CN106749562A - A kind of gene expression product BLSJ 1 of brucella diagnostic marker effect and preparation method thereof - Google Patents

A kind of gene expression product BLSJ 1 of brucella diagnostic marker effect and preparation method thereof Download PDF

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CN106749562A
CN106749562A CN201611077399.9A CN201611077399A CN106749562A CN 106749562 A CN106749562 A CN 106749562A CN 201611077399 A CN201611077399 A CN 201611077399A CN 106749562 A CN106749562 A CN 106749562A
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朱良全
丁家波
蒋卉
彭小薇
张磊
范学政
孙翠丽
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China Institute of Veterinary Drug Control
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Abstract

Gene expression product BLSJ 1 the present invention relates to a kind of effect of brucella diagnostic marker and preparation method thereof.The product is to number the gene prokaryotic that (GI) is 489054867 by brucella S2 pnca genes, and purifies acquisition through glutathione s-transferase (GST) affinity column and glutathione linear gradient elution method.Using the product as envelope antigen, can be used as the diagnostic antigen for distinguishing animal brucella vaccine immunity and natural infection Virus monitory.The antigen is carried out into Western blotting (Western blot) with vaccine immunity antibody and natural infection antibody respectively, difference is obvious.Using the antigens of BLSJ 1 as indirect ELISA method (ELISA) envelope antigen, hundreds of parts of cloth disease clinical serum samples of detection, antidiastole effect is significant.

Description

A kind of gene expression product BLSJ-1 of brucella diagnostic marker effect and its preparation Method
Gene expression product BLSJ-1 and its system that technical field is acted on the present invention relates to a kind of brucella diagnostic marker Preparation Method, belongs to veterinary microbiology diagnostic field.
Background technology
Brucellosis (Brucellosis is referred to as cloth disease) is the infecting both domestic animals and human caused by brucella (Brucella) Infectious disease.People's infection cloth disease mainly carrys out infected animal and products thereof.China's infection sources relevant with mankind's cloth disease is mainly trouble Sick sheep, ox, but with brucella melitensis harm the most serious (the full et al. microbiologies circulars of Zhu Liang, 2015 (01):171~ 177；Wu Qing people animal doctor guide, 2011 (09):46~47).
Serological method is the main method of Current Diagnostic animal cloth disease, and vaccine inoculation is the important of prevention animal cloth disease Means.The existing serodiagnosis of cloth disease is based primarily upon the anti-brucella lipopolysaccharides antibody of detection, due to the existing animal cloth of China Disease vaccine strain (S2, A19 and M5) belongs to smooth type bacterial strain with wild virus infection strain, induces body to produce lipopolysaccharide antibody resisting, Vaccine immunity and natural infection thus cannot be distinguished by existing serological method, and (Mao Kairong waits animal brucellosis to diagnose Technology Chinese agriculture publishing houses, 2014.).
But, there is notable difference in the serology antibody response after strong and weak poison Infected with Brucella host, is from serology Upper screening distinguishes natural infection and provides some backgrounds and clue with the antidiastole antigen of vaccine immunity.Table is such as studied It is bright, after strong poison infection brucella, 7 days detectable antibody, antibody level reaches highest within 15 days, and its agglutinating antibody titre reaches 1：3000, then begin to decline, antibody level when antibody level was already below 7 days after 60 days, and antibody is feminine gender after 240 days (Gao XL.et al Turkish Journal of Veterinary and Animal Sciences,2015,39:271~ 278).And after brucella vaccine S2 immune sheeps, start to detect antibody after 2 weeks, antibody level reaches highest within the 30th day, its Titre is 1:120, then begin to decline, to 180 days after antibody for feminine gender.Above-mentioned display it is immune and natural infection serum between Different information open new approaches for the screening of antidiastole point.
It is reported that memebrane protein occupies about the 30%~40% of pathogenic microorganism gross protein, be not only medicine and Where the major target class of vaccine development, and containing protective antigens composition, thus as brucella diagnostic antigen and medicine (Hu Jianfei waits disease surveillance, 2010 (05) to the key object of therapy target research:380~389；Wu Shuo, waits immunologys miscellaneous Will, 2008 (04):385~388).Because immunoproteomics method can show albumen serology response difference information, and can transport With bioinformatics technique as the technology platform of high flux screening, a large amount of possible antidiastole antigens are obtained from memebrane protein Information, so as to crack the problem of vaccine immunity and natural infection.
The content of the invention
The purpose of the present invention is directed to existing serological method and cannot be distinguished by brucella vaccine immune antiboidy and natural sense Dye antibody problem, there is provided the brucella gene expression product with differential diagnosis value, with immunological method, so as to realize The antidiastole of vaccine immunity antibody and natural infection antibody.
