CN103725697A - Chemically synthesized staphylococcus aureus surface protein FnBPA gene fragment and expression and application thereof - Google Patents

Chemically synthesized staphylococcus aureus surface protein FnBPA gene fragment and expression and application thereof Download PDF

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CN103725697A
CN103725697A CN201310750813.8A CN201310750813A CN103725697A CN 103725697 A CN103725697 A CN 103725697A CN 201310750813 A CN201310750813 A CN 201310750813A CN 103725697 A CN103725697 A CN 103725697A
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CN103725697B (en
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李越希
李丙军
许桂丽
马颖
张素芬
袁敬宇
徐悦玥
潘英
李素芹
陈乐如
蔡冉
周洁
尚彦红
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Eastern Theater Disease Prevention And Control Center Of Pla
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Abstract

The invention relates to a chemically synthesized staphylococcus aureus surface protein FnBPA gene fragment, as well as expression and application thereof, belonging to the fields of genetic engineering technologies, antibodies and kits. According to the chemically synthesized staphylococcus aureus surface protein FnBPA gene fragment provided by the invention, a strong antigen epitope, namely 133 amino acids in total from 745th amino acid to 877th amino acid in the staphylococcus aureus surface protein FnBPA are screened out through computer analysis, a codon preferred by prokaryotes is selected, a brand new gene sequence of the antigen epitope is chemically synthesized, the gene fragment is expressed by utilizing the genetic engineering technology, and the strong antigen epitope fragment of the staphylococcus aureus surface protein FnBPA is prepared. The expressed strong antigen epitope fragment of the staphylococcus aureus surface protein FnBPA can be used for detection of staphylococcus aureus antibodies and immune preparation of anti-staphylococcus aureus monoclonal antibodies, polyclonal antibodies and the like.

Description

The surface protein FnBPA gene fragment of the streptococcus aureus of chemosynthesis and expression thereof, application
Technical field
What the present invention relates to is the surface protein FnBPA gene fragment of streptococcus aureus of chemosynthesis and expression thereof, application, utilizes genetic engineering technique, prepares the surface protein FnBPA of recombination staphylococcus aureus.By Computer Analysis, filter out the FnBPA fragment containing strong antigen epi-position, select the codon of prokaryotic organism preference, the gene order that chemosynthesis is brand-new, utilize genetic engineering technique to express, the albumen of expressing can be used for the foundation of vaccine and streptococcus aureus method for quick etc., the present invention relates to genetic engineering technique and diagnostic reagent field.
Background technology
Streptococcus aureus is a kind of conditioned pathogen that can cause human and animal's disease.It parasitizes human skin and mucomembranous surface as normal microflora, accounts for the major part of hospital infection and Community Acquired Infections.Gold Portugal bacterium can cause Skin and soft tissue infection, and life-threatening transitivity complication, as pneumonia, microbemia, endocarditis, suppurative arthritis and osteomyelitis, it extensively retains in hospital, the host and the operation patients that cause immune system injury infect, and retain in medicine equipment.Streptococcus aureus is ubiquitous at occurring in nature, in the movement of air, water, dust and humans and animals, all can find, and therefore, the food such as milk, meat, egg, fish and goods thereof are subject to the chance of its pollution a lot.Streptococcus aureus is also one of pathogenic agent of health organs at different levels emphasis detection.Therefore, set up the method for quick of streptococcus aureus extremely important.
The method steps of tradition evaluation and SEPARATION OF GOLD staphylococcus aureus is more, and has certain limitation.First to sample, carry out enrichment culture according to detected pathogenic bacterium type, its qualification result generally can only be qualitative can not be quantitative, and required time is long, speed is slow, general 3 ~ 4d could estimation result, precise Identification needs 5 ~ 7 days.The experiment of most of immunological techniques such as fluorescent-labeled antibody, enzyme linked immune assay (ELISA), radioimmunoassay test etc. are all used widely, but their required costs are higher, consuming time and sample is carried out to complicated processing, limit its widespread use in life.Set up a kind of detection method simple, quick, that be applicable to the streptococcus aureus of rig-site utilization and have important meaning.
Summary of the invention
The present invention is the brand-new gene fragment of the staphylococcus aureus surface albumen FnBPA of chemosynthesis, utilizes genetic engineering technique, prepares the Partial Fragment of the FnBPA of recombination staphylococcus aureus.By Computer Analysis, filter out the Partial Fragment containing the staphylococcus aureus surface albumen FnBPA of strong antigen epi-position, 877 amino acid of the 745th amino acid-, totally 133 amino acid, select the codon of prokaryotic organism preference, the gene order that chemosynthesis is brand-new, utilizes genetic engineering technique in e. coli bl21, to express this gene.The albumen of expressing can be used for the preparation of monoclonal antibody and polyclonal antibody, for the foundation of detection methods of staphylococcus aureus is laid a good foundation.
The extracellular binding matrix albumen (ECMBP) that streptococcus aureus produces, comprises FnBP, ClfA and Ebp etc., and they are the protein ingredient of bacterium surface, have similar structure.FnbpA is a kind of of fibronectin binding protein (Fnbp), is the protein component of bacterium surface.By the double PCR of streptococcus aureus clinical separation strain Fnbp is detected, find that more than 95% bacterial strain exists HpaA gene FnbpA and FnbpB gene only has 10%, this research selects FnBPA as research object.
