CN1884302A - Staphylococcus aureus fusion protein of fibronectin and bindin, its preparation method and uses - Google Patents
Staphylococcus aureus fusion protein of fibronectin and bindin, its preparation method and uses Download PDFInfo
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- CN1884302A CN1884302A CN 200510077738 CN200510077738A CN1884302A CN 1884302 A CN1884302 A CN 1884302A CN 200510077738 CN200510077738 CN 200510077738 CN 200510077738 A CN200510077738 A CN 200510077738A CN 1884302 A CN1884302 A CN 1884302A
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Abstract
The invention discloses a fusion protein for Micrococcus pyogenes surface protein, and also discloses the method for preparing the same as well as its usage. The fusion protein is fused by Micrococcus pyogenes fnb and thioredoxin Trx on expression carrier, and the process comprises preparation of Micrococcus pyogenes surface protein fnb coding gene, colony of fnb coding gene into expression carrier and protein fusion. The fusion protein is soluble protein and retains immunity of natural protein. It is detected that said fusion protein is characterized by good response for humoral immunity and cell immunity, enlargement of protection range of protein vaccine and prevention of infection caused by most of Micrococcus pyogenes.
Description
Technical field
The present invention relates to a kind of fusion rotein, relate in particular to a kind of fusion rotein of streptococcus aureus fibronectin binding protein, also relate to the Preparation method and use of this fusion rotein.
Background technology
Gold Portugal bacterium is a modal pathogenic bacteria in the hospital infection, also is the The main pathogenic fungi that infects on a large scale in the society.Gold Portugal bacterium infects and can cause diseases such as endocarditis, osteomyelitis, septic arthritis, pneumonia and abscess.Bacterium also is the The main pathogenic fungi of important economic animal in the gold Portugal.The subject matter that causes after gold Portugal bacterium develops immunity to drugs to the such choice drug of penicillin is, the staphylococcus aureus strains of X-1497 resistance can be resisted the microbiotic that great majority are used clinically, and therefore the S.aureus of about 10-60% is isolating in the hospital; Especially troubling is, the most effectively microbiotic---the susceptibility of vancomycin reduces the MRSA bacterial strain day by day to the treatment staphylococcal infections, these appearance to the insensitive staphylococcus aureus strains of vancomycin make people produce the incurable fear of golden Portugal bacterium, therefore must search out a kind of effective methods of treatment.
Except eukaryotic cell, various pathogenic bacterias also are extracellular binding matrix (extracellular-matrix, ECM) the specific interactions of binding molecule and host's ECM generation by cell surface.The ECM binding molecule of pathogenic bacteria belongs to a histone, is called as adhesin or can discerns the cell surface component (MSCRAMMs) of sticking matrix; The interaction of they and ECM is considered to prerequisite (the 1.Juliano RL of infectation of bacteria, Haskill S.Signal transduction from the extracellularmatrix.J Cell Biol.1993,120 (3), 577-85.2.Yamada KM.Adhesive recognitionsequences.J Biol Chem.1991,266 (20), 12809-12.), otherwise bacterium just can't settle down and breed, and can not produce and discharge extracellular enzyme and extracellular toxin.Though stick the non-tissue damaged that directly causes, the selection of tissue and the severity of disease had significant effects.The adhesin of gold Portugal bacterium is also given the function that bacterium escapes host defense mechanism except that the mediation bacterial adhesion.This mainly is by these adhesins, the soluble E CM (as fiber adhesion element, fibrinogen) of body is combined in a large number the surface of bacterium, make the bacterium thalline fully by host ECM bag quilt, thereby do not discerned by host's immunity system, this also has material impact to the result that golden Portugal bacterium infects.These proteic common traits are the protein ingredients that are bacterium surface, and the structure that tool is similar does not form ancillary attachments such as pili; Their institute's bonded host compositions are all ECM; And extracellular binding matrix albumen of golden Portugal bacterium and the effect of ECM are protein-protein interactions, and carbohydrate participates in.For infection host, germ must tightly " be pasted the jail ", and tissue moves it to overcome external force.They are except using nonspecific hydrophobic force and electrostatic attraction and the host combines, and its surface protein and ECM and plasma proteins also have special avidity.These albumen are commonly referred to as extracellular binding matrix albumen (ECMBPs), though some among them do not interrelate with matrix structure in conjunction with plasma proteins.
Gold Portugal bacterium can be expressed several extracellular binding matrix albumen (ECMBPs), and these albumen all are positioned mycetocyte surface, golden Portugal, the feature of tool gram-positive microorganism surface molecular.They all can discern body ECM member, and the specificity combination takes place, and mediate golden Portugal bacterium according to this and attach to host tissue.Although ECM does not under normal circumstances generally expose, after histoorgan was by mechanical effect or chemical substance damage, ECM came out, and then golden Portugal bacterium is sticked on it, and causes infecting.In addition, in different animals, the structure of ECM is quite conservative, so golden Portugal bacterium can attach to the various tissues of different animals, and does not have tangible host specificity and organizes proneness.
