotide sequence of Brucella abortus outer membrane protein Omp22 claimed in claim 1.
For a primer pair for the sequence described in the claim 2 or 3 that increases, it is characterized in that, described primer pair sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4.
A kind of transformation cell lines containing carrier described in claim 5.
Described in claim 2 or 3, a cloning process for Brucella abortus outer membrane protein Omp22 gene, is characterized in that comprising the steps:
1) from Brucella abortus liquid culture, extract genomic dna as template,
2) the PCR primer of design B.abortus outer membrane protein Omp22 gene is as follows:
3) Omp22 gene PCR amplified reaction,
4) purifying of Omp22 gene PCR amplified production,
5) Omp22 gene PCR amplified production cut and be connected with enzyme cloning vector,
6) conversion of host cell,
7) screening and identification of transformation cell lines.
Described in claim 2 or 3, the application of gene, is characterized in that, described Omp22 gene clone can be used for the monitoring of brucellosis and the target as medicine research.
Particularly can be used for the diagnostic reagent that preparation contains claim 1～3 any one.
Particularly, be included in as brucellosis application, the test kit of the target of drug research, animal brucellosis diagnostic reagent, the sick investigation of epidemic situation of animal cloth, prepares brucella vaccine, prepares the application in brucella DNA vaccination.
" separation " of the present invention, " purifying " DNA refer to, this DNA or fragment are separated from the sequence that is arranged in its both sides from native state, also refer to this DNA or fragment with native state under the component of the nucleic acid followed separate, and separate with the protein of following it in cell.
Said carrier can be selected various carrier known in the art in the present invention, as commercially available carrier, comprises plasmid, clay etc.Cloning vector is the carrier of high copy mostly; it is generally protokaryon bacterium; needs clone's gene is connected with the plasmid of cloning vector; import again in protokaryon bacterium; plasmid can be in protokaryon bacterium massive duplication; form a large amount of gene clones, the gene being cloned not necessarily can be expressed, but necessarily by massive duplication.Cloning vector object is to copy abundant target plasmid, thus normal with stronger self-replacation element, as replication origin etc., often in thalline, there is multiple copied, can extract out a lot of so take out plasmid.But do not possess Expression element.Preferred plasmid of the present invention, particularly preferably PGEM-7Zf vector is that empty carrier is prepared cloning vector.
-7Zf (+) and
-7Zf (-) Vector is by carrier
-3Zf (+) is derivative, the replication orgin that comprises filobactivirus f1.These two kinds of carriers can be used as standard cloning vector, also can be as the template of in-vitro transcription and preparation ring-type ssDNA.In the α-peptide-coding region of the beta-galactosidase enzymes on carrier, have multiple clone site, and then multiple clone site also contains SP6 and t7 rna polymerase promotor.Select suitable E.coli bacterial strain (such as JM109), the recombinant clone that just can α-peptide be inserted to inactivation by the method for blue/white screening directly identifies.Multiple clone site region is unique, and contains some restriction sites, as ApaI, and AatII, SphI, XbaI, XhoI, EcoRI, KpnI, SmaI, Csp45I, ClaI, HindIII, BamHI, SacI, BstXI and NsiI.These constructional features are beneficial to massive duplication and the screening of the object of the invention gene.
In the time producing outer membrane protein Omp22 of the present invention, brucella albumen Omp22 encoding sequence operationally can be connected in and copy after initiating sequence, thereby form brucella outer membrane protein Omp22 encoding gene cloning vector.Said " being operationally connected in " refers to so a kind of situation, and some part of linear DNA sequence can affect the activity of same other parts of linear DNA sequence.
Described " host cell " is prokaryotic cell prokaryocyte.Conventional prokaryotic host cell is intestinal bacteria (E.coli) or subtilis etc., preferably E.coli DH5 α, E.coli BL21 and E.coli TOP10 etc., particularly preferably E.coli DH5 α.