By computer software analysis, filter out the specific surfaces albumen FnPFA epitope enrichment region of streptococcus aureus, select the codon optimized gene order of prokaryotic organism preference, the gene order that chemosynthesis is brand-new, utilizes genetic engineering technique to express.The albumen of expressing has good antigenicity and specificity, can be used for the preparation of monoclonal antibody and polyclonal antibody, for the foundation of streptococcus aureus method for quick lays the foundation.
133 amino acid whose gene fragments of the staphylococcus aureus surface albumen FnBPA of chemosynthesis and expression thereof, application take following steps to realize:
1. the screening of staphylococcus aureus surface albumen FnBPA epitope and the chemosynthesis of gene fragment thereof:
Utilize the softwares such as ANTHEWIN, DNAStar, by the full amino acid sequence of the Computer Analysis gold bacterium surface protein FnBPA of Portugal, the N end (the 745th amino acid-877th amino acid) of the bacterium surface protein FnBPA of Fa Jin Portugal contains stronger antigenic determinant.Select the codon of prokaryotic organism preference, the gene order that chemosynthesis is brand-new, and increased at 5' end bamHi restriction enzyme site (lower setting-out part), 3' end increased terminator codon (TGA) and xhoi restriction enzyme site (lower setting-out part), makes this gene fragment be easy to be cloned into plasmid pGEX4T-2's bamHi and xhoin I restriction enzyme site.
Epitope aminoacid sequence (the 877th aa of the 745th aa-) in the staphylococcus aureus surface albumen FnBPA of screening:
Gly Gln Asn Ser Gly Asn Gln Ser Phe Glu Glu Phe Thr Glu Glu Asp Lys Pro Lys Tyr Glu Gln Gly Gly Asn Ile Val Asp Ile Asp Phe Asp Ser Val Pro Gln Ile His Gly Gln Asn Lys Gly Asn Gln Ser Phe Glu Glu Asp Thr Glu Lys Asp Lys Pro Lys Tyr Glu His Gly Gly Asn Ile Ile Asp Ile Asp Phe Asp Ser Val Pro His Ile His Gly Phe Asn Lys His Thr Glu Ile Ile Glu Glu Asp Thr Asn Lys Asp Lys Pro Ser Tyr Gln Phe Gly Gly His Asn Ser Val Asp Phe Glu Glu Asp Thr Leu Pro Lys Val Ser Gly Gln Asn Glu Gly Gln Gln Thr Ile Glu Glu Asp Thr Thr Pro Pro Ile Val
The DNA sequence (414bp) of the staphylococcus aureus surface albumen FnBPA antigen epitope genes of chemosynthesis is as follows:
GGATCC GGT CAG AAC AGC GGT AAC CAG TCG TTT GAA GAA GAC ACG GAA GAA GAT AAG CCG AAG TAT GAA CAG GGT GGC AAC ATT GTG GAC ATT GAT TTT GAC AGT GTC CCG CAG ATT CAT GGC CAA AAC AAA GGT AAT CAG TCC TTC GAA GAA GAC ACC GAA AAA GAT AAG CCG AAA TAT GAA CAC GGC GGT AAC ATT ATC GAT ATT GAC TTT GAT AGC GTT CCG CAT ATC CAC GGC TTC AAC AAG CAT ACC GAA ATT ATC GAA GAA GAC ACG AAT AAG GAT AAA CCG AGC TAC CAA TTT GGC GGT CAC AAT TCT GTG GAC TTC GAA GAA GAT ACG CTG CCG AAA GTT TCC GGT CAG AAC GAA GGC CAG CAG ACG ATT GAA GAA GAC ACG ACG CCG CCG ATT GTT TGA CTCGAG
2. express the structure of streptococcus aureus FnBPA fragment recombinant plasmid:
Extract plasmid pGEX4T-2, use bamHi and xhoi double digestion, reclaims the plasmid large fragment that enzyme is cut after 1.0% agarose gel electrophoresis, be dissolved in deionized water.Same using bamhI and xhothe streptococcus aureus FnBPA fragment of I double digestion chemosynthesis, reclaims object fragment after 1.0% agarose gel electrophoresis, be dissolved in deionized water.
DNA fragmentation after above-mentioned two kinds of enzymes of the volumetric molar concentration such as getting are cut with the connection of spending the night of 16 ℃ of T4 DNA ligase enzymes, is inserted in vector pGEX 4T-2 streptococcus aureus FnBPA gene fragment in same centrifuge tube bamh I and xhobetween I site, consistent with the initiator codon translation framework on carrier, express a fusion rotein.