The binding characteristic that basic feature of the isolate of S.aureus is a fibronectin.Many bacterial strains are expressed two kinds of relevant fibronectin binding proteins, FnBPA and FnBPB; The two is (the Greene C by two close genes encodings, McDevitt D, Francois P, Adhesion properties of mutants ofStaphylococcus aureus defective in fibronectin-binding proteins and studies on theexpression of fnb genes.Mol Microbiol.1995,17 (6), 1143-52.).Protein fragments by homeotic mutation body and reorganization shows that these protein mediated bacterial adhesions are attached to plasma clot and are gone to stick the host as external biomaterial to the cell walls of intravital fibronectin and mediation S.aureus.Therefore, they are important factors for initial infection.
The animal experiment study of nearest staphylococcosis shows; based on the protein-bonded recombinant protein vaccine of aureus cell epimatrix body is had more provide protection (1.rennermalm A; Li YH; BohaufsL; et al.Antibodies against a truncated Staphylococcus aureus fibronectin-bindingproteins protect against dissemination of infection in therat.Vaccine; 2001; 19 (25-26): 3376.2.Lee JC.Development of anti-Staphylococcalvaccines.Curr.Infect.Dis.2001,3 (6): 517-524.).
Have not yet to see the report of fibronectin binding protein FnBP and Trx fusion rotein.
Summary of the invention
The invention discloses a kind of fusion rotein, (Thioredoxin protein Trx) merges and forms this fusion rotein by staphylococcus aureus surface albumen fnb and Trx.This fusion rotein is that the encoding gene by staphylococcus aureus surface albumen fnb forms with the encoding gene amalgamation and expression that is arranged in the Trx Trx of expression vector.
The invention also discloses a kind of recombinant expression vector of expressing above-mentioned fusion rotein, this carrier is made up of the encoding gene of expression vector that contains the Trx encoding gene and staphylococcus aureus surface albumen fnb, and the encoding gene of staphylococcus aureus surface albumen fnb is arranged in after the encoding gene of expression vector Trx.
The invention also discloses a kind of recombinant expression vector trx-fnb-pET-32a (+) that expresses above-mentioned fusion rotein, this carrier is made up of the encoding gene of expression vector pET-32a (+) and staphylococcus aureus surface albumen fnb.
The invention also discloses the preparation method of above-mentioned fusion rotein, comprise the steps:
(1) preparation staphylococcus aureus surface albumen fnb encoding gene, the preparation method can pass through PCR method, is that template amplification obtains with the streptococcus aureus;
(2) staphylococcus aureus surface albumen fnb encoding gene is cloned into expression vector, place after the expression vector Trx;
(3) staphylococcus aureus surface albumen fnb is with the formal representation of fusion rotein.
The used fusion expression vector of the present invention can be prokaryotic expression carrier or carrier for expression of eukaryon, contains the Trx encoding gene, preferred pET-32a (+) expression vector.
Fusion rotein after the expression carries out purifying, and purification process can adopt affinity chromatography, preferably adopts metal chelating and medium affinity chromatography.
The experimental result demonstration has obtained the amalgamation and expression albumen of staphylococcus aureus surface albumen fnb gene in intestinal bacteria, and is solubility expression, the expression amount height.
The invention also discloses the application of above-mentioned fusion rotein in preparation streptococcus aureus vaccine.
Fusion rotein behind the purifying is carried out the research of protein immunization effect test.At first prepare golden Portugal mycoprotein vaccine immunity animal, carry out mensuration, spleen lymphocyte proliferation experiment, the antibody-mediated conditioning phagocytosis and the immunoprotection of the humoral immune function of animal then and test.Test-results shows that fusion rotein of the present invention has kept its natural structure preferably, has higher immunogenicity, can be used as effective vaccine and uses.
Because the molecular weight of the more non-fusion rotein of fusion rotein increases, immunogenicity strengthens thereupon, thereby has improved vaccine protection effect, has significant values in the research of new generation vaccine.Up to the present, fusion rotein of the present invention is not seen relevant report at home and abroad as yet.
Description of drawings
Fig. 1 is fnb gene PCR amplification collection of illustrative plates.1:fnb gene (345bp) pcr amplification band wherein; M: be DNAmarker DL2000.
Fig. 2 is a fnb-T recombinant plasmid restriction enzyme mapping.1:pET-32a (+) wherein; 2:pET-32a (+) (BamHI and XholI double digestion); M is DNA marker DL2000; 3:fnb-T plasmid (BamHI and HindIII double digestion).
Fig. 3 expression plasmid trx-fnb-pET-32a (+) double digestion is identified collection of illustrative plates.M wherein: be DNAmarkerDL2000,1:pET-32a (+); 2: plasmid trx-fnb-pET-32a (+) (BamHI/Hind III); 3: plasmid trx-fnb-pET-32a (+) (Nde I/Hind III)
Fig. 4 is that trx-fnb-pET-32a (+) reorganization bacterium IPTG abduction delivering SDS-PAGE analyzes collection of illustrative plates.1:pET-32a (+) abduction delivering result wherein; The full bacterium SDS-PAGE of 2:trx-fnb-pET-32a (+) reorganization bacterium IPTG abduction delivering; Supernatant after the full bacterium ultrasonication of 3:trx-fnb-pET-32a (+) reorganization bacterium abduction delivering; 4; The full bacterium ultrasonication postprecipitation of trx-fnb-pET-32a (+) reorganization bacterium abduction delivering; M is a standard molecular weight albumen, and size is followed successively by 97,000Da, 66,000Da, 45,000Da, 30,000Da, 20,100Da, 14,000Da.
