CN103773779A - Gene segment of chemosynthetic streptococcus pneumoniae surface adhesin A as well as expression and application of gene segment - Google Patents

Gene segment of chemosynthetic streptococcus pneumoniae surface adhesin A as well as expression and application of gene segment Download PDF

Info

Publication number
CN103773779A
CN103773779A CN201310752891.1A CN201310752891A CN103773779A CN 103773779 A CN103773779 A CN 103773779A CN 201310752891 A CN201310752891 A CN 201310752891A CN 103773779 A CN103773779 A CN 103773779A
Authority
CN
China
Prior art keywords
gaa
aag
streptococcus pneumoniae
ctg
aaa
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201310752891.1A
Other languages
Chinese (zh)
Other versions
CN103773779B (en
Inventor
李越希
蔡冉
徐悦玥
许桂丽
周洁
李素芹
潘英
李丙军
陈乐如
张素芬
马颖
袁敬宇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eastern Theater Disease Prevention And Control Center Of Pla
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to CN201310752891.1A priority Critical patent/CN103773779B/en
Publication of CN103773779A publication Critical patent/CN103773779A/en
Application granted granted Critical
Publication of CN103773779B publication Critical patent/CN103773779B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Peptides Or Proteins (AREA)

Abstract

The invention provides a gene segment of chemosynthetic streptococcus pneumoniae surface adhesin A as well as expression and application of the gene segment, and relates to the fields of a genetic engineering technology and diagnostic reagents. A bran-new gene sequence of an epitope is chemically synthesized by screening out a strong antigen epitope and total 197 amino acids from the 84th amino acid to the 280th amino acid in streptococcus pneumoniae adhesin A through computer analysis and selecting a codon which a procaryotic organism prefers. The genetic engineering technology is used for expressing the gene segment and preparing a strong antigen epitope segment of the streptococcus pneumoniae adhesin A. Expressed protein can be used for vaccine research and development, detection of streptococcus pneumoniae infection antibodies and preparation of monoclonal antibodies and polyclonal antibodies.

Description

The gene fragment of chemosynthesis streptococcus pneumoniae surface adhesion element A and expression, application
Technical field
What the gene fragment of chemosynthesis streptococcus pneumoniae surface adhesion element A of the present invention and expression, application related to is a kind of by the brand-new gene fragment of chemosynthesis streptococcus pneumoniae surface adhesion element A (PsaA albumen), utilize genetic engineering technique, prepare recombinant protein.By Computer Analysis, filter out the fragment containing strong antigen epi-position streptococcus pneumoniae PsaA albumen, 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 detection of streptococcus pneumoniae infection antibody and the preparation of monoclonal antibody etc., the present invention relates to genetic engineering technique and detection reagent field.
Background technology
Streptococcus pneumoniae (Streptococcus pneumoniae) is called for short streptococcus pneumoniae (pneumococcus).For gram's staining positive bacteria.Streptococcus pneumoniae can cause different types of disease, includes acute sinusitis, otitis media, meningitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis and cerebral abscess.
Have for the main detection method of streptococcus pneumoniae infection at present: bacteriology partition method, euzymelinked immunosorbent assay (ELISA) (ELISA), DNA probe and round pcr etc.But it is long that these methods more or less all exist experimental period, is not suitable for the shortcomings such as field quick detection.In recent years, immune-gold labeled chromatographic technique is widely used in the each field of biomedicine, for the detection of streptococcus pneumoniae provides new thinking.Screening streptococcus pneumoniae adventitia specific antigens, and prepare highly purified specific antigens, be the important prerequisite of setting up this technology.
Streptococcus pneumoniae surface adhesion element A (PsaA) is that the total one heredity of various Sp is conservative, and species specific surface bonding lipoprotein is relevant with aggressive and the virulence of bacterium.PsaA albumen is positioned at surface of cell membrane, is wrapped in by pod membrane layer closely.PsaA has the conservative property of height on gene order and coded protein sequence.The all or part of cell surface that is exposed to of PsaA antigenic determinant, has good antigenicity, after organism infection Sp, can produce anti-PsaA specific antibody.Therefore, this albumen is likely as the antigen of streptococcus pneumoniae candidate albumen vaccine, simultaneously also likely for detection of corresponding antibody in patients serum, for the quick diagnosis of streptococcus pneumoniae infection provides foundation.PsaA coding open reading frame is 930bp, and molecular weight is about 37kDa.