3. the screening of recombinant plasmid and evaluation:
By recombinant plasmid transformed e. coli bl21 (DE3), coating is dull and stereotyped containing penbritin (100 μ g/ml) LB, puts 37 ℃ and spends the night.Next day, random picking transformed bacterium colony, was seeded to the in vitro jolting containing 4ml LB substratum (containing penbritin 100 μ g/ml), extracted recombinant plasmid, used bamHi and xhothe checking of I double digestion, 1.0% agarose gel electrophoresis result shows, cuts the object fragment of about 414bp.The plasmid that contains foreign gene is carried out to DNA sequencing analysis (Nanjing Jin Sirui company helps through), and sequencing result confirms that recombinant plasmid contains streptococcus aureus FnBPA gene fragment, and sequence is entirely true:
GGT CAG AAC AGC GGT AAC CAG TCG TTT GAA GAA GAC ACG GAA GAA GAT AAG CCG AAG TAT GAA CAG GGT GGC AAC ATT GTG GAC ATT GAT TTT GAC AGT GTC CCG CAG ATT CAT GGC CAA AAC AAA GGT AAT CAG TCC TTC GAA GAA GAC ACC GAA AAA GAT AAG CCG AAA TAT GAA CAC GGC GGT AAC ATT ATC GAT ATT GAC TTT GAT AGC GTT CCG CAT ATC CAC GGC TTC AAC AAG CAT ACC GAA ATT ATC GAA GAA GAC ACG AAT AAG GAT AAA CCG AGC TAC CAA TTT GGC GGT CAC AAT TCT GTG GAC TTC GAA GAA GAT ACG CTG CCG AAA GTT TCC GGT CAG AAC GAA GGC CAG CAG ACG ATT GAA GAA GAC ACG ACG CCG CCG ATT GTT
The expression of recombinant plasmid streptococcus aureus FnBPA gene fragment (133 amino acid) building, has merged 226 amino acid on carrier at its N end, 359 amino acid of total length, and its aminoacid sequence is as follows:
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg Gly Ser
4. the Screening and Identification of expressed fusion protein engineering bacteria:
To contain the positive transformant of recombinant plasmid, be seeded to containing 4ml LB substratum (containing penbritin 100 μ g/ml) in vitro, 37 ℃ of shaking culture 4h, preserve bacterial classification.Remaining bacterium liquid adds IPTG to final concentration 0.2mmol/L, continue shaking culture induction 4h, centrifugal collection thalline carries out SDS-PAGE detection, recon is expressed the streptococcus aureus FnBPA albumen that relative molecular weight is about 42 kDa, expression amount is about 20%, and contrast bacterium pGEX4T-2 is without this protein band.
5. express the purifying of streptococcus aureus FnBPA albumen:
1) express the ultrasonic degradation of the golden bacterium FnBPA of Portugal protein engineering bacterium
By the engineering bacteria of abduction delivering fusion rotein 8000 rpm, 4 ℃, centrifugal 20 mins, thalline is resuspended in the lysate (20mmol/L PB pH8.0,10 mmol/L EDTA, 1mmol/L DTT, 5% glycerine) of original fluid 1/10 volume, the broken bacterium of ice-bath ultrasonic 75 times, 8000 rpm, 4 ℃, centrifugal 20 mins, abandon precipitation, collect supernatant.The supernatant of collecting is for next step affinitive layer purification.
2) express the purifying of streptococcus aureus FnBPA albumen
Supernatant solution adds counter-balanced High-Affinity GSH Resin 10ml, and 4 ℃ of combinations are spent the night, and loading is collected and penetrated liquid.With 1 * PBS washing pillar of ten times of column volumes, then with GSH elutriant (50 mmol/L Tris-HCl pH8.0+10 mmol/L GSH) minute three wash-out target proteins of 50ml high density, be the streptococcus aureus FnBPA protein fragments of purifying.
6. the streptococcus aureus FnBPA protein fragments of expressing is for streptococcus aureus vaccine.
7. by the streptococcus aureus FnBPA protein fragments of expressing, for immune Balb/C mouse, prepare monoclonal antibody specific.
8. by the streptococcus aureus FnBPA protein fragments of expressing, for immune new zealand white rabbit, prepare polyclonal antibody antibody.
9. by monoclonal antibody and the how anti-assembling colloid gold reagent bar of preparation, for scene, the rapid detection of streptococcus aureus.
The gene fragment order of the staphylococcus aureus surface albumen FnBPA of described chemosynthesis, utilizes bacterium, yeast cell, insect cell, mammalian cell and genetically modified animals and plants to carry out recombinant expressed, preparation.
Staphylococcus aureus surface albumen FnBPA fragment prepared by above-described method, for streptococcus aureus antibody test and monoclonal antibody and how anti-preparation, and for the assembling of colloid gold reagent bar.
the present invention compared with prior art has advantages of
The staphylococcus aureus surface albumen FnBPA fragment that the present invention expresses, has more advantages:
1. current, lack streptococcus aureus vaccine.Vaccine inoculation can lifting body internal specific antibody titers, also increases Cellular Immunity power simultaneously, have synergy, and can reduce the selective pressure that microbiotic produces with antibiosis, delays the generation of resistant organism.But because streptococcus aureus vaccine is deactivation whole-bacterial-vaccine, except protective immunity is former, also contain a lot of incoherent and poisonous compositions, toxic side effects is larger, makes clinical application limited.Investigators are also in the development of being actively devoted to new generation vaccine.Surface protein FnBPA exists in more than 95% streptococcus aureus, and we have selected its strong antigen epi-position, utilizes genetic engineering technique to express preparation, for development recombinant vaccine lays the foundation.Recombinant vaccine safety, cost are low.
2, current, the method for shortage fast detecting Staphylococcus aureus.The present invention selects staphylococcus aureus surface albumen FnBPA epitope enrichment region, and immune animal, prepares specific antibody, and assembling colloid gold reagent bar, improves the recall rate of streptococcus aureus.
3. according to the streptococcus aureus FnBPA fragment aminoacid sequence that filters out, select the codon of prokaryotic organism preference, the gene order that chemosynthesis is brand-new, this gene suitable in prokaryotic cell prokaryocyte high expression level.