Fig. 5 is that trx-fnb-pET-32a (+) reorganization bacterium IPTG abduction delivering SDS-PAGE thin layer scanning is analyzed collection of illustrative plates.Wherein peak 10 is the target protein peak.
Fig. 6 is the SDS-PAGE collection of illustrative plates of Trx-FnBP protein purification.
Fig. 7 is the 2nd all titration results behind the mouse immune (n=8, bag is by Trx-FnBP albumen).
Fig. 8 is the 4th all titration results behind the mouse immune (n=8, bag is by Trx-FnBP albumen).
Fig. 9 for FN group mouse immune after the 6th all titration results (bag is by Trx-FnBP albumen)
Figure 10 (1) is the 6th all titration results behind the CN-FN group mouse immune (bag is by Trx-FnBP albumen).
Figure 10 (2) is the 6th all titration results behind the CN-FN group mouse immune (bag is by Trx-Cna albumen).
Figure 11 is each each time of immune group mouse immunity back antibody titers variation diagram.
Figure 12 is Fn group mice spleen lymphocytes proliferation variation diagram.
Figure 13 organizes mouse NBT test figure as a result for each.
Figure 14 is the proteic Western Blot of FnBP figure as a result.
Embodiment
Embodiment one staphylococcus aureus surface albumen fnb gene exists
Clonal expression in the intestinal bacteria, purifying
One, material
1, bacterial strain: streptococcus aureus (S.aureus) FRI 1169 is the international standard strain, and is commercial.
2, carrier and host bacterium: cloning vector PMD 18-T Vector is that Takara company product, expression vector pET-32a (+) Vecter are the Novagen product, bacillus coli DH 5 alpha, and e. coli bl21 (DE3) is the Stratagene product.
3, substratum: LB substratum, preparation according to a conventional method.
4, toolenzyme: restriction endonuclease BamH I, Nde I, Hind III, Xhol I; Ligase enzyme Ligation SolusionI is Takara company product.
5, main agents and test kit: PCR reagent: available from Shanghai Sangon, X-gal, IPTG, DNAMarker DL2000 is a Takara company product, narrowing azoles is the QIAGEN product, sodium lauryl sulphate (SDS) is the SIGMA product, acrylamide, N, N '-methylene-bisacrylamide, the lower molecular weight standard protein is an Amersham Biosciences product, and Wizard Plus SV Minipreps DNAPurification System is a Promega company product, and it is the vast sci-tech product in Beijing that dna fragmentation glass milk reclaims test kit.Other reagent are analytical pure, and are commercial.
7, damping fluid: TE commonly used, TAE, 5 * Tris-glycine buffer, PBS, 6 * DNA sample loading buffer, SDS-PAGE gel dye liquor, SDS-PAGE gel destainer, Lysis Buffer, Wash Buffer, Elution Buffer, albumen transfering buffering liquid, Wester blotting solution etc. all according to document (J. Sa nurse Brooker, D.W. Russell work. Huang Peitang etc. translate. molecular cloning experiment guide [M]. Beijing: Science Press .) preparation.
8, instrument
PCR instrument GeneAmp PCR System 2400, PERKIN ELMER; High speed low temperature centrifugal machine AllegraTM 21R Centrif μ ge, BECKMAN COMLTER; The polyacrylamide gel electrophoresis instrument is the BIORAD product; Enzyme linked immunological absorption instrument SPECTRA MAX PLUS, software is MR4PLUS ELISA-FLEXTM; Protein purification analyser AKTA FPLC, Amersham PharmaciaBiotech; Low-temperature freeze drying machine FREEZE DRY SYSTEM, LABCONCOR; ST-1 type half dry type electrophoretic blotting groove, Dalian compete to step biotechnology company
Two, method
1, primer design and synthetic
The coding gene sequence of the S.aureus that has delivered according to NCBI uses the auxiliary primer that has designed gene fnb respectively of DNAClub software, and Bo Ya biotech company in Shanghai is synthetic, and primer sequence is:
fnb up：5’-GC
GGATCC CATATG GGCCAAAATAGCGGTAAC-3’
fnb down：5’-GC
AAGCTT TAAGCTTACTTTTGGAAGTGT-3’
Wherein
GGATCCBe BamH I restriction enzyme site,
AAGCTTBe Hind III restriction enzyme site.
2, pcr template DNA's obtains
Scrape with transfering loop and to get the S.aureus bacterium colony in 100 μ l physiological saline, boil 10min in the boiling water, ice bath 5min again, so repeatedly several times, this liquid can be used as pcr template and uses.
3, pcr amplification goal gene
Amplification condition is: 98 ℃ of pre-sex change 10min; 94 ℃ of sex change 30s, 45 ℃ of annealing 30s, 72 ℃ are extended 45s, 30 circulations; 72 ℃ are extended 10min.1% agarose gel electrophoresis is checked amplified production.
4, the structure of fnb recombinant clone plasmid and evaluation
(1) fnb PCR product carry out agarose gel electrophoresis (180v, 6min).
(2) use the vast company in Beijing dna fragmentation glass milk to reclaim test kit and reclaim target DNA fragment.
(3) the fnb PCR product that reclaims connects cloning vector PMD 18-T Vector, and condition of contact is 16 ℃, 1-2hr.