The clone of Brucella abortus outer membrane protein Omp22 encoding gene full length sequence of the present invention or its fragment can use pcr amplification method, recombination method or screening libraries to obtain conventionally, particularly preferably pcr amplification method.For pcr amplification method, can design primer by relevant nucleotide sequence disclosed according to the present invention, and with brucella as template, amplification and must relevant sequence.
In brucella Brucella abortus outer membrane protein Omp22 encoding gene cloning process of the present invention, the preparation method of template is simple, only by the centrifugal easy steps with boiling, draw supernatant.Design of primers is introduced BamH I and Xho I restriction enzyme site at upstream and downstream primer respectively, and the sticky end of formation is conducive to produce efficient enzyme and connects effect.
Once obtain relevant sequence, just can obtain in large quantity relevant sequence with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated and obtains relevant sequence from the host cell propagation by ordinary method.
In addition, also can synthesize relevant sequence by the synthetic method of artificial chemistry.Before the application, prior art completely can be by first synthetic multiple polynucleotide small segments, and then connect and obtain the nucleotide sequence of code book invention outer membrane protein Omp22.Then, can be by various existing DNA moleculars (as carrier) and cell in this nucleotide sequence introducing this area.In addition, also can will suddenly change and introduce in protein sequence of the present invention by chemosynthesis.
The encoding gene cloning vector of the Brucella abortus outer membrane protein Omp22 that technique scheme obtains provides sufficient material for the present patent application people has carried out deep bioinformatic analysis to clone.
The invention has the beneficial effects as follows:
1. adopt the inventive method to can be mass the encoding gene of Brucella abortus outer membrane protein Omp22, preparation method is simple simultaneously, and template extraction does not need to expend chemical reagent; Simultaneously the present invention can efficient replication goal gene, has advantages of environmental protection and economic.
2. there is good application prospect diagnostic monitoring and prevention and control aspect that the encoding gene of the product Brucella abortus recombinant bacterial strain outer membrane protein Omp22 that technical scheme of the present invention obtains is cloned in brucellosis, be included in as brucellosis application, the test kit of the target of drug research, animal brucellosis diagnostic reagent, the sick investigation of epidemic situation of animal cloth, prepares brucella vaccine, prepares the application in brucella DNA vaccination.
3. brucella and the host research for application and development of interactional mechanism and protein function research and outer membrane protein Omp22 expression in pathogenic course is understood in the coding gene sequence information of the product Brucella abortus outer membrane protein Omp22 that technical scheme of the present invention obtains and cloning vector horn of plenty and in-depth provides continuable feedstock production process, therefore has important society and commercial value.
4. utilize the physico-chemical property of information biology instrument and Network System Analysis Brucella abortus outer membrane protein Omp22, as the power of the size of the size of iso-electric point, molecular weight, absorbancy and length amino acid sequence.Be that wetting ability and snappiness are higher region by the analysis 56-62 amino acids of our above information biology instrument.These analyses and prediction results all will play directive function to research and the analysis of Brucella abortus outer membrane protein Omp22.This contributes to us, and next step takes reasonably strategy to express outer membrane protein OMP22, improves the expression of OMP22 outer membrane protein, and expects active recombinant protein, for diagnosis, medicine and the vaccine research of brucellosis provide material base.