Technical solution of the present invention
1. a kind of expression product BLSJ-1 of brucella diagnostic marker effect gene, it is characterised in that the product is by cloth Shandong Salmonella S2 pnca genes numbering (GI) is 489054867 gene, is expressed as " general stress protein (universal stress protein)”；The mrna length：450bp；The molecular weight of albumen of its expression product about 15.5kDa.Its gene order is sequence 1：
2. the preparation method of the expression product BLSJ-1 of a kind of brucella diagnostic marker effect gene of the present invention, It is characterized in that the method is comprised the following steps：
(1) genescreen：From brucella S2 pnca gene groups, by immunoproteomics method, on Screening and Identification goes out State the gene of right 1；
(2) gene expression：By the primer (sequence 2 and sequence 3) for designing, by Standard PCR clonal expansion, it is placed on Induced expression in pGEX6p-1 carriers；
(3) expression product purifying：By glutathione s-transferase (GST) affinity column and glutathione gradient elution Method purified expression product, antigen needed for obtaining, is named as BLSJ-1.
3. the application of the expression product BLSJ-1 of a kind of brucella diagnostic marker effect gene of the present invention, it is special Levy is that, by the gene PCR clonal expansion, used as antigen, the antigen can be used as differentiation animal brucella vaccine for its expression product Immune and natural infection Virus monitory antigen.
Detailed description of the invention
1. antidiastole identifies the screening of gene
(1) immunoproteomics technology, the most widely used brucella vaccine strain S2 of selection China and China's cloth are used The serious sheep blood serum of disease influence is research object, and antidiastole antigen gene information is filtered out from S2 plants of memebrane protein.
(2) it is each with 30 parts of cloth disease negative healthy sheep (Nm), 30 parts of S2 immune sheeps (Sm), and 30 parts of clinical cloth disease infection sheep Positive pooled serum (Zm), carries out Western blotting (Western-blot) with S2 plants of memebrane protein two dimensional electrophoresis glue (2DE) respectively, seeks S2 memebrane proteins and different conditions (infection/immune/negative) sheep serum Low Response dissimilarity are looked for, 113 differential proteins are searched out altogether Point.30 protein sites that selection immune response differs greatly carry out substance assistant laser desorpted ionized-flight time (MALDI- TOF) Mass Spectrometric Identification and bioinformatic analysis, identify 14 albumen altogether.
(3) 3 kinds of pooled serums (infection/immune/negative) are carried out into co-immunoprecipitation (IP) with S2 memebrane proteins respectively, and it is right IP conjugates carry out high flux Mass Spectrometric Identification (Q-Exactive mass spectrographs) and biological information analysis, obtain 182 candidate's eggs White point.Functional analysis is carried out to all albumen that 2 kinds of methods are identified, 8 candidate targets of external antidiastole are picked out altogether.
(4) 8 candidate's target protein prokaryotic expression plasmids are built, expressing protein is immunized with 3 kinds of pooled serums respectively Trace (Western-blot) and enzyme linked immunosorbent assay (ELISA) (ELISA), screen a gene with antidiastole effect (general stress protein, universal stress protein) antidiastole effect is obvious.
2. the expression of gene outcome
(1) S2 plants of brucella of culture breeding, extracts its genome.
(2) upstream and downstream primer is respectively with sequence 2 and sequence 3, conventional PCR method carries out gene magnification.
Upstream EcoRI GAATTC：5 '-gggGAATTCa tgtataagag aatcctgatc-3 ' 30 (sequence 2)
Downstream XhoI CTCGAG：5 '-tttCTCGAGc ttacttgata accagcacc-3 ' 29 (sequence 3)
(3) gene magnification band, detects that fragment is in the same size with expected through in 1% agarose gel electrophoresis, and will The recombinant plasmid of acquisition send company to be sequenced and is carried out with the gene order announced in American National Biotechnology Information center (NCBI) Compare, coincidence rate is 100%.
(4) after the positive recombinant plasmid for screening is transformed into E.coli BL21 (DE3) competent cell of commercialization, use 1mM isopropyl-β-D-thiogalactosides (IPTG) induced expression, then carries out sodium dodecyl sulfate polyacrylamide gel Electrophoresis (SDS-PAGE), its recombinant protein molecular size range is basically identical with theoretical size respectively.
3. the purifying of expression product
(1) albumen of induced expression, is accredited as through SDS-PAGE (SDS-PAGE) Inclusion body.
(2) by the inclusion body through gradient urea element (4.5M, 3.5M, 2.5M, 2M, 1.5M, 1M, 0.5M, 0M) dialysis renaturation Buffer solution carries out gradient dialysis renaturation according to a conventional method in 4 DEG C.