Summary of the invention
The present invention, by the brand-new gene fragment of the PsaA albumen of the brachymemma of chemosynthesis, utilizes genetic engineering technique, preparation PsaA albumen high expression level fragment.By Computer Analysis, filter out the PsaA protein extracellular fragment containing strong antigen epi-position, 280 amino acid of the 84th amino acid-, totally 197 amino acid, the codon of selecting prokaryotic organism preference, the gene order that chemosynthesis is brand-new, utilizes genetic engineering technique to express this gene.The albumen of expressing can be used for the detection of streptococcus pneumoniae infection antibody or the preparation of monoclonal cell strain.
the gene fragment of chemosynthesis streptococcus pneumoniae surface adhesion element A and expression, application take following steps to realize:
The screening of 1.PsaA Protein Epitopes and the chemosynthesis of gene fragment thereof:
Utilize the softwares such as ANTHEWIN, by the aminoacid sequence of Computer Analysis, find that PsaA albumen contains stronger antigenic determinant, selects 280 amino acid of the 84th amino acid-as research object.Select the codon of prokaryotic organism preference, the gene order that chemosynthesis is brand-new, and increased at 5' end bamhI restriction enzyme site (underscore part), 3' end increased terminator codon (TAA) and ecorI restriction enzyme site (underscore part), makes this gene fragment be easy to be cloned in plasmid pGEX4T-2 bamhI and ecoin RI restriction enzyme site.
The PsaA protein fragments aminoacid sequence (280 aa of the 84th aa-) of screening:
The DNA sequence (606 bp) of the PsaA gene fragment of chemosynthesis:
GGATCC?CTG?ATC?TTC?TAC?AAT?GGT?ATC?AAT?CTG?GAA?ACG?GGT?GGT?AAC?GCC?TGG?TTC?ACG?AAA?CTG?GTC?GAA?AAT?GCC?AAG?AAA?ACG?GAA?AAC?AAA?GAC?TAT?TTT?GCA?GTC?AGC?GAT?GGC?GTG?GAC?GTT?ATT?TAC?CTG?GAA?GGC?CAG?AAC?GAA?AAA?GGC?AAG?GAA?GAT?CCG?CAT?GCA?TGG?CTG?AAC?CTG?GAA?AAT?GGT?ATT?ATC?TTT?GCG?AAG?AAC?ATC?GCC?AAA?CAA?CTG?TCT?GCT?AAG?GAC?CCG?AAC?AAC?AAG?GAA?TTC?TAC?GAA?AAG?AAC?CTG?AAG?GAA?TAC?ACC?GAT?AAA?CTG?GAC?AAG?CTG?GAT?AAG?GAA?TCA?AAG?GAT?AAG?TTC?AAC?AAG?ATT?CCG?GCG?GAA?AAG?AAA?CTG?ATC?GTG?ACG?TCG?GAA?GGC?GCC?TTT?AAG?TAT?TTC?AGT?AAA?GCA?TAC?GGT?GTT?CCG?TCC?GCT?TAT?ATT?TGG?GAA?ATC?AAT?ACC?GAA?GAA?GAA?GGC?ACG?CCG?GAA?CAG?ATT?AAA?ACC?CTG?GTT?GAA?AAA?CTG?CGT?CAA?ACG?AAA?GTC?CCG?TCC?CTG?TTC?GTC?GAA?AGC?TCT?GTG?GAT?GAC?CGC?CCG?ATG?AAG?ACC?GTG?TCA?CAG?GAT?ACC?AAC?ATT?CCG?ATT?TAT?GCC?CAG?ATT?TTT?ACC?GAC?TGA? CTCGAG
2. express the structure of PsaA protein extracellular fragment recombinant plasmid:
Extract plasmid pGEX4T-2, use bamhI and ecor I double digestion, reclaims the plasmid large fragment that enzyme is cut after electrophoresis, be dissolved in deionized water.Same using bamhI and ecothe PsaA gene fragment of R 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, connect with T4 DNA ligase enzyme, PsaA gene fragment is inserted in vector pGEX 4T-2 bamh I and ecobetween R 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. colibL21 (DE3), coating, containing penbritin (100 μ g/ml) LB flat board, is put 37 ℃ and is spent the night.Next day, random picking transformed bacterium colony and 1 contrast bacterium (plasmid pGEX4T-2 transformed bacteria), extract respectively plasmid, take extract plasmid as template, pcr amplification PsaA protein extracellular gene fragment, containing the positive recombinant plasmid of PsaA gene fragment, should amplify the gene fragment that is about 591 bp.The plasmid that contains foreign gene is carried out to DNA sequence analysis, and sequential analysis confirms that recombinant plasmid contains synthetic PsaA gene fragment, and sequence is entirely true:
CTG?ATC?TTC?TAC?AAT?GGT?ATC?AAT?CTG?GAA?ACG?GGT?GGT?AAC?GCC?TGG?TTC?ACG?AAA?CTG?GTC?GAA?AAT?GCC?AAG?AAA?ACG?GAA?AAC?AAA?GAC?TAT?TTT?GCA?GTC?AGC?GAT?GGC?GTG?GAC?GTT?ATT?TAC?CTG?GAA?GGC?CAG?AAC?GAA?AAA?GGC?AAG?GAA?GAT?CCG?CAT?GCA?TGG?CTG?AAC?CTG?GAA?AAT?GGT?ATT?ATC?TTT?GCG?AAG?AAC?ATC?GCC?AAA?CAA?CTG?TCT?GCT?AAG?GAC?CCG?AAC?AAC?AAG?GAA?TTC?TAC?GAA?AAG?AAC?CTG?AAG?GAA?TAC?ACC?GAT?AAA?CTG?GAC?AAG?CTG?GAT?AAG?GAA?TCA?AAG?GAT?AAG?TTC?AAC?AAG?ATT?CCG?GCG?GAA?AAG?AAA?CTG?ATC?GTG?ACG?TCG?GAA?GGC?GCC?TTT?AAG?TAT?TTC?AGT?AAA?GCA?TAC?GGT?GTT?CCG?TCC?GCT?TAT?ATT?TGG?GAA?ATC?AAT?ACC?GAA?GAA?GAA?GGC?ACG?CCG?GAA?CAG?ATT?AAA?ACC?CTG?GTT?GAA?AAA?CTG?CGT?CAA?ACG?AAA?GTC?CCG?TCC?CTG?TTC?GTC?GAA?AGC?TCT?GTG?GAT?GAC?CGC?CCG?ATG?AAG?ACC?GTG?TCA?CAG?GAT?ACC?AAC?ATT?CCG?ATT?TAT?GCC?CAG?ATT?TTT?ACC?GAC?TGA
The expression of recombinant plasmid PsaA protein extracellular fragment (197 amino acid) building, has merged 226 amino acid on carrier at its N end, 423 amino acid of total length, and its aminoacid sequence is as follows:
Figure 38398DEST_PATH_IMAGE002
4. the Screening and Identification of expressed fusion protein engineering bacteria:
The positive transformant of recombinant plasmid will be contained, be seeded to containing 3ml LB substratum (containing penbritin 100 μ g/ml) in vitro, 37 ℃ of shaking culture 3h, add IPTG to final concentration 1.0mmol/L, continue shaking culture induction 4h, centrifugal collection thalline carries out SDS-PAGE detection, and recon is expressed the Flic albumen that relative molecular weight is about 40 kDa, expression amount is about 30%, and contrast bacterium pGEX4T-2 is without this protein band.
5. express the purifying of PsaA albumen:
1) ultrasonic degradation of expression PsaA protein engineering bacterium
Centrifugal the engineering bacteria of abduction delivering fusion rotein (8000 rpm, 20 min, 4 ℃) are received to bacterium, thalline is resuspended in the lysate (50 mmol/L Tris-HCl pH8.0,10 mmol/L EDTA, 10 mmol/L DTT) of original fluid 1/10 volume, ice-bath ultrasonic is broken bacterium 10 min, centrifugal (8000 rpm, 20 min, 4 ℃) collect supernatant, abandon precipitation.The supernatant of collecting is for next step affinitive layer purification.
2) purifying of expression PsaA albumen
Supernatant solution adds counter-balanced Glutathione Sepharose 4B gel 3ml, and room temperature is in conjunction with 60min, and loading, collects and penetrate 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) point three wash-out target proteins of 15ml high density, be the PsaA protein extracellular fragment of purifying.
6. the ELISA of the PsaA albumen of purifying detects:
By preliminary purification obtain fusion rotein press 1:1000～1:32 000 doubling dilution, and by negative control with same concentration doubling dilution, detection expressing protein antigenicity (concrete operation method is shown in test kit specification sheets).Result shows that this amalgamation and expression albumen has good antigenicity and specificity.