(4) preparation of competent cell: adopt CaCl
2Method.
(5) connect product Transformed E .coli DH5 α competent cell.
(6) screening of recon (α-Hu Bu) and evaluation
The white colony of growing on the picking flat board carries out PCR and identifies that the PCR condition is: 98 ℃ of pre-sex change 10min.94 ℃ of sex change 30s, 45 ℃ of annealing 30s, 72 ℃ are extended 45s, 25 circulations.72 ℃ are extended 10min.1% agarose gel electrophoresis is checked amplified production.
Inoculation is accredited as the male bacterium colony in the LB substratum that contains penbritin (100 μ g/ml) through PCR, and 37 ℃ of shaking bath overnight incubation are pressed Wizard
Plus SV Minipreps DNA PurificationSystem kit method extracts plasmid, recombinant plasmid called after fnb-T.
(7) double digestion of recombinant plasmid is identified: the fnb-T plasmid is carried out the double digestion evaluation with Nde I and Hind III and BamH I and Hind III.Enzyme tangent condition: 37 ℃ of 3-4hr.1% agarose gel electrophoresis inspection enzyme is cut product.
(8) order-checking: select PCR, enzyme is cut the positive reorganization bacterium of qualification result, extract and the plasmid purification order-checking.Sequencing primer is M13 primers, is checked order by Shanghai Bo Ya biotech company.
5, the structure of recombinant expression plasmid fnb-pET-32a (+)
(1) recombinant plasmid cut of above-mentioned enzyme carries out using the vast company in Beijing dna fragmentation glass milk to reclaim test kit and reclaiming after agarose gel electrophoresis separates.Simultaneously expression vector pET-32a (+) is carried out the double digestion in corresponding site.Each enzyme is cut system at 37 ℃ of thermostat water bath internal reaction 3-4hr.1% agarose gel electrophoresis inspection enzyme is cut product.Using the vast company in Beijing dna fragmentation glass milk to reclaim test kit reclaims.
(2) connect: the fnb-T plasmid that enzyme is cut is connected 16 ℃ of 1-2hr with the corresponding expression vector pET-32a (+) that enzyme cuts.
(3) transform: get 5 μ l connection product and change 200 μ l E.coli BL21 (DE3) competent cells over to.Get the LB flat board that 200 μ l coating contains penbritin (100 μ g/ml).
(4) screening of recon and evaluation
The bacterium colony of growing on the picking flat board carries out the PCR evaluation as template one by one.Inoculation PCR is accredited as the male bacterium colony in LB substratum (containing penbritin 100 μ g/ml), and 37 ℃ of shaking table overnight incubation are pressed Wizard
Plus SV Minipreps DNA Purification System kit method extracts plasmid.Carrying out corresponding double digestion respectively identifies.
6, the abduction delivering of reorganization bacterium
To recombinate colony inoculation in 5ml LB substratum (containing penbritin 100 μ g/ml), and 37 ℃ of shaking tables are cultured to logarithmic growth early stage (the OD value is about 0.6), add 1mol/l IPTG and induce 3-4hr.Carry out SDS-PAGE analysis expression after getting the centrifugal collection of an amount of bacterium liquid bacterium.The albumen called after FnBP (fibronectin-binding protein) of fnb genetic expression.
All in broken supernatant, this just makes protein purification comparatively easy to expressed proteins, cultivates in a large number, prepares purifying.
7, SDS-PAGE protein electrophoresis
Get cultured bacterium liquid 1.5mL, with centrifugal 5 minutes collection of 8000rpm room temperature bacterium, abandon supernatant, 200 μ l pure water have hanged precipitation, get 25 μ l suspension and add 25 μ l 2XSDS gel loading buffers, boil 10 minutes, get 20 μ l and carry out the SDS-PAGE analysis.Voltage: 180V, about 1-2hr.
Dye, decolour.After gel-colored 30 minutes, decolour and analyze after 1 hour.
8, the purifying of expressing protein-Ni-NTA affinity chromatography.
(1) pre-treatment of new Ni post: with the deionization washing post of at least 1 times of column volume; 100mM NiSO4 with 2 times of volumes washes post; Deionization washing post with 2 times of column volumes;
(2) purge process: with the Lysis buffer balance pillar of 5 times of column volumes, flow velocity＜2mL/min; Institute collection bacterium hang with Lysis buffer, after the ultrasonication (ultrasonication 30 times, sound 20s, interval 10s), 4 ℃, 8000, centrifugal 15, the collection supernatant; Supernatant further after the dilution, divides upward sample 3-5 time, and collection is penetrated liquid with Lysis buffer; 3. use the Lysis buffer of 5-10 times of column volume to wash post, collect washing lotion; Use the Wash buffer of 5-10 times of column volume to wash post, collect washing lotion; Use the Elution buffer eluted protein of 5-10 times of column volume, every 2mL collects 1 pipe, carries out mark; Use SDS-PAGE to detect in collected sample.
(3) after purifying finishes, use 15L 0.2M acetate successively, 15mL 30% glycerine, 15mL pure water, 15mL 30% Ethanol Treatment.Be stored in standby with ethanol 3 at last.
9, protein quantification
(1) BCA method protein quantification: the BCA that uses PIERCE company
TMProtein Assay Kit operation.