(3) purified by its specification method with glutathione s-transferase (GST) fusion protein purification post.
4. expression product BLSJ-1 as diagnostic antigen application
By destination protein BLSJ-1 --- general stress protein (universal stress protein) uses glutathione S Transferase (GST) affinity column after purification, as enzyme-linked immunosorbent assay (ELISA) envelope antigen, is infected 30 parts Serum and 656 parts of immune sheep blood serum samples are detected that the enzyme-linked immunosorbent assay (ELISA) that it is set up is to infection sample This recall rate is 93%.
Brief description of the drawings
Fig. 1 S2 memebrane protein two-phase electrophoresis and the Western blotting (Western-blot) from different immune state sheep serums In result figure：Figure A is S2 memebrane protein two-phase electrophoresis results, and figure B-D is followed successively by memebrane protein and is mixed with cloth disease negative healthy sheep respectively Close serum (Nm), S2 immune sheeps pooled serum (Sm), the Western blotting (Western- of natural infection sheep pooled serum (Zm) Blot) result.Figure E is had an overlay analysis result by figure B-D.
Fig. 2 S2 memebrane protein co-immunoprecipitations result 1 is the IP mixtures of negative serum (Nm)；2 is the IP of S2 immune serums Mixture (Sm)；3 is the IP mixtures of natural infection serum (Zm).
Noted in S2 plants of antigen gene PCR amplification figure of Fig. 3 Brucella suis：Swimming lane M：Marker.Swimming lane 1-8 is volume The gene of number 1-8.
In 8 kinds of recombinant protein induced expression collection of illustrative plates figures of Fig. 4：M, protein molecular weight standard (Protein molecular weightmarker)；1-8, is 1-8# candidate albumens；U, does not induce mycoprotein；W, induces whole bacterial protein；S, supernatant；P, sinks Form sediment
6 kinds of Fig. 5 restructuring candidate albumens respectively with three kinds of Western blottings of serum (Western-blot)
The dodecane of the general stress protein BLSJ-1 (universal stress protein) of Fig. 6 2# restructuring purifying In base sodium sulphate polyacrylamide gel electrophoresis (SDS-PAGE) result figure：M：Protein molecular weight standard；2,2# candidate albumens； P, precipitation；Pu, the albumen of purifying.
The present invention relates to biomaterial resource information
The microbial resources being related in the present invention are S2 plants of pig kind brucella low virulent strains (CVCC70502 plants), by China Veterinary medicament supervision is identified, keeping and supply (are protected see see China Veterinery Drug Inspection Office, Chinese veterinary microorganism strain Hide administrative center to write, Chinese animal doctor's strain catalogue (second edition), Scientia Agricultura Sinica technology publishing house, p145 in 2002).
The positive effect of the present invention
Gene expression product the present invention relates to a kind of effect of brucella diagnostic marker and preparation method thereof.The product is By the gene prokaryotic that brucella S2 pnca genes numbering (GI) are 489054867, and through glutathione s-transferase (GST) Affinity column is purified with glutathione linear gradient elution method and obtained.Using the product as envelope antigen, can be used as differentiation animal cloth The diagnostic antigen of Shandong Salmonella vaccine immunity and natural infection Virus monitory.By the antigen respectively with vaccine immunity antibody and natural sense Dye antibody carries out Western blotting (Western-blot), and difference is obvious.Using the antigen as indirect ELISA method (ELISA) envelope antigen, hundreds of parts of cloth disease clinical serum samples of detection, antidiastole effect is significant.
Embodiment
Following examples are not limited the invention to better illustrate the present invention.
Embodiment 1
--- the screening study of antidiastole antigen protein point
Antidiastole antigen is screened from S2 plants of memebrane protein using immunoproteomics method.It is each negative strong with 30 parts of cloth diseases Kang Yang, 30 parts of S2 immune sheeps, and 30 parts of clinical cloth disease infection sheep positive pooled serums, respectively with S2 plants of memebrane protein two dimensional electrophoresis Glue (2D) carries out Western blotting (Western-blot), finds S2 memebrane proteins continuous from different cloth diseased states (infection/immune/negative) Sheep blood serum response difference, searches out 113 differential protein spots altogether.30 protein sites that selection immune response differs greatly carry out base Matter assisted laser desorption ionisation-flight time (MALDI-TOF) Mass Spectrometric Identification and bioinformatic analysis, identify 14 eggs altogether In vain, these albumen undertake 13 quasi-molecule functions, participate in composition 6 class cellular components and 13 quasi-biology processes.By 3 kinds of pooled serums (infection/immune/negative) carries out immunoprecipitation (IP) with S2 memebrane proteins respectively, and high flux mass spectrum (Q- is carried out to conjugate Exactive) identify, obtain 182 candidate albumen points, these albumen exercise 129 quasi-molecule functions, participate in 91 class cells of composition Component and 137 quasi-biology processes.Functional analysis is carried out to all albumen that 2 kinds of methods are identified, 8 external mirror are picked out altogether The candidate targets (numbering 1#-8#) not diagnosed.