4. the engineering bacteria of the expression staphylococcus aureus surface albumen FnBPA building, expression amount can reach the more than 20% of tropina, and what express is soluble proteins, is easy to purifying, can prepare this albumen by large-scale purification.
Accompanying drawing explanation
Below with reference to accompanying drawing, the invention will be further described:
Fig. 1 is the full length amino acid sequence Characterization of antigenic epitopes result of molecular biology software to staphylococcus aureus surface albumen FnBPA.Result shows, from 877 amino acid of the 745th amino acid to the, contains strong wetting ability epitope, the position that in figure, arrow indicates at N end.
Fig. 2 is the construction of recombinant plasmid schema of expressing staphylococcus aureus surface albumen FnBPA.
Fig. 3 detects the double digestion figure of recombinant plasmid with 1.0% sepharose.M:DNA marker DL5000,1: recombinant plasmid pGEX4T-2- fnbpawarp bamHi and xhoi double digestion cuts the object fragment of about 414bp, the position that in figure, arrow indicates.
Fig. 4 is the SDS-PAGE analytical results of expressing staphylococcus aureus surface albumen FnBPA recombinant bacterium.
M: albumen marker (Quan Shijin); C: contrast bacterium is containing pGEX4T-2 plasmid; 1,2: recombinant bacterium, two recons are all expressed the fusion rotein that relative molecular weight is about 42kDa, the position that in figure, arrow indicates.
Fig. 5 is the SDS-PAGE analytical results of expressing after staphylococcus aureus surface albumen FnBPA purifying.M: albumen marker (Quan Shijin); Staphylococcus aureus surface albumen FnBPA after 1:High-Affinity GST Resin affinity chromatography column purification, OD 280=1.4, the about 1.2mg/m of concentration; 2:BSA(1mg/ml).
Fig. 6 is the result that ELISA detects rabbit anti-serum.The staphylococcus aureus surface albumen FnBPA immunity new zealand white rabbit of expressing is prepared antiserum(antisera), and serum titer reaches 1:400000.
Embodiment
The detailed description of embodiment of the present invention:
the analysis of staphylococcus aureus surface albumen FnBPA epitope, gene synthesize and express
By the full length amino acid sequence of Computer Analysis staphylococcus aureus surface albumen FnBPA, filter out the strong antigen epi-position in staphylococcus aureus surface albumen FnBPA, select the codon of prokaryotic organism preference, translate into corresponding nucleotide sequence, the brand-new gene order of chemosynthesis staphylococcus aureus surface albumen FnBPA strong antigen epi-position.By gene fragment clone in plasmid pGEX4T-2 bamHi and xhobetween I site, consistent with the translation framework of initiator codon on carrier, express a fusion rotein.By recombinant plasmid transformed e. coli bl21 (DE3), screening has obtained the engineering bacteria of high efficient expression staphylococcus aureus surface albumen FnBPA, and the staphylococcus aureus surface albumen FnBPA of expression accounts for 20% left and right of tropina total amount, and solvable.
materials and methods
1. bacterial classification and plasmid: e. coli bl21 (DE3) and expression vector pGEX4T-2 are preserved by military medical research institute of Nanjing Military Command medicine bioengineering.
2. molecular biology reagent: restriction enzyme bamHi, xhoi and T4 DNA ligase are TaKaRa company product.Plasmid purification test kit is Promega company product.DTT and IPTG are BIOSHARP company product.Other reagent is import or domestic analytical pure.
3. gene fragment is synthetic: by Nanjing Genscript Biotechnology Co., Ltd., helped synthetic.
4. the enzyme of gene clone method: DNA is cut, is connected; The extraction of plasmid, conversion; The general molecular cloning methods such as the SDS-PAGE analysis of albumen carry out according to a conventional method.Other test kit by specification operates.
5. DNA sequence analysis: with Promega company plasmid purification test kit plasmid purification, with the full-automatic sequenator order-checking of DNA.