(2) double wave regular way: behind the diluted sample, survey the OD value of 280nm and 260nm.The substitution formula:
(1.55 * OD
280-0.75 * OD
260) * extension rate
10, the albumen behind the purifying uses low-temperature freeze drying machine FREEZE DRY SYSTEM, LABCONCO
RFinished product is made in freeze-drying, and experimentation on animals is standby.
Three, result
1, clonal expression, the purification result of staphylococcus aureus surface protein fnb gene in intestinal bacteria
(1) amplification of goal gene: extract S.aureus J72, the chromosomal DNA of 1169 strains carries out pcr amplification as template, capable 1% agarose gel electrophoresis of amplified production.There is a macula densa at fnb gene 360bp place, consistent with expected results (Fig. 1).
(2) structure of fnb-T plasmid: agarose gel electrophoresis reclaims (glass milk reclaims) fnb, cna gene fragment, is connected with cloning vector PMD 18-T Vector.Transformed into escherichia coli DH5 α competent cell, coating contains the LB flat board (containing the ammonia Bian) of X-gal and IPTG.After 37 ℃ of overnight incubation, the picking white colony carries out PCR as template to be identified.Positive bacterium colony carries out double digestion with BamH I and Hind III, 1% agarose gel electrophoresis (Fig. 2) after extracting the fnb-T plasmid.
2, sequencing
Select fnb-T one of them through being accredited as male reorganization bacterium colony, extract plasmid, carry out dna sequencing with universal primer M13 primers behind the purifying.With sequencing result is fnb full length gene 345bp, 115 amino acid of encoding.Submit to NCBI to carry out homology search (advanced BLAST) analysis sequencing result, determine to have obtained the fnbB gene.
3, the structure of expression plasmid trx-fnb-pET-32a (+): use Nde I respectively, Hind III and BamHI are behind Hind III double digestion pET-32a (+) and the fnb-T plasmid.After enzyme is cut the product connection, Transformed E .coliBL21 (DE3) competence.After the incubated overnight, the bacterium colony of growing on the picking flat board carries out PCR and identifies.The picking positive findings extracts plasmid (trx-fnb-pET-32a (+) plasmid) respectively, carries out the double digestion in corresponding site and identifies (Fig. 3).
4, the expression of reorganization trx-fnb-pET-32a (+): the engineering bacteria that inoculation contains trx-fnb-pET-32a (+) is in LB substratum (containing penbritin 100 μ g/ml), and 37 ℃ of shaking tables are cultured to logarithmic growth after date adding 1mol/l IPTG and induce 3-4hr.Get the capable SDS-PAGE protein electrophoresis of 1ml nutrient solution, the result shows and uses BamH I, a newly-increased protein band appears in trx-fnb-pET-32a (+) the reorganization bacterium of Hind III double digestion between molecular weight 43KD and 67KD, pET-32a (+) carrier bacterium after inducing does not then have this protein band and produces (Fig. 4).Centrifugal after the full bacterium ultrasonication of abduction delivering, separate supernatant, the demonstration of post precipitation SDS-PAGE protein electrophoresis, target protein is mainly with the soluble form secreting, expressing.
Trx-fnb-pET-32a (+) reorganization bacterium IPTG abduction delivering SDS-PAGE analytical results shows that the reorganization bacterium obtains high expressing quantity, analyzes 52.8% (Fig. 5) that the expression amount of target protein as can be known accounts for the whole bacterial protein total amount through thin layer scanning.
5, protein purification result: Trx-FnBP albumen uses protein purification analyser KTA FPLC, carries out Ni-NTA affinity chromatography (HiTrap Chelating HP).Use Lysis Buffer (50mMNaH
2PO
4.2H
2O, 300mM NaCL, the 20mM imidazoles is transferred pH to 8.0 with NaOH) behind the balance pillar; With Wash Buffer (50mM NaH2PO4,300mM NaCL, the 20mM imidazoles, transfer pH to 8.0 with NaOH) and Elution Buffer (50mM NaH2PO4.2H2O, 300mM NaCL, the 250mM imidazoles is transferred pH to 8.0 with NaOH) do linear wash-out, collect protein peak, carry out SDS-PAGE and analyze purification effect (Fig. 6).
6, purifying protein is quantitative: purified protein B CA method protein quantification (uses the BCA of PIERCE company
TMProtein Assay Kit) result is: Trx-FnBP albumen can reach 1.25mg/ml.
Embodiment two Trx-FnBP protein immunization effect studies
One, material
1, recombinant protein: Trx-FnBP albumen is the low-temperature freeze drying goods after the purified albumen dialysis.
2, immunological adjuvant: FREUND ' S ADJUVANT Complete and FREUND ' SADJUVANT Incomplete are SIGMA company product.
3, other reagent: MUCIN (Type II:Crude; From Porcine Stomach) be the SIGMA product; Ficoll-paque is the Pharmacia product; CellTiter 96
RAqueous Non-RadioactiveCell Proliferation Assay is a Promega company test kit; Thioglycolate Broth withResazurine is the Fluka product; Goat anti-mouse igg-HRP Beijing Zhong Shan biotech company product; ELISA reagent is analytical pure.
4, laboratory animal: 5-6 week Kunming is that secondary female mice and Balb/c female mice are all purchased White Army's thing Academy of Medical Sciences Experimental Animal Center.