8 candidate's target protein prokaryotic expression plasmids are built, except 1# (transport protein/transporter) and 4# (cell divisions Albumen FtsH/cell division protein FtsH) outward, 6 albumen successful expressions.Expressing protein is mixed with 3 kinds respectively Closing serum carries out Western blotting (Western-blot) and enzyme linked immunosorbent assay (ELISA) (ELISA) detection, filters out 2#BLSJ-1 (general stress protein, universal stress protein) antidiastole effect is obvious.
1. brucella S2 plants of bacterium solution prepare, Membrane protein extraction and quantitative
Brucella S2 plants of seed liquor prepares reference《People's Republic of China's regulations》((Ministry of Agriculture beast With the People's Republic of China (PRC) of biological products Rule Committee regulations (two 〇 〇 〇 editions) Beijing:Chemical industry Publishing house, 2000, the present invention is hereinafter referred to as《Code》) method carries out, Membrane protein extraction carries out (bent with reference to recommendation methods such as bent Qing Qing, waits Jilin University journal (medicine), 2009 (05):805~811).Membrane protein concentration is quantified, according to Coomassie brilliant blue (Bradford) protein quantification (Thermo Pierce article No.s：23236 protein quantification kits) method carries out.
2.S2 plants of memebrane protein two-phase electrophoresis and Western blotting (western-blot) result
Gel protein Point matching rate between the experiment of 2 different batches is up to more than 80%.First using solid in preliminary experiment Surely changing the adhesive tape of the gradients of pH 3~10 carries out one to separation, it is found that albumen is all mainly centrally located at pH 4~7.Therefore, select The adhesive tape of the gradient of immobilization pH 4~7 carries out one to separation, uses 12.5% SDS-PAGE (SDS-PAGE) dielectrophoresis (Figure 1A) is carried out, with the Western blotting (Western-blot) (Figure 1B -1D) of different sera incubations. Electrophoretogram passes through the softwares of PDquest 8.0 after being scanned into figure, by the Western blotting (Western-blot) of different sera incubations The figure of exposure is compared, to determine the difference of immune-reactive protein.According to Fig. 1 E results, with reference to the software analysis knots of PDquest 8.0 Really, 262 protein diversity points can be told altogether.30 obvious protein sites of grey value difference are chosen, through in-gel digestion and matter Spectrum identification.
3. two-phase electrophoresis (2DE) and substance assistant laser desorpted ionized-flight time (MALDI-TOF) mass spectral analysis is identified Data list
The gray scale value list (table 1) reacted in 30 points is as follows.Nm is immune with normal sheep serum anti-memebrane protein The gray value answered, Sm is the gray value of memebrane protein and immune sheep seroreaction, and Zm is memebrane protein and natural infection sheep serum Gray value.Ratio is the ratio of the immune gray value and natural infection between.
The grey value difference information list of 1 30 protein sites of table seroreaction different from 3 kinds
/:No data
4. substance assistant laser desorpted ionized-flight time (MALDI-TOF) Mass Spectrometric Identification result
Result shows, 29 points is identified altogether, wherein there is 9 points (to be directed to transcriptional regulatory activity 3, DNA replication dna 3 Individual, kytoplasm large ribosomal subunit 1 does not have functional 2), because C.I. values are insincere less than 90.20 points are in credible Area, it can be divided into 3 classes, molecular function (Molecular Function) 7 points, cellular component by bioinformatics body point (Cellular Component) 3 points, 12 points of biological process (Biological Process).Wherein there are 2 points to relate to And redox enzyme activity function can not only be classified as molecular function but also can be classified as biological process classification in.
5. co-immunoprecipitation result
Can be with IgG antibody F according to albumin A/G magnetic beads (protein A/G beads)CDuan Jinhang non-specificity covalent bonds Characteristic, 3 kinds of different serum antibodies that mix are respectively incorporated in albumin A/G magnetic beads (protein A/G beads), Ran Houzai Said mixture is combined with S2 memebrane proteins, according to antigen-antibody reaction principle, 3 kinds of different serum respectively with memebrane protein combination shape Resulting mixture, its electrophoretogram is shown in Fig. 2.