result
1. staphylococcus aureus surface albumen FnBPA epitope screening and gene fragment is synthetic:
Utilize ANTHEWIN, DNAStar equimolecular biological software, by the full length amino acid sequence (GeneBank of Computer Analysis staphylococcus aureus surface albumen FnBPA, ACCESSION:P14738), filter out the strong antigen epi-position in staphylococcus aureus surface albumen FnBPA, from 877 amino acid of the 745th amino acid to the, see Fig. 1, its aminoacid sequence is as follows:
Gly Gln Asn Ser Gly Asn Gln Ser Phe Glu Glu Phe Thr Glu Glu Asp Lys Pro Lys Tyr Glu Gln Gly Gly Asn Ile Val Asp Ile Asp Phe Asp Ser Val Pro Gln Ile His Gly Gln Asn Lys Gly Asn Gln Ser Phe Glu Glu Asp Thr Glu Lys Asp Lys Pro Lys Tyr Glu His Gly Gly Asn Ile Ile Asp Ile Asp Phe Asp Ser Val Pro His Ile His Gly Phe Asn Lys His Thr Glu Ile Ile Glu Glu Asp Thr Asn Lys Asp Lys Pro Ser Tyr Gln Phe Gly Gly His Asn Ser Val Asp Phe Glu Glu Asp Thr Leu Pro Lys Val Ser Gly Gln Asn Glu Gly Gln Gln Thr Ile Glu Glu Asp Thr Thr Pro Pro Ile Val
According to the epitope aminoacid sequence in the staphylococcus aureus surface albumen FnBPA of screening, select the codon of prokaryotic organism preference, the gene order that chemosynthesis is brand-new, and increased at 5' end bamHi restriction enzyme site (lower setting-out part), 3' end increase terminator codon (TGA) and xhoIi restriction enzyme site (lower setting-out part), makes this gene fragment be easy to be cloned in plasmid pGEX4T-2 bamHi and xhoin I restriction enzyme site.The DNA sequence (414bp) containing staphylococcus aureus surface albumen FnBPA antigen epitope genes of chemosynthesis is as follows:
GGATCC GGT CAG AAC AGC GGT AAC CAG TCG TTT GAA GAA GAC ACG GAA GAA GAT AAG CCG AAG TAT GAA CAG GGT GGC AAC ATT GTG GAC ATT GAT TTT GAC AGT GTC CCG CAG ATT CAT GGC CAA AAC AAA GGT AAT CAG TCC TTC GAA GAA GAC ACC GAA AAA GAT AAG CCG AAA TAT GAA CAC GGC GGT AAC ATT ATC GAT ATT GAC TTT GAT AGC GTT CCG CAT ATC CAC GGC TTC AAC AAG CAT ACC GAA ATT ATC GAA GAA GAC ACG AAT AAG GAT AAA CCG AGC TAC CAA TTT GGC GGT CAC AAT TCT GTG GAC TTC GAA GAA GAT ACG CTG CCG AAA GTT TCC GGT CAG AAC GAA GGC CAG CAG ACG ATT GAA GAA GAC ACG ACG CCG CCG ATT GTT TGA CTCGAG
2. express the structure of staphylococcus aureus surface albumen FnBPA fragment recombinant plasmid:
Extract plasmid pGEX4T-2, use bamHi and xhoi double digestion, reclaims the plasmid large fragment that enzyme is cut after 1.0% agarose gel electrophoresis, be dissolved in deionized water.Same using bamHi and xhothe gene fragment of I double digestion chemosynthesis, electrophoresis is dissolved in deionized water after reclaiming.
Above-mentioned two kinds of enzymes of volumetric molar concentration such as get and cut rear DNA fragmentation, in same centrifuge tube, with 16 ℃ of T4 DNA ligase enzymes, the connection of spending the night, is inserted in vector pGEX 4T-2 staphylococcus aureus surface albumen FnBPA gene fragment bamHi and xhobetween I site, consistent with the initiator codon translation framework on carrier, express a fusion rotein, specifically build flow process, see Fig. 2.
3. the evaluation of recombinant plasmid:
The recombinant plasmid transformed that upper step is connected, to e. coli bl21 (DE3), containing on the solid LB substratum of penbritin (100 μ g/ml), is put 37 ℃ of overnight incubation by converted product coating.2 transformant bacterium colonies of random choose next day, be labeled as respectively 1, No. 2, choose the contrast bacterium that 1 empty plasmid pGEX4T-2 transforms simultaneously, be labeled as C, be inoculated into respectively containing 4ml liquid LB substratum (containing penbritin 100 μ g/ml) in vitro, put 37 ℃ of shaking culture 5h, extract recombinant plasmid.With bamHi and xhoi double digestion, detects with 1.0% sepharose.Recombinant plasmid cuts out the goal gene fragment of 414bp, and does not cut out this gene fragment containing the contrast bacterium of plasmid pGEX4T-2, sees Fig. 3.Tentative confirmation, the gene fragment that transformant contains staphylococcus aureus surface albumen FnBPA.
Extract the plasmid of recon, send Nanjing Genscript Biotechnology Co., Ltd.'s order-checking, DNA sequence analysis confirmation, recombinant plasmid contains synthetic staphylococcus aureus surface albumen FnBPA gene fragment, and sequence is entirely true:
GGT CAG AAC AGC GGT AAC CAG TCG TTT GAA GAA GAC ACG GAA GAA GAT AAG CCG AAG TAT GAA CAG GGT GGC AAC ATT GTG GAC ATT GAT TTT GAC AGT GTC CCG CAG ATT CAT GGC CAA AAC AAA GGT AAT CAG TCC TTC GAA GAA GAC ACC GAA AAA GAT AAG CCG AAA TAT GAA CAC GGC GGT AAC ATT ATC GAT ATT GAC TTT GAT AGC GTT CCG CAT ATC CAC GGC TTC AAC AAG CAT ACC GAA ATT ATC GAA GAA GAC ACG AAT AAG GAT AAA CCG AGC TAC CAA TTT GGC GGT CAC AAT TCT GTG GAC TTC GAA GAA GAT ACG CTG CCG AAA GTT TCC GGT CAG AAC GAA GGC CAG CAG ACG ATT GAA GAA GAC ACG ACG CCG CCG ATT GTT TGA
The expression of recombinant plasmid staphylococcus aureus surface albumen FnBPA fragment (133 amino acid) building, has merged 226 amino acid on carrier at its N end, 359 amino acid of total length, and its aminoacid sequence is as follows:
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg Gly Ser Gly Gln Asn Ser Gly Asn Gln Ser Phe Glu Glu Phe Thr Glu Glu Asp Lys Pro Lys Tyr Glu Gln Gly Gly Asn Ile Val Asp Ile Asp Phe Asp Ser Val Pro Gln Ile His Gly Gln Asn Lys Gly Asn Gln Ser Phe Glu Glu Asp Thr Glu Lys Asp Lys Pro Lys Tyr Glu His Gly Gly Asn Ile Ile Asp Ile Asp Phe Asp Ser Val Pro His Ile His Gly Phe Asn Lys His Thr Glu Ile Ile Glu Glu Asp Thr Asn Lys Asp Lys Pro Ser Tyr Gln Phe Gly Gly His Asn Ser Val Asp Phe Glu Glu Asp Thr Leu Pro Lys Val Ser Gly Gln Asn Glu Gly Gln Gln Thr Ile Glu Glu Asp Thr Thr Pro Pro Ile Val
4. express the Screening and Identification of staphylococcus aureus surface albumen FnBPA engineering bacteria:
By the positive transformant that contains recombinant plasmid and 1 contrast bacterium that empty plasmid pGEX4T-2 transforms, be seeded to containing 4ml LB substratum (containing penbritin 100 μ g/ml) in vitro, 37 ℃ of shaking culture 5h, preserve after bacterial classification, adding final concentration induces to the IPTG of 0.2mmol/L, 25 ℃ are continued shaking culture 4h, centrifugal collection thalline carries out SDS-PAGE detection, recon is expressed the staphylococcus aureus surface albumen FnBPA that relative molecular weight is about 42kDa, expression amount is about 20%, and contrast bacterium is without this protein band, see Fig. 4.