7. by the PsaA protein fragments of expressing, prepare anti-PsaA monoclonal antibody and how anti-etc. for the detection of streptococcus pneumoniae infection antibody and for immunity.
8. synthetic PsaA protein gene fragment is connected with other gene fragment, expresses, prepare with the form of fusion rotein.
The gene fragment order of the streptococcus pneumoniae PsaA protein extracellular of described chemosynthesis, utilizes bacterium, yeast cell, insect cell, mammalian cell and genetically modified animals and plants to carry out recombinant expressed, preparation.
Streptococcus pneumoniae PsaA protein extracellular fragment prepared by method described above, for detection and monoclonal antibody and the how anti-preparation of streptococcus pneumoniae infection antibody.
the present invention compared with prior art has advantages of
The PsaA protein fragments that the present invention expresses, has more advantages:
1. adopt gene optimization technical optimization goal gene with solubility expression target protein;
2. according to the PsaA protein extracellular 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;
3. the engineering bacteria of the expression streptococcus pneumoniae PsaA albumen that the present invention builds, expression amount can reach 30% of tropina, is easy to purifying, can prepare this albumen by large-scale purification.Have no at present the report of expressing this section of albumen;
4. in experiment, the PsaA albumen of screening is streptococcus pneumoniae specific antigens, chooses this antigen as research object, can avoid other similar pathogenic agent to the interference detecting.
Accompanying drawing explanation
Below with reference to accompanying drawing, the invention will be further described:
Fig. 1 is that molecular biology software carries out analytical results to the epitope of PsaA albumen.Result shows, from 280 amino acid of the 84th amino acid to the of total length, contains strong antigenic determinant.
Fig. 2 is the construction of recombinant plasmid schema of expressing PsaA albumen.
Fig. 3 is the Agarose gel detection recon plasmid double digestion product with 1.0%.M:DNA marker DL5000 tM; 1: negative control; 2～5:4 the equal double digestion of transformant goes out the goal gene fragment of 590bp left and right.
Fig. 4 is the SDS-PAGE analytical results of expressing PsaA Protein reconstitution bacterium.M:Marker; 1～3:1～No. 3 recombinant bacterium, 3 recons are all expressed the fusion rotein that relative molecular weight is about 45KDa.
Fig. 5 is the SDS-PAGE analytical results after the PsaA protein purification of expressing.M:Marker; After 1 ~ 2:Glutathione Sepharose 4B affinity chromatography column purification PsaA albumen, purifying has obtained the fusion rotein that molecular weight is about 45kDa.
Fig. 6 is that ELISA detects immune sero-fast result schematic diagram.
embodiment
The detailed description of embodiment of the present invention:
analysis, the gene of streptococcus pneumoniae surface adhesion element Staphylococal Protein A epitope synthesize and express
By whole aminoacid sequences of Computer Analysis streptococcus pneumoniae PsaA albumen, filter out the strong antigen epi-position in PsaA albumen, select the codon of bacterium preference, the brand-new gene fragment of chemosynthesis PsaA albumen strong antigen epi-position.By gene fragment clone in plasmid pGEX4T-2 bamhI/ ecorI site, consistent with the translation framework of the initiator codon on carrier, can express a fusion rotein.By recombinant plasmid transformed e. coli bl21 (DE3), screening has obtained the engineering bacteria of high efficient expression PsaA albumen, and the PsaA albumen of expression accounts for 30% left and right of tropina total amount.
materials and methods
1. bacterial classification and plasmid: e. colibL21(DE3) and expression vector pGEX4T-2 be that preserve in this laboratory.
2. molecular biology reagent: restriction enzyme bamhI, ecorI and T4 DNA ligase are TaKaRa company product.Plasmid purification test kit and be TaKaRa company product from the test kit that reclaims DNA fragmentation in sepharose.DTT and IPTG are Promega company product.Other reagent is import or domestic analytical reagent.
3. gene fragment is synthetic: helped synthetic by Dalian TaKaRa company.