6. immunoprecipitation obtains mass spectrum point information and the possibility diagnosis target spot information obtained through bioinformatics functional analysis
Immunological response is carried out by natural antibody and non denatured antigen, the point that it catches is more, totally 284 points.Compare Abundance of the protein site identified in different sero-immunities precipitate (IP) mixture, wherein 54 protein sites and negative serum (Nm) there is nonspecific reaction.In remaining 230 protein sites with negative serum in the absence of nonspecific reaction, there are 50 Protein site only reacts with S2 immune serums (Sm)；There are 63 protein sites only to be reacted with natural infection serum (Zm)；With it is immune and from So infection serum has the protein site 117 of reaction, wherein the ratio between abundance in S2 serum and natural infection serum I P mixtures (Sm/Zm)>1.5 protein site has 12, Sm/Zm<0.67 protein site has 57.It can be seen that identifying possible antidiastole altogether Antigen protein 182.
By the retrieval of Uniprot gene ontologies (Gene Ontology) database, immunoprecipitation/high flux mass spectrum (IP/QE) 182 possible antidiastole antigen proteins of identification, by the gene ontology (Gene of its bioinformatics Ontology) dividing can be divided into 3 classes.It can be seen that, 182 albumen exercise the different molecular function (Molecular of 129 classes Function), 91 class cellular components of composition (Cellular Component) is participated in, 137 kinds of different biological processes are participated in (Biological Process).The possibility diagnosis target spot information list obtained through function and location information analysis is shown in Table 2.
Table 2 diagnoses target spot information list by the possibility that immunoprecipitation/high flux mass spectrum (IP/QE) is analyzed
*, carry out sequence alignment acquisition by with Brucella suis S2.
8 albumen are filtered out, its function may be mutual with the immunogenicity of brucella, virulence, brucella and host Effect, cell outer membrane protein transhipment are relevant (being shown in Table 2).Thus this 8 albumen are selected, is carried out as possible antidiastole target spot Further checking.
According to two dimensional electrophoresis/Western blotting/(MALDI-TOF) mass spectrum of substance assistant laser desorpted ionized-flight time and The possible antidiastole antigen protein that immunoprecipitation/high flux mass spectrum is obtained respectively, primarily determines that 8 protein sites are expected to turn into The target spot of in-vitro diagnosis, wherein two dimensional electrophoresis/Western blotting/substance assistant laser desorpted ionized-flight time (MALDI-TOF) Mass spectrum approach obtains 1 point, is transport protein；Immunoprecipitation/high flux mass spectrum approach obtains 8 points, respectively transport protein, logical With stress protein, memebrane protein (26kDa pericentral siphons chamber immune protein), Cyclin FtsH, cell membrane biological synthetic proteins OmpA, imaginary albumen (lipoprotein), memebrane protein (peptide glycan associated lipoprotein), agglutinin.Wherein immunoprecipitation/high flux mass spectrum Two points for being obtained and two dimensional electrophoresis/Western blotting/substance assistant laser desorpted ionized-flight time (MALDI-TOF) matter Two obtained points of spectrum are repeated, i.e. transport protein and general stress protein.
Embodiment 2
--- the determination of antidiastole antigen protein point gene information
The candidate albumen point that 2 kinds of methods are obtained, with the announcement of American National Biotechnology Information center (NCBI) website Expressing protein is compared in S2 genomic informations, is obtained each candidate albumen point correspondence gene information and is shown in Table 3 (containing Gene Name letter Breath, gene numbering, mrna length, molecular weight of albumen etc.), then separately design primer carries out gene cloning with PCR, obtains former Nuclear expression product (table 3, Fig. 3, Fig. 4), respectively with brucella immune serum and brucella natural infection serum Diagnosis of Sghistosomiasis Mark (Western-blot) and indirect ELISA (ELISA) verify its differential diagnosis value (Fig. 5, table 4).
1.8 antidiastole antigens (table 3) of candidate, numbering #1-#8, is numbered with albumen in stating hereinafter and replaced respectively Detailed protein name.
The details and upstream and downstream primer of 38 candidate gene target spots of table
2.PCR is expanded and prokaryotic expression
With S2 genomic DNAs template, 8 PCR primers of gene of amplification are obtained.Fig. 3 electrophoresis results show 8 genes There is specific amplification band, purpose fragment is in the same size with expected, the recombinant plasmid of acquisition send company to be sequenced after with U.S. NCBI of state (compare, and coincidence rate is 100%, its insertion by the respective gene order provided on NCBI Direction it is correct, can be used for further) induced expression.
After the positive recombinant plasmid for screening is transformed into E.coLi BL21 (DE3) competent cell of commercialization, 1mM is used IPTG induced expressions, after collected thalline is through ultrasonication, supernatant precipitation is taken respectively carries out SDS-PAGE, as a result finds 1# Do not expressed with 4# candidate albumens, remaining 6 recombinant protein is all expressed with inclusion bodies.Each recombinant protein molecular size range point (Fig. 4) not basically identical with theoretical size.