express the purifying of staphylococcus aureus surface albumen FnBPA
According to the aminoacid sequence of expressing staphylococcus aureus surface albumen FnBPA, analyze its physicochemical property, determine suitable purification process.We expressed staphylococcus aureus surface albumen FnBPA merges the GST albumen on carrier, gst fusion protein has been expressed glutathione S has simultaneously been turned enzyme when expressing, can be easily separated with GST sepharose FF, therefore determine to adopt affinity chromatography, with High-Affinity GST Resin, carry out purifying.Concrete steps are as follows:
Materials and methods
1. main agents:
High-Affinity GSH Resin is Nanjing Genscript Biotechnology Co., Ltd.'s product, IPTG, DTTWei BIOSHARP company product.Other reagent is domestic or Import Analysis is pure.
2. express the ultrasonic degradation of staphylococcus aureus surface albumen FnBPA engineering bacteria:
The engineering bacteria of the expression staphylococcus aureus surface protein FnBPA of cultivation is poured in Centrifuge Cup, 8000rpm, 4 ℃, centrifugal 20mins, abandon supernatant, thalline is resuspended in the lysate (20mmol/L PB pH8.0,10 mmol/L EDTA, 1mmol/L DTT, 5% glycerine) of original fluid 1/10 volume, and ice-bath ultrasonic is broken bacterium 10mins, 8000rpm, 4 ℃, centrifugal 20mins, collects supernatant, abandons precipitation.The supernatant of collecting is for affinitive layer purification.
3. express the purifying of staphylococcus aureus surface albumen FnBPA:
Supernatant solution adds counter-balanced High-Affinity GSH Resin gel 10ml, and 4 ℃ of combinations are spent the night, and loading is collected and penetrated liquid.With 1 * PBS(of ten times of column volumes, contain 1mM pmsf) washing pillar, then use the GSH elutriant of 50ml high density, elutriant is 50 mmol/L Tris-HCl pH8.0+10 mmol/L GSH, divides three wash-out target proteins, is the staphylococcus aureus surface protein fragments of purifying.
Result
The albumen of wash-out from High-Affinity GSH Resin post is carried out to SDS-PAGE analysis, and result demonstration, obviously gives expression to FnBPA/GST fusion rotein through inducing, and expression product is mainly present in supernatant liquor, and the about 42kDa of molecular weight, is shown in Fig. 5.After wash-out, record OD 280=1.4, calculate the about 1.2mg/ml of concentration, SDS-PAGE Explicit Expression product purity is more than 90%.
the application of the staphylococcus aureus surface albumen FnBPA of purifying
The staphylococcus aureus surface albumen FnBPA antigen that purifying is obtained, immune new zealand white rabbit, prepares polyvalent antibody.Adopt indirect elisa method to detect antiserum titre, result confirmation, the staphylococcus aureus surface albumen FnBPA antigen that purifying obtains has good immunogenicity and antigenicity.
materials and methods
1. main raw:
New zealand white rabbit is purchased from Nanjing Hua Bukang biological products company, 96 hole elisa plates are Shenzhen Jin Canhua company product, complete Freund's adjuvant and incomplete Freund's adjuvant are sigma company product, Goat Anti-rabbit IgG HRP is Beijing Bo Aosen company product, and TMB nitrite ion is beyotime company product.Other reagent are domestic or Import Analysis is pure.
2. animal immune and antiserum(antisera) preparation:
The Healthy female new zealand white rabbit of two 2kg left and right of picking, auricular vein is got the about 1ml of blood, and normal serum before preparation immunity, as negative control.The staphylococcus aureus surface albumen FnBPA that first immunisation obtains purifying and Freund's complete adjuvant by volume 1:1 push into emulsion in syringe, carry out the subcutaneous multi-point injection in back.The FnBPA that 2w, 4w obtain purifying after initial immunity and Freund's incomplete adjuvant by volume 1:1 push into emulsion in syringe, carry out the subcutaneous multi-point injection in back.At the pure antigen of 6w injection FnBPA, after 1w, get blood examination and survey serum titer.Carotid artery is got blood, and room temperature is placed 1h, and 4 ℃ of standing over night, with the centrifugal 30min of 2500g, get supernatant ,-20 ℃ of preservations.