Two, experimental technique
1, the golden Portugal of preparation mycoprotein vaccine
The Trx-FnBP albumen that freeze-drying is preserved is dissolved in PBS, and (0.03mol/l, pH7.2), concentration is 100 μ g/100 μ l.Each protein 10 0 μ l is used for animal immune respectively with after FREUND ' the S ADJUVANT Complete of 100 μ l and the emulsification of FREUND ' S ADJUVANT Incomplete adjuvant thorough mixing.
2, mouse immune program and dosage
5-6 week Kunming is female mice or Balb/c female mice random packet.Three (0,2,3 week) dosage of each group immunity are 100 μ g.Adopt for the first time subcutaneous abdomen multidigit point injection (100 μ g albumen/every.Freund's complete adjuvant emulsification); Adopt for the second time abdominal injection (100 μ g albumen/every.Freund's incomplete adjuvant emulsification); Abdominal injection (100 μ g albumen are dissolved in PBS) for the third time.A week after the last immunity (around) is taked laboratory sample.
3, the mensuration of immune mouse humoral immune function
Blood sample is taked in each time immunity back (2,3,4 week) docking, indirect elisa method mensuration antibody titer (referring to Zhu Liping, the Chen Xueqing chief editor. immunology common experimental method [M]. Beijing: People's Medical Officer Press .).
The Trx-FnBP albumen of 1 μ g is wrapped by elisa plate with 100 μ l/ holes, 4 ℃ of coating buffers that spend the night → abandon, PBST gives a baby a bath on the third day after its birth in 120 μ l/ holes inferior, 3min/ time → adding confining liquid (3%BSA of PBST dilution), 37 ℃ of sealing 40min → abandon confining liquids, PBST gives a baby a bath on the third day after its birth in 120 μ l/ holes inferior, 3min/ time → adding immune serum (mice serum carried out 10 times of gradient dilutions in white 1: 10), by 100 μ l/ hole application of samples, give a baby a bath on the third day after its birth time in 37 ℃ of effect 1hr → PBST120 μ l/ holes, 3min/ time → add enzyme mark goat anti-mouse igg (dilution in 1: 1000), 37 ℃ act on 40min → PBST 120 μ l/ holes and wash four times, 3min/ time → adding A, B liquid colour developing 4min adds 2NH
2SO
4Stop buffer → OD
450The nm measured value.
4, immune mouse spleen lymphocyte proliferation experiment
(1) preparation of splenocyte
Aseptic separating spleen is put to death in one week of the Balb/c female mice back of last immunity.Spleen is extruded splenocyte after screen cloth grinds, wash cell twice with the RPMI-1640 cell culture fluid, and adjusting cell count again is 5 * 10
6Cell/ml.
(2) lymphocyte proliferation assay (MTS method), referring to Zhu Liping, the Chen Xueqing chief editor. immunology common experimental method [M]. Beijing: People's Medical Officer Press.
The every hole of 96 porocyte plates adds 100 μ l enchylema → get each purifying protein to add respectively in each hole, make final concentration be respectively 40 μ g/ml, 20 μ g/ml, 10 μ g/ml, 5 μ g/ml, 2.5 μ g/ml, 1.25 μ g/ml, 0.65 μ g/ml (doubling dilution), every hole 100 μ l, cumulative volume are 200 μ l/ holes.Put 37 ℃, 5%CO
2Incubator 72hr, and establish cell control well → each hole and add the 20 μ l colour developing liquid (CellTiter of Promega company 96
RAqueous Non-Radioactive Cell Proliferation Assay test kit), hatch 2.5hr → with the zeroing of RPMI-1640+20 μ l colour developing liquid, 590nm place survey OD value.
5, antibody-mediated conditioning phagocytosis
The conditioning phagocytosis of neutrophil leucocyte (NBT test) (referring to Zhu Liping, the Chen Xueqing chief editor. immunology common experimental method [M]. Beijing: People's Medical Officer Press .)
Healthy three HASs, the anti-blood coagulation of the heparin of adding 5%.With 3% contain the dextran salts solution, 37 ℃ of effect 30min are with the precipitation red corpuscle.Separate neutrophil leucocyte with Ficoll-paque (Pharmacia) density gradient centrifugation, the RPMI-1640 cell culture fluid is washed cell twice, and adjusting cell count again is 5 * 10
6Cell/ml.Bacterium through the antibody conditioning can stimulate neutrophil leucocyte.The super-oxide that produces can make colourless four to make azoles nitroblue (NBT) and be degraded into blue first (month praise) product.
Experimental procedure is: 20 μ l S.aureus strain S-6 (1 * 10
9CFU) mix with 30 μ l immune serums (doubling dilution 1: 2 to 1: 32), add 96 porocyte plates, hatch 60min for 37 ℃; The neutrophil leucocyte (5 * 10 of 100 μ l new systems
6Cell/ml) and the NBT liquid of 50 μ l, 0.1% filtration sterilization add cell hole altogether, hatch 60min for 37 ℃; 0.5M HCl termination reaction with 50 μ l; First (moon praises) product is carefully inhaled and is abandoned liquid 200 μ l in the cell hole with the centrifugal 10min of 1200rpm; Use DMSO 200 μ l/ holes resuspended again; The 540nm place surveys the OD value.