3. recombinant protein and three kinds of Western blottings of serum (Western-blot)
By 6 kinds of recombinant proteins in addition to 1# and 4#, centrifugation is contained into 6 kinds of precipitations of recombinant protein (P) point Not with cloth disease feminine gender pooled serum (Nm), the immune pooled serum (S of S2m) and natural infection pooled serum (Zm) carry out Western- Blot (Fig. 5), it is found that wherein 2# candidate albumens have stronger difference with the response intensity of immune serum and natural infection serum.
4. recombinant protein and the three kinds of enzyme linked immunosorbent assay (ELISA) of serum (ELISA) reactions
In addition to using Western blotting (Western-blot) detection candidate albumen and three kinds of immunoreactivities of serum, Also further verified using enzyme linked immunosorbent assay (ELISA) (ELISA) method.
The different coated antigen protein blood serum sample testing results of table 4
As seen from Table 4,6 kinds of albumen in addition to 1# and 4#, i.e. centrifugation are contained into 6 kinds of precipitations of recombinant protein (P) dissolved with 6M urea and suspended, 1 μ g/mL are diluted to pH9.6 carbonate buffer solutions, carry out indirect ELISA Method detection finds 2# in the immune negative serum reaction ODs sick with cloth of S2450nmIt is basically identical, and it is bright with natural infection serum difference It is aobvious, show that 2# albumen has antidiastole effect.Therefore filter out to be immunized with differentiation and play mirror with natural infection serum Other diagnostic gene identification information is shown in Table 5.
The antidiastole that table 5 is filtered out identifies the details table of gene
Embodiment 3
--- 2# (BLSJ-1, general stress protein, universal stress protein) antidiastole compliance test result
1. using glutathione s-transferase (GST) affinity column and glutathione linear gradient elution method purification of recombinant proteins 8#, obtains the purifying destination protein (Fig. 6) of purity more than 90%.
2. 2# recombinant proteins (BLSJ-1) cloth disease antibody ELISA immunosorbent assay (ELISA) testing result for purifying
The 2# recombinant proteins that will be purified are with normal condition：Optional 3 kinds single part of feminine genders/immune/infection serum and 1 part of feminine gender/ , be diluted to for the recombinant protein antigen of purifying with the carbonate buffer solution of pH9.6 respectively by immune/infection serum pooled serum 100ng/mL, overnight, 10% defatted milk closes 2h to 4 DEG C of coatings as confining liquid, and serum is made into 1: 50 with PBS dilutes, and uses phosphoric acid Salt buffer (PBS) carries out 1: 5000 dilution to ELIAS secondary antibody, and then the same conventional program of other steps carries out OD450nmDetection.Inspection Survey the results are shown in Table 6
Enzyme linked immunosorbent assay (ELISA) result of the 2# recombinant proteins of table 6 to different serum
As seen from Table 6,2# recombinant proteins BLSJ-1 has obvious difference really in immune and between infection reaction.And Response value between negative and S2 immune serums is relatively low, and the value of natural infection reaction is higher.
Embodiment 4
--- the foundation of indirect ELISA method and clinical serum pattern detection
1. the optimization of reaction condition
(1) coating buffer, envelope antigen concentration and serum dilution optimum results
It is antigen by 2# restructuring purifying proteins BLSJ-1, its coating buffer is set to the phosphate buffer (PBS) of neutrality, alkali Acid carbonate buffer solution, acid citrate buffer solution.Antigen concentration is set to 20ng/mL, 100ng/mL, 500ng/mL etc. 3 Individual dilution factor, serum-concentration is set to 1: 50,1: 100 and 1: 200.Tested by chessboard and screened, it is minimum with background value, and And infection/immune ratio is maximum, is defined as optimum concentration.The 2# recombinant protein antigens of purifying its antigen coat liquid, antigen coat Concentration and serum dilution condition optimizing the results are shown in Table 7.
The 2# antigen proteins coating buffer of table 7, antigen concentration and antibody dilution the selection result
As seen from Table 7,2# protein Bs LSJ-1 is adapted to coating buffer phosphate buffer (PBS), and its antigen coat concentration is 100ng/mL, serum dilution is 1: 100.
(2) confining liquid and ELIAS secondary antibody dilution factor optimum results
According to the above results, further confining liquid and ELIAS secondary antibody concentration are optimized, be respectively provided with bovine serum albumin (BSA), defatted milk and isinglass ELIAS secondary antibody are made 1: 10000 and 1: 20000 dilution respectively, by chess as confining liquid in vain Disk experiment is screened, minimum with background value, and infection/immune ratio is maximum, is defined as optimum concentration.Its concrete outcome is shown in Table 8.