Brief description of the drawings
Fig. 1 .Omp22 gene amplification result (M.DL2000DNA Marker; 1,2.PCR product)
The enzyme of Fig. 2 .Omp22 gene recombination plasmid is cut qualification (M.DL2000DNA Marker; 1.BamHI single endonuclease digestion product; 2.BamHI and Xho I double digestion empty carrier PGEM-7Zf product .3,4.BamHI and Xho I double digestion recombinant plasmid PGEM-7Zf-Omp22 product)
Fig. 3. the molecular flexibility analysis of Bacillus brucellae Omp22 albumen
Fig. 4. the hydrophilicity analysis of Bacillus brucellae Omp22 albumen
Fig. 5. the linear epitope analysis of B cell of outer membrane protein Omp22
Fig. 6. the Characterization of antigenic epitopes of outer membrane protein Omp22
Fig. 7. locus (the 1.N-glycosylation site of the 3D structure of outer membrane protein Omp22 and structural domain; 2. protein kinase C phosphorylation site; 3. casein kinase ∏ phosphorylation site)
Accompanying drawing 8. predictions and the interactional protein network system of outer membrane protein Omp22
Embodiment
The clone of embodiment 1 Brucella abortus Omp22 gene
Bacterial strain and plasmid
B.abortus 544A is separating obtained by Xarachin District; Bacillus coli DH 5 alpha, preserve in this laboratory.Cloning vector PGEM-7Zf vector is purchased from PROMEGA company
Main agents and equipment
Restriction enzyme BamH I, Xho I, Taq polysaccharase, toolenzyme, the DNA standard molecule mass M arker such as T4DNA ligase enzyme are all purchased from the precious biotech firm in Dalian; Plasmid extraction kit is purchased from Qiagen company, and DNA glue reclaims test kit purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd; Gel imaging system is purchased from (France); PCR instrument (U.S.); Cryogenic thermostat circulator (sky, Ningbo perseverance); Other reagent are domestic analytical pure.
1. the cultivation of original Omp22 B.abortus strain and genomic dna preparation
By streak culture the B.abortus separating from Horqin Region, northeast 3%TSB solid medium, growth 48h chooses single bacterium colony and is placed in 3%TSB liquid nutrient medium, 200r/min shake 48h gets 2ml bacterium liquid and is placed in respectively two aseptic 1.5ml centrifuge tubes, 12000r/min is centrifugal, add aseptic distillation water washing 4 times, boil 10min, centrifugal absorption supernatant, be DNA profiling, be placed in-20 DEG C of Refrigerator stores after packing for subsequent use.
2.Omp22 full length gene clone
(1) design of primers is with synthetic
The B.abortus strain AM712379.1 including with reference to GenBank and the coding region sequence of AM712380.1 gene, in conjunction with reading frame, the multiple clone site of cloning vector PGEM-7Zf, the PCR primer of application Primer5.0 design B.abortus outer membrane protein Omp22 gene is as follows:
Upstream 5`-GA
gGGATCcGGAATGTTCAAGCGTTCTATCACC-3;
Downstream 5`G
cTCGAGcTAGAATTTGTAGTTCAG-3.
This primer sequence is synthetic by the Shanghai biological company limited of raw work, and introduces BamH I and Xho I restriction enzyme site at upstream and downstream primer respectively.
(2) pcr amplification
Pcr amplification system is 50ul; Wherein dNTP mixture6ul, TaKaRa Ex Taq 0.5ul (1.25U), Bacillus brucellae DNA profiling 1ul, Ex Taq Buffer5ul, the each 1ul of upstream and downstream primer, last MilliQ H2O is to cumulative volume.Pcr amplification condition: 95 DEG C of denaturation 10min, 94 DEG C of sex change 1min, 55 DEG C of annealing 1min, 72 DEG C are extended 2min, circulate 30 times, finally at 72 DEG C of total elongation 10min.
(3) pcr amplified fragment reclaims
PCR product 5ul mixes with sample-loading buffer, electrophoresis in 1.0g/L sepharose (containing EB), and gel image analysis instrument is taken pictures.Product electrophoretic analysis in 1% sepharose finds there is a band in 639bp left and right, and size conforms to the size of estimating, sees Fig. 1.
(4) structure of recombinant plasmid and qualification
By PCR product, after BamHI and Xho I double digestion, directed insertion in the cloning vector PGEM-7Zf of same double digestion, obtains recombinant plasmid called after PGEM-7Zf-Omp22.Recombinant clone PGEM-7Zf-Omp22 plasmid is cut with BamHI and the single, double enzyme of Xho I respectively, and electrophoresis in 1% sepharose, is shown in Fig. 2 simultaneously, all obtains the big or small goal gene fragment of expection, shows Omp22 gene clone success.