3. the ELISA of immune serum detects:
The staphylococcus aureus surface albumen FnBPA that purifying is obtained is diluted to the coated 96 hole elisa plates of 1 μ g/ml, and 4 ℃ are spent the night.With PBST washing 3 times, every hole adds 20% calf serum confining liquid 130 μ l, 37 ℃ of sealing 2h.Rabbit anti-serum starts dilution from 1:1000, and negative control dilutes in proportion, 37 ℃ of reaction 1h.After PBST washing 3 times, add the IgG (1:5000) of the goat antirabbit of HRP mark, 37 ℃ of reaction 30min.After PBST washing 5 times, add TMB nitrite ion, 37 ℃ of lucifuge colour developing 10min, with 1mol/L HCl termination reaction, detect A by microplate reader 450value.Using the antibody greatest dilution of P/N >=2.1 as the final value of tiring.
Result
Adopt ELISA method to detect sero-fast tiring, rabbit anti-serum starts dilution from 1:1000, get serum before not immunity as negative control simultaneously, ELISA result shows, after the pure antigen booster immunization of FnBPA, serum titer is greater than 1:400000, negative control can not be shown in Fig. 6 in conjunction with colour developing, illustrates that the staphylococcus aureus surface albumen FnBPA antigen that purifying obtains has good immunogenicity and antigenicity.
the gene fragment order table of the FnBPA epitope enrichment region of chemosynthesissee appendix document: Nucleotide or the readable carrier of aminoacid sequence list machine.
Figure IDA0000452083740000011
Figure IDA0000452083740000031

Claims (4)

1. the gene fragment of the staphylococcus aureus surface albumen FnBPA of a chemosynthesis, this gene fragment coding is containing the staphylococcus aureus surface albumen FnBPA gene fragment of strong antigen epi-position, i.e. 877 amino acid of the 745th amino acid to the, totally 133 amino acid, have increased at the 5' of this gene fragment end bamHi restriction enzyme site (underscore part), 3' end increased terminator codon TGA and xhoi restriction enzyme site (underscore part), the gene order total length 414bp of chemosynthesis, sequence is as follows:
GGATCC GGT CAG AAC AGC GGT AAC CAG TCG TTT GAA GAA GAC ACG GAA GAA GAT AAG CCG AAG TAT GAA CAG GGT GGC AAC ATT GTG GAC ATT GAT TTT GAC AGT GTC CCG CAG ATT CAT GGC CAA AAC AAA GGT AAT CAG TCC TTC GAA GAA GAC ACC GAA AAA GAT AAG CCG AAA TAT GAA CAC GGC GGT AAC ATT ATC GAT ATT GAC TTT GAT AGC GTT CCG CAT ATC CAC GGC TTC AAC AAG CAT ACC GAA ATT ATC GAA GAA GAC ACG AAT AAG GAT AAA CCG AGC TAC CAA TTT GGC GGT CAC AAT TCT GTG GAC TTC GAA GAA GAT ACG CTG CCG AAA GTT TCC GGT CAG AAC GAA GGC CAG CAG ACG ATT GAA GAA GAC ACG ACG CCG CCG ATT GTT TGA CTCGAG。
2. the gene fragment of the staphylococcus aureus surface albumen FnBPA of chemosynthesis claimed in claim 1, adopt genetic engineering technique to express the staphylococcus aureus surface albumen FnBPA gene fragment of this gene order coding, the protein fragments that purifying is expressed, concrete grammar is as follows:
The structure of staphylococcus aureus surface albumen FnBPA gene fragment recombinant plasmid:
With bamHi and xhothe staphylococcus aureus surface albumen FnBPA gene fragment of I double digestion plasmid pGEX4T-2 and chemosynthesis, after electrophoresis reclaims, connects with T4 DNA ligase enzyme, and staphylococcus aureus surface albumen FnBPA gene fragment is inserted in vector pGEX 4T-2 bamh I and xhobetween I site, consistent with the initiator codon translation framework on carrier, express a fusion rotein, 359 amino acid of total length, this fusion rotein N end has merged 226 amino acid on carrier, C end comprises 877 amino acid of the 745th amino acid to the in staphylococcus aureus surface albumen FnBPA, and full length amino acid sequence is as follows:
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys
Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg Gly Ser Gly Gln Asn Ser Gly Asn Gln Ser Phe Glu Glu Phe Thr Glu Glu Asp Lys Pro Lys Tyr Glu Gln Gly Gly Asn Ile Val Asp Ile Asp Phe Asp Ser Val Pro Gln Ile His Gly Gln Asn Lys Gly Asn Gln Ser Phe Glu Glu Asp Thr Glu Lys Asp Lys Pro Lys Tyr Glu His Gly Gly Asn Ile Ile Asp Ile Asp Phe Asp Ser Val Pro His Ile His Gly Phe Asn Lys His Thr Glu Ile Ile Glu Glu Asp Thr Asn Lys Asp Lys Pro Ser Tyr Gln Phe Gly Gly His Asn Ser Val Asp Phe Glu Glu Asp Thr Leu Pro Lys Val Ser Gly Gln Asn Glu Gly Gln Gln Thr Ile Glu Glu Asp Thr Thr Pro Pro Ile Val
The Screening and Identification of expressed fusion protein engineering bacteria:
By recombinant plasmid transformed e. coli bl21, coating is dull and stereotyped containing the LB of 100 μ g/ml penbritins, puts 37 ℃ and spends the night, next day, random picking transformed bacterium colony and containing the contrast bacterium of plasmid pGEX4T-2, extract plasmid, double digestion is verified, cuts out the object band of 414bp; The plasmid that contains foreign gene is carried out to DNA sequence analysis, and sequential analysis confirms that recombinant plasmid contains streptococcus aureus fnbpa gene fragment, and sequence is entirely true; The positive transformant of recombinant plasmid will be contained, be seeded to containing in the LB substratum of penbritin 100 μ g/mL, 37 ℃ of shaking culture 4h, add IPTG to final concentration 0.2 mmol/L, continue shaking culture induction 4h, centrifugal collection thalline carries out SDS-PAGE detection, and recon is expressed the golden bacterium surface protein FnBPA of Portugal that relative molecular weight is about 42 kD, expression amount is about 20%, and the contrast bacterium that contains empty plasmid pGEX4T-2 is without this protein band;
Express the purifying of staphylococcus aureus surface albumen FnBPA:
The engineering bacteria of abduction delivering fusion rotein is centrifugal, receive bacterium, thalline is resuspended in lysate (20mmol/L PB pH8.0,10 mmol/L EDTA, 1mmol/L DTT, 5% glycerine), the broken bacterium of ice-bath ultrasonic 75 times, centrifugal collection supernatant, supernatant solution adds counter-balanced High-Affinity GSH Resin gel 10ml, and 4 ℃ of combinations are spent the night, loading, collects and penetrates liquid; With 1 * PBS washing pillar of ten times of column volumes, then with GSH elutriant (50 mmol/L Tris-HCl pH8.0+10 mmol/L GSH) minute three wash-out target proteins of 50ml high density, be the golden bacterium FnBPA of the Portugal protein fragments of purifying.
3. the gene fragment order of the staphylococcus aureus surface albumen FnBPA of chemosynthesis claimed in claim 1, utilizes bacterium, yeast cell, insect cell, mammalian cell and genetically modified animals and plants to carry out recombinant expressed, preparation.
4. the staphylococcus aureus surface albumen FnBPA fragment that prepared by claim 2 or method claimed in claim 3, for streptococcus aureus antibody test and monoclonal antibody and how anti-preparation, and for the assembling of colloid gold reagent bar.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106478777A (en) * 2016-09-29 2017-03-08 安徽农业大学 Staphylococcus aureus FnBPA A albumen mimic epitope peptide, mimic epitope peptide combinations and its application with immune protective
CN106544352A (en) * 2016-10-30 2017-03-29 吉林农业大学 A kind of intrusion type lactic acid bacteria based on FnBPA albumen
CN111423516A (en) * 2020-04-01 2020-07-17 广州佰斯伦医疗器械有限公司 Protein and application thereof in wound repair and bacteriostasis
CN111909279A (en) * 2020-08-14 2020-11-10 石河子大学 Staphylococcus aureus and streptococcus agalactiae immunogen fusion expression and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102847168A (en) * 2012-07-10 2013-01-02 新疆农业大学 Design and construction of nucleic acid vaccine PV-Fn for preventing cow mastitis

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102847168A (en) * 2012-07-10 2013-01-02 新疆农业大学 Design and construction of nucleic acid vaccine PV-Fn for preventing cow mastitis

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FABRIZIA CASOLINI 等: "Antibody Response to Fibronectin-Binding Adhesin FnbpA in Patients with Staphylococcus aureus Infections", 《INFECTION AND IMMUNITY》, vol. 66, no. 11, 31 December 1998 (1998-12-31), pages 5433 - 5442, XP002222372 *
SIGNAS,C.等: "RecName: Full=Fibronectin-binding protein A; Flags: Precursor，UniProtKB/Swiss-Prot: P14738.1", 《GENBANK》, 13 November 2013 (2013-11-13) *
高翔 等: "金黄色葡萄球菌FnbpA、ClfA、Ebps 抗原表位的串联表达及多克隆抗体的制备", 《中国畜牧兽医》, vol. 38, no. 5, 31 December 2011 (2011-12-31) *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106478777A (en) * 2016-09-29 2017-03-08 安徽农业大学 Staphylococcus aureus FnBPA A albumen mimic epitope peptide, mimic epitope peptide combinations and its application with immune protective
CN106544352A (en) * 2016-10-30 2017-03-29 吉林农业大学 A kind of intrusion type lactic acid bacteria based on FnBPA albumen
CN111423516A (en) * 2020-04-01 2020-07-17 广州佰斯伦医疗器械有限公司 Protein and application thereof in wound repair and bacteriostasis
CN111423516B (en) * 2020-04-01 2022-02-11 广州佰斯伦医疗器械有限公司 Protein and application thereof in wound repair and bacteriostasis
CN111909279A (en) * 2020-08-14 2020-11-10 石河子大学 Staphylococcus aureus and streptococcus agalactiae immunogen fusion expression and application thereof
CN111909279B (en) * 2020-08-14 2022-06-24 石河子大学 Staphylococcus aureus and streptococcus agalactiae immunogen fusion expression and application thereof

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