4. the enzyme of gene clone method: DNA is cut, connection, electrophoresis; 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 TAKARA company plasmid purification test kit plasmid purification, with the full-automatic sequenator order-checking of DNA.
result
1. the screening of streptococcus pneumoniae PsaA Protein Epitopes and gene fragment is synthetic:
Utilize the softwares such as ANTHEWIN, by whole aminoacid sequences of Computer Analysis streptococcus pneumoniae PsaA albumen, filter out the strong antigen epi-position (Fig. 1) in streptococcus pneumoniae PsaA albumen,, from 280 amino acid of the 84th amino acid to the, its aminoacid sequence is as follows:
Figure 381917DEST_PATH_IMAGE003
According to the epitope aminoacid sequence in the streptococcus pneumoniae PsaA albumen 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 (underscore part), 3' end increased terminator codon (TGA) and eco((underscore part) makes this gene fragment be easy to be cloned in plasmid pGEX4T-2 to RI restriction enzyme site bamhI and ecoin RI restriction enzyme site.The DNA sequence (606 bp) containing streptococcus pneumoniae PsaA Protein Epitopes gene of chemosynthesis is as follows:
GGATCC?CTG?ATC?TTC?TAC?AAT?GGT?ATC?AAT?CTG?GAA?ACG?GGT?GGT?AAC?GCC?TGG?TTC?ACG?AAA?CTG?GTC?GAA?AAT?GCC?AAG?AAA?ACG?GAA?AAC?AAA?GAC?TAT?TTT?GCA?GTC?AGC?GAT?GGC?GTG?GAC?GTT?ATT?TAC?CTG?GAA?GGC?CAG?AAC?GAA?AAA?GGC?AAG?GAA?GAT?CCG?CAT?GCA?TGG?CTG?AAC?CTG?GAA?AAT?GGT?ATT?ATC?TTT?GCG?AAG?AAC?ATC?GCC?AAA?CAA?CTG?TCT?GCT?AAG?GAC?CCG?AAC?AAC?AAG?GAA?TTC?TAC?GAA?AAG?AAC?CTG?AAG?GAA?TAC?ACC?GAT?AAA?CTG?GAC?AAG?CTG?GAT?AAG?GAA?TCA?AAG?GAT?AAG?TTC?AAC?AAG?ATT?CCG?GCG?GAA?AAG?AAA?CTG?ATC?GTG?ACG?TCG?GAA?GGC?GCC?TTT?AAG?TAT?TTC?AGT?AAA?GCA?TAC?GGT?GTT?CCG?TCC?GCT?TAT?ATT?TGG?GAA?ATC?AAT?ACC?GAA?GAA?GAA?GGC?ACG?CCG?GAA?CAG?ATT?AAA?ACC?CTG?GTT?GAA?AAA?CTG?CGT?CAA?ACG?AAA?GTC?CCG?TCC?CTG?TTC?GTC?GAA?AGC?TCT?GTG?GAT?GAC?CGC?CCG?ATG?AAG?ACC?GTG?TCA?CAG?GAT?ACC?AAC?ATT?CCG?ATT?TAT?GCC?CAG?ATT?TTT?ACC?GAC?TGA? CTCGAG
2. express the structure of PsaA protein extracellular fragment recombinant plasmid:
Extract plasmid pGEX4T-2, use bamhI and ecor I double digestion, reclaims the plasmid large fragment that enzyme is cut after electrophoresis, be dissolved in deionized water.Same using bamhI and ecothe Flic protein extracellular gene fragment of R 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, connect with T4 DNA ligase enzyme, PsaA protein extracellular gene fragment is inserted in vector pGEX 4T-2 bamh I and ecobetween R I site, consistent with the initiator codon translation framework on carrier, express a fusion rotein (build flow process and see Fig. 2).
3. the screening of recombinant plasmid and evaluation:
The recombinant plasmid transformed that upper step is connected arrives e. colibL21 (DE3), containing on the solid LB substratum of penbritin (100 μ g/ml), puts 37 ℃ of overnight incubation by converted product coating.4 transformant bacterium colonies of random choose next day (being labeled as respectively No. 1-2), are inoculated into respectively containing 3 ml liquid LB substratum (containing penbritin 100 μ g/ml) and in vitro, put 37 ℃ of shaking culture 5h, get bacterium liquid 1ml, centrifugal receipts bacterium.Use the plasmid extraction kit of TAKARA company to extract plasmid, after 1.0% Agarose gel detection plasmid extraction success, use restriction enzyme BamHI and XhoI double digestion plasmid 4h under 37 ℃ of water-baths.Get double digestion product 1.0% Agarose gel detection, result, 4 equal enzymes of transformant cut out the goal gene fragment of about 590bp, and these 4 transformants of tentative confirmation all contain PsaA gene fragment (seeing Fig. 3).