(5) structure of recombinant bacterial strain
I. the conversion of competent cell preparation and recombinant plasmid
Bacillus coli DH 5 alpha nutrient solution is transferred to the EP pipe of an ice-cold 1.5ml, 4 DEG C, 4000rpm × 5min; Abandon supernatant, in precipitation, add ice-cold 0.1mol/L CaCl
2300 μ l, resuspended thalline, ice bath 20min; 4 DEG C, 4000rpm × 5min abandons supernatant, and pipe is inverted in to 1min on filter paper, makes dried liquid stream clean; In precipitation, add ice-cold 0.1mol/L CaCl
2100 μ l, resuspended thalline; Ice bath 10min; Every pipe adds plasmid 5 μ l, rotates gently test tube, mixes mixture, places 20min test tube and put into the water-bath of 42 DEG C on ice bath, places 90s, does not shake test tube, rapidly test tube is transferred to ice bath, cooling 1～2min; Take out after test tube, every pipe adds LB substratum 800 μ l, is placed in 37 DEG C of shaking tables (100～150r/min), gentle concussion 45min; Spread bacterium device with aseptic elbow glass 200 μ l bacterium liquid are laid on to the agar plate surface containing Amp; Room temperature is placed 20min, makes liquid be absorbed (sweating); Be inverted plate, 37 DEG C of cultivations, 12 hours visible colony growths;
Ii. the screening of recombinant cloning and qualification: blue hickie experiment
Experiment reagent
X-gal:2% mother liquor (with dimethyl formamide preparation, wrap destroyedly to prevent being subject to illumination with aluminium foil or black paper ,-20 DEG C save backup), working concentration 20ul/20ml flat board;
Penbritin (Amp): be mixed with 100mg/ml mother liquor with sterilized water, put-20 DEG C of Refrigerator stores.Working concentration 100ug/ml;
IPTG: mother liquor 100mmol/L ,-20 DEG C of Refrigerator stores.Working concentration 40ul/20ml flat board;
LB liquid nutrient medium: 1% peptone, 0.5% yeast extract, 1%NaCl, with NaOH tune pH to 7.2,121 DEG C of sterilizing 20min are for subsequent use.Solid LB substratum adds 1.5%～2% agar in LB liquid nutrient medium, for subsequent use after sterilizing;
LB solid medium containing Amp: by being cooled to 60 DEG C of left and right after the LB solid medium autoclaving preparing, add Amp storage liquid, making final concentration is 100ug/ml, shakes up rear bed board;
Escherichia coli DH5a.
Experimental procedure
(1), get 4ul and connect product and add in 100ul competent cell, blow and beat gently evenly ice bath 30min with rifle.
(2), pipe is placed in to 42 DEG C of water-bath heat shock 90sec of water-bath, place at once 5min on ice.
(3), add the LB substratum of 1ml37 DEG C of preheating, mix.
(4), pipe is placed in to 37 DEG C of shaking culture 1h on constant-temperature table.
(5), pipe is placed in to the centrifugal 5min of whizzer 3000rpm.
(6), abandon 1ml supernatant, remaining about 100ul supernatant, blow gently with rifle even, for coated plate.
(7), one containing on the LB flat board of penbritin, add 20 μ l20mg/ml X-gal and 40 μ l100mmol/L IPTG.
(8), glass spatula is overdoed after sterilizing and is stretched in culture plate, even spread plate after it is cooling, the glass spatula sterilizing that overdoes is placed in alcohol for subsequent usely, and it is dull and stereotyped that to place 30min in room temperature for subsequent use.
(9), by aforementioned gained containing the bacterium liquid of recon be drawn to prepare containing on the flat board of X-gal, be evenly coated with glass spatula.Flat board is placed in after the positive 30min of placement of biochemical cultivation case, then is inverted, in 37 DEG C of overnight incubation, select White strain.