6, immunoprotection experiment
5-6 week Kunming is female mice, attacks the poison experiment after the last immunity week.S.aureus strain S-6 (5 * 10
7CFU, 5% MUCIN) (referring to document: Ali I.Fattom, Jawad Sarwar, AlbertoOrtiz, et al.A Staphylococcus aureus capsular), 1ml/ only behind the abdominal injection, observes protection ratio.Each organized survival mice after four days, got viscera tissue (heart, liver, spleen, lung and kidney) after alive the killing and did pathological section, and pathological change is observed in row HE dyeing.
7, Western Blot testing goal albumen
(1) when the SDS-PAGE electrophoresis closes to an end, cleans graphite cake with distilled water;
(2) be with gloves, cut 6 filter paper and 1 nitrocellulose filter.The size of filter paper and film equates or fully less than gel, otherwise the edge contact of two filter paper can cause short circuit current, reaches to reduce the efficient that shifts greatly;
(3) nitrocellulose membrane is floated on the dish deionization water surface, borrow wicking action to make film moistening from the bottom up after, film is intact not in water, soak 5 minutes to remove bubble residual on the filter membrane;
(4) in a shallow pallet, add a small amount of transfering buffering liquid, 6 filter paper are immersed in wherein;
(5) the electrophoretic blotting groove is installed:
Keep flat the base of Graphite Electrodes, this graphite cake is an anode; Place 3 through shifting the filter paper that liquid soaked on graphite cake, roll on filter paper with a glass pipet in the alignment back, to get rid of residual bubble; Nitrocellulose filter is placed on the filter paper heap, guarantees accurate alignment, avoid between filter paper and nitrocellulose filter, bubble being arranged; Take out the SDS-PAGE gel from electrophoresis chamber, transfer in the dish deionized water slightly that rinsing accurately lies against on the nitrocellulose filter then to gel once, on the mark angle of gel lower left corner as for nitrocellulose filter, the band gloves are got rid of all bubbles; 3 filter paper of another group are placed on the gel top, must guarantee that equally the accurate heap of each layer is neat and avoid bubble; The electrophoretic blotting groove upper cap is anchored on Graphite Electrodes-transfer film glue complex body, connects power supply, note positive and negative electrode, according to gel area 0.65-1.0mA/cm2 making current, electrotransfer 0.5-2h; Deenergization is also extracted plug on the groove, unloads transfer device one by one, takes out gel and carries out antibody labeling; The nitrocellulose filter that has shifted closed with the 3%BSA/B fluid-tight spend the night; Abandon confining liquid, liquid washing 3 times, 5min/ time at interval; Add an anti-anti-TSST-1 (dilution in 1: 500), with nitrocellulose filter room temperature effect 1 hour; Abandon one and resist, with washing 5 times, 5min/ time at interval; Add goat anti-rabbit antibody (dilution in 1: 1000), room temperature effect 30 minutes; Abandon two and resist, with washing 5 times, 5min/ time at interval; 3mg DAB is added on 5mLA+10 μ l H
2O
2, fully react with nitrocellulose filter then; With the distilled water termination reaction, colour developing is observed.
Three, result
1, immune Balb/c female mice serum antibody titer changes
5-6 week Balb/c female mice is divided into two groups (FN group and control groups), 8 every group at random.Administration situation: FN group, each 100 μ of Trx-FnBP albumen g/ time; Control group, 100 μ l PBS/ time.Each time immunity back (the 2nd, 3,4 week) docking is got blood (each mice serum mixes) and is carried out ELISA survey antibody titer.
(1) (the 2nd week) serum antibody titer mensuration (2003-4-14) is seen Fig. 7 after the immune mouse immunity for the first time
(2) (the 4th week) serum antibody titer mensuration (2003-4-28) is seen Fig. 8 behind the immune mouse booster immunization second time
(3) (the 6th week) serum antibody titer mensuration (2003-5-15) is seen Fig. 9,10 (1), 10 (2), 11 after the immune mouse immunity for the third time.
2, immune mouse spleen lymphocyte proliferation experiment
Table 1 Fn group mice spleen lymphocytes proliferation situation
| Antigen (FnBP) concentration (μ g/ml) | Fn organizes (A490) | Control organizes (A490) |
| 0.195 0.39 0.78 | 0.594 0.391 0.457 | 0.097 0.097 0.172 |
| 1.56 3.12 6.25 12.5 25 | 0.345 0.491 0.322 0.33 0.423 | 0.131 0.153 0.161 0.247 0.167 |
T check P=0.000249 shows the splenic lymphocyte that stimulates Fn group, Control group with antigen FnBP respectively in pairs, and the two propagation has significant difference (P＜0.001), Figure 12.