The different confining liquids of table 8, ELIAS secondary antibody concentration screening result
As seen from Table 8, the most suitable confining liquid of 2# albumen is 2% bovine serum albumin(BSA), and its most suitable ELIAS secondary antibody concentration is respectively 1 ∶20000。
(3) optimization of reaction time and developing time is shown in Table 9 and table 10 respectively.
0.5h will be set to the reaction time and 1h will be compared, will separately develop the color 15min, 20min and 30min will be compared, Selection Zm/Sm higher values, as most suitable parameter.
The reaction time of table 9 optimizes
The developing time of table 10 optimizes
Find out from table 9 and table 10,2# recombinant proteins enzyme linked immunosorbent assay (ELISA) reaction 0.5h is basically identical with reaction 1h, separately It is slightly higher that 0.5h reacts its Zm/Sm；2# recombinant proteins colour developing 30min, its effect is better than 15min and 20min.
(4) antidiastole antigen indirect enzyme-linked immunosorbent assay for measuring detects the critical value (Cut-off) of immune serum
The indirect ELISA method set up as antigen with 2# recombinant proteins, detects that 30 parts of S2 exempt from respectively Epidemic disease serum, calculates its average value and variance.
The indirect ELISA method that the 2# recombinant antigens of table 11 are set up
2. the application in clinical sheep serum
30 parts are used for clinical sense known to immunoproteomics with the indirect ELISA method set up Contaminate single part of pattern detection and detected from immune 4 sheep, 656 parts of samples.The results are shown in Table 12.
The clinical detection serum sample of table 12
Note：Denominator is detection sample size；Molecule is to judge positive quantity.It is judged to for antidiastole purifying antigen The positive, as clinical infection sample；Indirect ELISA method for anti-grease polysaccharide is judged to the positive, is as immunized Or natural infection serum.
As seen from Table 12, the recall rate of 30 parts of serum of the known infection of 2# recombinant proteins detection reaches 93.3%.
Sequence table
　　<110>China Veterinery Drug Inspection Office
　　<120>A kind of gene expression product BLSJ-1 of brucella diagnostic marker effect and preparation method thereof
　　<130>
　　<160> 17
　　<170> Patentin version 3.5
　　<210> 1
　　<211> 450
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Brucella S2 plants（GI）489054867 gene
　　<400> 1
ATGTATAAGA GAATCCTGAT CGCAACCGAT GGATCGGAAC TTGCTGACAA GGGCGTGAAG 060
CAAGGTGTGA CGCTTGCCAA GGAAACGGGC GCCGGCGTGG TGTTTGTTTC CGTGACGGAA 120
CTGTTGCCAT CTTATGGCAT CGTGGTTGCT GCCGAATGGG CATCGAGCCC GGCGGCTTTC 180
CAGGAATATC GTGAGGCAAT TTCCAAGGCT GCGGCTGATA TTCTGCACAA GGCCAAGGCG 240
GAAGCCGAAG CGGCCGGTAT CAATGCCGAG ACGGTGCATG TGGAGAATCA GTCTGCGGCG 300
CAAGGTATCG TCGAGGCGGC GAAAGCGAAG GACTGCGATC TGATTGTGAT TGCTTCGCAC 360
GGGCGGCGCG GCGTCAACAA GCTGCTTCTG GGCAGCCAGG CGGCGGAAGT CCTGTCACTC 420
AGCACGGTTC CGGTGCTGGT TATCAAGTAA 450
　　<210> 2
　　<211> 30
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Amplification（GI）The sense primer EcoRI GAATTC of 489054867 genes
　　<400> 2
　　gggGAATTCa tgtataagag aatcctgatc 30（Sequence 2）
　　<210> 3
　　<211> 29
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Amplification（GI）The anti-sense primer XhoI of 489054867 genes: CTCGAG
　　<400> 3
　　tttCTCGAGc ttacttgata accagcacc 29（Sequence 3）
　　<210> 4
　　<211> 30
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the upstream BamHI of 489056667 genes：GGATCC
　　<400> 4
　　ggGGATCCat gaggtacacg gtgttcaaag 30（Sequence 4）
　　<210> 5
　　<211> 29
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the anti-sense primer XhoI of 489056667 genes：CTCGAG
　　<400> 5
　　aaaCTCGAGt cagcggccat caggcgtac 29（Sequence 5）
　　<210> 6
　　<211> 34
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the sense primer EcoRI of 489054409 genes: GAATTC
　　<400> 6
　　aatGAATTCa tgaacactcg tgctagcaat tttc 34（Sequence 6）
　　<210> 7
　　<211> 34
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the anti-sense primer XhoI of 489054409 genes: CTCGAG
　　<400> 7
　　cctCTCGAGt tacttgattt caaaaacgac attg 34（Sequence 7）
　　<210> 8
　　<211> 30
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the sense primer SalI of 490822055 genes: GTCGAC
　　<400> 8