Embodiment 2 B.abortus bacterial strain Omp22 gene sequencing and sequential analysis and bioinformatic analysis
B.abortus bacterial strain Omp22 gene sequencing standard sequence used:
Show through order-checking, B.abortus recombinant bacterial strain outer membrane protein Omp22 mrna length 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 this gene, applying biological information software (danman and Vector NTI) is derived 212 amino-acid residues of Omp22 coded by said gene, and molecular weight is 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 sequencing: the DNA sequence dna of the B.abortus Omp22 gene that successfully to have obtained length be 639bp through checking order, utilize on-line analysis tools BLAST ORF Finder (http://blast.ncbi.nlmg.nih.goV/Blast.cgi) and http://blast.ncbi.nlmg.nih.goV/gorf/orfig.cgi in NCBI) compare with ORF and search discovery, in this sequence, CDS sequence length is 639bp, as SEQ No.4; According to the nucleotide sequence of this gene, applying biological information software (danman and Vector NTI) is derived 212 amino-acid residues of Omp22 coded by said gene, and molecular weight is about 22kDa; On nucleotide level, 11-645 sequence in the whole coding sequence (GenBank Accession No.AM712379) of it and standard brucella (Brucella microti CCM 4915) outer membrane protein Omp22 has 88% homogeny (in table 1), and table 2 is the homology comparison (BLAST) of the aminoacid sequence (GenPept Accession No.AM712380) of brucella outer membrane protein of the present invention and standard brucella (Brucella microti CCM4916) outer membrane protein Omp22.On amino acid levels, the amino-acid residue of it and standard brucella outer membrane protein Omp22 has high homogeny and similarity (in table 2).Wherein, identical amino acid marks with amino acid monocase between two sequences, and similar amino acid marks with "+".
The Nucleotide BLAST result of the encoding gene of table 1. Brucella abortus recombinant bacterial strain of the present invention outer membrane protein Omp22 and the outer membrane protein Omp22 encoding gene of reference culture
The BLAST result of the aminoacid sequence of table 2. Brucella abortus recombinant bacterial strain of the present invention outer membrane protein Omp22 and the outer membrane protein Omp22 aminoacid sequence of reference culture
Find that by BLAST we separate the coded nucleotide sequence of the outer membrane protein Omp22 that obtains from milk cow emulsion and the outer membrane protein OMP22 of reference culture has higher homology.Result is bright, and this albumen is outer membrane protein.
(2) chemical feature of Omp22 protein molecular
Utilize the main chemical feature of bioinformatics software (danman and Vector NTI) and database (prosite and PDB) analysis Bacillus brucellae outer membrane protein Omp22 molecule in table 3, Fig. 3 and Fig. 4
The main chemical property of table 3 B.abortus outer membrane protein Omp22 protein molecular
Utilize the dependency of physico-chemical property and B cell antigen epi-position can analyze and predict by phenomenological theory the B cell antigen epi-position of Brucella abortus OMP22, B cell antigen epi-position region is mostly Brucella abortus outer membrane protein Omp22 wetting ability and the high amino acid section of snappiness score value, is that wetting ability and snappiness are higher region by the analysis 56-62 amino acids of our above information biology instrument.These analyses and prediction results all will play directive function to research and the analysis of Brucella abortus outer membrane protein Omp22.
The displaying of Vector NTI suite software to protein steric structure, provides theoretical and supports for we understand the structure of Brucella abortus outer membrane protein Omp22 and the relation of function and the pathogenesis of bacterium in depth.By the modification on gene level to Brucella abortus OMP22 structural domain, or the sudden change of signal peptide cutting site, as by potential N-glycosylation site deletion mutantion, may change to the immunne response of Brucella abortus outer membrane protein Omp22 and host cell, the function provider who can be further investigation outer membrane protein Omp22 to.Obtain according to prediction in prosite database simultaneously with the interactional protein network system of outer membrane protein Omp22, this provides certain theory support for research interactions between protein.