3, the conditioning phagocytosis of neutrophil leucocyte
Healthy three HASs, the anti-blood coagulation of the heparin of adding 5%.With 3% contain the dextran salts solution, 37 ℃ of effect 30min are with the precipitation red corpuscle.Separate neutrophil leucocyte with Ficoll-paque (Pharmacia) density gradient centrifugation, the RPMI-1640 cell culture fluid is washed cell twice, and adjusting cell count again is 5 * 10
6Cell/ml.Bacterium through antibody (antiserum(antisera)) conditioning can stimulate neutrophil leucocyte.The super-oxide that produces can make colourless four to make azoles nitroblue (NBT) and be degraded into blue first (month praise) product.See Figure 13
4, immunoprotection experiment
Table 4 immunoprotection experiment related parameter arranged
| Group | Antigen | The laboratory animal number | S.aureus strain S-6 inoculum size | The surviving animals number | Survival |
| FN contrast | |||||
| 1 | FnBP is not immune | 8 8 | 5×10
7CFU， 5％MUCIN 5×10
7CFU， 5 | 4 2 | 50％ 25％ |
After the test group mouse was attacked poison, its survival rate showed tentatively that apparently higher than control group albumen that we obtain has the better protecting effect.But, because sample number is few, also need increases number of samples and further confirm its effect.
5, Western Blot analyzes
Trx-FnBP albumen carries out the film transfer printing after SDS-PAGE separates, carry out antigen-antibody binding reaction.One is anti-for each group mouse immune antiserum(antisera), sees Figure 14.
Claims (9)
1, the proteic fusion rotein of a kind of staphylococcus aureus surface is characterized in that being formed by streptococcus aureus fibronectin binding protein fnb and Trx Trx fusion.
2, fusion rotein according to claim 1 is characterized in that the encoding gene of staphylococcus aureus surface albumen fnb and the encoding gene amalgamation and expression of Trx Trx form.
3, fusion rotein according to claim 2 is characterized in that the encoding gene of Trx is arranged in expression vector.
4, express the recombinant expression vector of the described fusion rotein of claim 1, it is characterized in that forming by the encoding gene of expression vector that contains the Trx encoding gene and staphylococcus aureus surface albumen fnb.
5, recombinant expression vector according to claim 4 is characterized in that the encoding gene of staphylococcus aureus surface albumen fnb is arranged in after the expression vector Trx encoding gene.
6, recombinant expression vector according to claim 4, the expression vector that it is characterized in that containing the Trx encoding gene is pET-32a (+).
7, the preparation method of the described fusion rotein of claim 1 is characterized in that comprising the steps:
(1) preparation staphylococcus aureus surface albumen fnb encoding gene;
(2) staphylococcus aureus surface albumen fnb encoding gene is cloned into expression vector, place after the expression vector Trx;
(3) staphylococcus aureus surface albumen is with the formal representation of fusion rotein.
8, method according to claim 7 is characterized in that used expression vector is pET-32a (+).
9, the application of the described fusion rotein of claim 1 in preparation streptococcus aureus vaccine.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102070719A (en) * | 2010-11-24 | 2011-05-25 | 中国人民解放军第四军医大学 | Soluble leukemia stem cell targeting proteins TrxHis-hCD47 |
| CN102977214A (en) * | 2012-09-29 | 2013-03-20 | 重庆原伦生物科技有限公司 | Recombinant protein HF2 used for methicillin-resistant staphylococcus aureus (MRSA) vaccine, and preparation method and application thereof |
| CN102993278A (en) * | 2012-09-29 | 2013-03-27 | 重庆原伦生物科技有限公司 | Purification method of methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein antigen FnbA1 |
| CN103314008A (en) * | 2010-09-09 | 2013-09-18 | 芝加哥大学 | Methods and compositions involving protective staphylococcal antigens |
| CN105713096A (en) * | 2016-03-29 | 2016-06-29 | 黑龙江八一农垦大学 | Preparation and application of ATT fusion protein for preventing infection of staphylococcus aureus |
| CN103314008B (en) * | 2010-09-09 | 2016-12-14 | 芝加哥大学 | The method and composition of being related to protection of property Staphylococcal antigen |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103314008A (en) * | 2010-09-09 | 2013-09-18 | 芝加哥大学 | Methods and compositions involving protective staphylococcal antigens |
| US9095540B2 (en) | 2010-09-09 | 2015-08-04 | The University Of Chicago | Methods and compositions involving protective staphylococcal antigens |
| CN103314008B (en) * | 2010-09-09 | 2016-12-14 | 芝加哥大学 | The method and composition of being related to protection of property Staphylococcal antigen |
| CN102070719A (en) * | 2010-11-24 | 2011-05-25 | 中国人民解放军第四军医大学 | Soluble leukemia stem cell targeting proteins TrxHis-hCD47 |
| CN102977214A (en) * | 2012-09-29 | 2013-03-20 | 重庆原伦生物科技有限公司 | Recombinant protein HF2 used for methicillin-resistant staphylococcus aureus (MRSA) vaccine, and preparation method and application thereof |
| CN102993278A (en) * | 2012-09-29 | 2013-03-27 | 重庆原伦生物科技有限公司 | Purification method of methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein antigen FnbA1 |
| CN102977214B (en) * | 2012-09-29 | 2014-03-12 | 重庆原伦生物科技有限公司 | Recombinant protein HF2 used for methicillin-resistant staphylococcus aureus (MRSA) vaccine, and preparation method and application thereof |
| CN105713096A (en) * | 2016-03-29 | 2016-06-29 | 黑龙江八一农垦大学 | Preparation and application of ATT fusion protein for preventing infection of staphylococcus aureus |
| CN105713096B (en) * | 2016-03-29 | 2019-01-01 | 黑龙江八一农垦大学 | A kind of preparation and application of the ATT fusion protein preventing infection of staphylococcus aureus |
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