　　gggGTCGACa tgaatccgaa ctatcgcaac 30（Sequence 8）
　　<210> 9
　　<211> 30
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the anti-sense primer NotI of 490822055 genes：GCGGCCGC
　　<400> 9
　　tttGCGGCCG Cttattgcgg ctgcggtttc 30（Sequence 9）
　　<210> 10
　　<211> 30
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Primer expands the sense primer EcoRI of 490824872 genes: GAATTC
　　<400> 10
　　ggGAATTCat gctgaagaaa accggtattg 30（Sequence 10）
　　<210> 11
　　<211> 26
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the anti-sense primer downstream XhoI of 490824872 genes: CTCGAG
　　<400> 11
　　tttCTCGAGt caggctccgc gtagcg 26（Sequence 11）
　　<210> 12
　　<211> 28
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the sense primer EcoRI of 489053693 genes: GAATTC
　　<400> 12
　　gaaGAATTCa tgcggaaaat ggcgcttg 28（Sequence 12）
　　<210> 13
　　<211> 32
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the anti-sense primer XhoI of 489053693 genes: CTCGAG
　　<400> 13
　　tttCTCGAGc tatcggcggc agcgtgctcg ag 32（Sequence 13）
　　<210> 14
　　<211> 31
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the sense primer EcoRI of 489055761 genes: GAATTC
　　<400> 14
　　ccGAATTCat gaacagcttc aggaaaactt g 31（Sequence 14）
　　<210> 15
　　<211> 30
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the anti-sense primer XhoI of 489055761 genes: CTCGAG
　　<400> 15
　　ccgCTCGAGt ttaacgagaa taaggcgaac 30（Sequence 15）
　　<210> 16
　　<211> 30
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the sense primer EcoRI of 489056820 genes: GAATTC
　　<400> 16
　　ttGAATTCat gcgccgtatc cagtcgattg 30（Sequence 16）
　　<210> 17
　　<211> 28
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the anti-sense primer XhoI of 489056820 genes: CTCGAG
　　<400> 17
　　tttCTCGAGt taccgtccgg ccccgttg 28（Sequence 17）
1

Claims (3)

1. a kind of expression product BLSJ-1 of brucella diagnostic marker effect gene, it is characterised in that the product is by cloth Lu Shi Bacterium S2 pnca genes numbering (GI) are 489054867 gene, are expressed as " general stress protein (universal stress protein)”；The mrna length：450bp；The molecular weight of albumen of its expression product about 15.5kDa；Its gene order is sequence 1.
2. a kind of preparation side of the expression product BLSJ-1 of brucella diagnostic marker effect gene as claimed in claim 1 Method, it is characterised in that the preparation method comprises the following steps：(1) genescreen：From brucella S2 pnca gene groups, by exempting from Epidemic disease proteomics methodology, Screening and Identification goes out the gene of aforesaid right 1；(2) gene expression：By the primer (sequence 2 and the sequence that design Row 3), by Standard PCR clonal expansion, it is placed on induced expression in pGEX6p-1 carriers；(3) expression product purifying：Pass through Glutathione s-transferase (GST) affinity column and glutathione linear gradient elution method purified expression product, antigen needed for obtaining, It is named as BLSJ-1.
3. a kind of application of the expression product BLSJ-1 of brucella diagnostic marker effect gene as claimed in claim 1, its Be characterised by the gene prokaryotic product as antigen, its antigen can as distinguish animal brucella vaccine immunity and from So infect Virus monitory antigen.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101363862A (en) * 2008-05-26 2009-02-11 北京庄笛浩禾生物医学科技有限公司 Test paper strip for rapidly detecting brucellosis IgM antibody colloidal gold
CN101362800A (en) * 2008-05-26 2009-02-11 北京庄笛浩禾生物医学科技有限公司 Test strip for rapid detection of brucella
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101363862A (en) * 2008-05-26 2009-02-11 北京庄笛浩禾生物医学科技有限公司 Test paper strip for rapidly detecting brucellosis IgM antibody colloidal gold
CN101362800A (en) * 2008-05-26 2009-02-11 北京庄笛浩禾生物医学科技有限公司 Test strip for rapid detection of brucella
CN104166000A (en) * 2014-07-03 2014-11-26 中国疾病预防控制中心传染病预防控制所 A method of indentifying brucella natural infection or immunifaction for livestock
CN104845949A (en) * 2014-08-20 2015-08-19 重庆医科大学 RGD-recombinatn staphylokinase-human alpha microglobulin fusion protein, and preparation method and application thereof

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* Cited by examiner, † Cited by third party
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