(3) analysis on immunological characterization
(i) by B cell epitope on-line analysis instrument
Predict that this albumen contains 12 potential B cell antigen linear epitopes.Wherein the longest B cell linear epitope is made up of 26-71 amino acids.Always have 44 amino acid and form, can be used as potential antigen target, see Fig. 5, Fig. 6.
(ii) predict that by epitope on-line analysis instrument this albumen contains 9 potential epitopes.Wherein the longest epitope is by 21 Amino acid profiles, between 157-177 amino acid, can pay the utmost attention to as antigen target.
(iii) transformylase domain analyses
Analytical results: Swiss-model outer membrane protein Omp22 mates with the protein three-dimensional structure in protein structure database, the tomograph of defeated time simulation, file is opened this protein structure file in vector NTI suite software package, and the structural domain of outer membrane protein is marked on Fig. 7.See Fig. 8 according to protein-interacting neural network forecast and the interactional protein of outer membrane protein Omp22.
Claims (10)
1. a Brucella abortus (Bovine brucellosis) outer membrane protein Omp22, it is characterized in that, by the protein that a) aminoacid sequence shown in SEQ ID NO.1 forms, or b) the aminoacid sequence shown in SEQ ID NO.1 be substituted, lack or add one or several amino acid and have same isoreactivity by a) derivative protein.
2. coding Brucella abortus outer membrane protein Omp22 gene claimed in claim 1.
3. Brucella abortus outer membrane protein Omp22 gene according to claim 2, its be following a) or b):
A) nucleotide sequence is as shown in SEQ ID NO.2; Or
B) be substituted, lack by nucleotide sequence shown in SEQ ID NO.2 or add one or several Nucleotide obtain encoding nucleotide sequence of Brucella abortus outer membrane protein Omp22 claimed in claim 1.
4. for a primer pair for the gene described in the claim 2 or 3 that increases, it is characterized in that, described primer pair sequence, as shown in SEQ ID NO.3 and SEQ ID NO.4, is respectively:
Upstream 5`-GAGGGATCCGGAATGTTCAAGCGTTCTATCACC-3;
Downstream 5`-GCTCGAGCTAGAATTTGTAGTTCAG-3
Described primer pair is introduced BamH I and Xho I restriction enzyme site at upstream and downstream primer respectively.
5. containing the cloning vector of gene described in claim 2 or 3, it is characterized in that the empty carrier being connected with described gene is plasmid or cosmid vector.
6. containing the transformation cell lines of cloning vector described in claim 5.
7. a cloning process for Brucella abortus outer membrane protein Omp22 gene described in claim 2 or 3, is characterized in that, comprises the steps:
1) pyrolysis method extracts genomic dna as template from Brucella abortus liquid culture,
2) the PCR primer pair sequence of design B.abortus outer membrane protein Omp22 gene is as shown in SEQ ID NO.3 and SEQ ID NO.4,
3) pcr amplification Omp22 gene
4) purifying of Omp22 gene PCR amplified production
5) Omp22 gene PCR amplified production on empty carrier, form cloning vector with being operationally connected in of cloning vector,
6) conversion of host cell,
7) screening and identification of transformation cell lines.
8. the preparation method of Omp22 gene as claimed in claim 7, is characterized in that, described empty carrier is plasmid or cosmid vector, and described prokaryotic cell prokaryocyte is bacillus coli DH 5 alpha.
9. the preparation method's of Omp22 gene as claimed in claim 7 preparation method, is characterized in that, in step 2) in, described host cell is prokaryotic cell prokaryocyte.
10. the application of Omp22 albumen or encoding gene described in claim 1～3 any one, it is characterized in that, be included in the test kit as the target of the monitoring of brucellosis and medicine research, animal brucellosis diagnostic reagent, the sick investigation of epidemic situation of animal cloth, prepare brucella vaccine, prepare the application in brucella DNA vaccination.
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