Extract the plasmid of No. 1 recon, measure the PsaA protein gene sequence in plasmid, DNA sequence analysis confirmation, recombinant plasmid contains synthetic streptococcus pneumoniae PsaA protein gene fragment, and sequence is entirely true:
CTG?ATC?TTC?TAC?AAT?GGT?ATC?AAT?CTG?GAA?ACG?GGT?GGT?AAC?GCC?TGG?TTC?ACG?AAA?CTG?GTC?GAA?AAT?GCC?AAG?AAA?ACG?GAA?AAC?AAA?GAC?TAT?TTT?GCA?GTC?AGC?GAT?GGC?GTG?GAC?GTT?ATT?TAC?CTG?GAA?GGC?CAG?AAC?GAA?AAA?GGC?AAG?GAA?GAT?CCG?CAT?GCA?TGG?CTG?AAC?CTG?GAA?AAT?GGT?ATT?ATC?TTT?GCG?AAG?AAC?ATC?GCC?AAA?CAA?CTG?TCT?GCT?AAG?GAC?CCG?AAC?AAC?AAG?GAA?TTC?TAC?GAA?AAG?AAC?CTG?AAG?GAA?TAC?ACC?GAT?AAA?CTG?GAC?AAG?CTG?GAT?AAG?GAA?TCA?AAG?GAT?AAG?TTC?AAC?AAG?ATT?CCG?GCG?GAA?AAG?AAA?CTG?ATC?GTG?ACG?TCG?GAA?GGC?GCC?TTT?AAG?TAT?TTC?AGT?AAA?GCA?TAC?GGT?GTT?CCG?TCC?GCT?TAT?ATT?TGG?GAA?ATC?AAT?ACC?GAA?GAA?GAA?GGC?ACG?CCG?GAA?CAG?ATT?AAA?ACC?CTG?GTT?GAA?AAA?CTG?CGT?CAA?ACG?AAA?GTC?CCG?TCC?CTG?TTC?GTC?GAA?AGC?TCT?GTG?GAT?GAC?CGC?CCG?ATG?AAG?ACC?GTG?TCA?CAG?GAT?ACC?AAC?ATT?CCG?ATT?TAT?GCC?CAG?ATT?TTT?ACC?GAC?TGA
The expression of recombinant plasmid streptococcus pneumoniae PsaA protein fragments (197 amino acid) building, has merged 226 amino acid on carrier at its N end, 423 amino acid of total length, and its aminoacid sequence is as follows:
Figure 2013107528911100002DEST_PATH_IMAGE005
4. express the Screening and Identification of PsaA protein engineering bacterium:
By 3 positive transformants that contain recombinant plasmid and 1 contrast bacterium (plasmid pGEX4T-2 transformed bacteria), be seeded to containing 3ml LB substratum (containing penbritin 100 μ g/ml) in vitro, 37 ℃ of shaking culture 3h, add IPTG to final concentration 1.0mmol/L, continue shaking culture induction 4h, centrifugal collection thalline carries out SDS-PAGE detection, and recon is expressed the PsaA albumen that relative molecular weight is about 45 kDa, and expression amount is about 30%(and sees Fig. 4).
express the purifying of PsaA albumen
According to expressing streptococcus pneumoniae PsaA Argine Monohydrochloride sequence, analyze its physicochemical property, determine suitable purification process.We have the GST albumen on carrier by expressed PsaA protein fusion, and the gst fusion protein of expression can be easily separates with GSH sepharose FF, and therefore we determine to adopt affinity chromatography, carry out purifying with Glutathione Sepharose 4B gel.Purifying has obtained PsaA albumen, and concrete steps are as follows:
Materials and methods
1. main agents:
Glutathione Sepharose 4B gel is GE Heathcare company product, and IPTG, DTT are Promega company product.Other reagent is domestic or Import Analysis pure reagent.
2. express the ultrasonic degradation of PsaA albumen:
By the centrifugal (8000rpm of engineering bacteria of the expression PsaA albumen of cultivating, 20mins, 4 ℃), abandon supernatant, thalline is resuspended in the lysate (50mmol/L Tris-HCl pH8.0,10mmol/L EDTA, 10mmol/L DTT) of original fluid 1/10 volume, and ice-bath ultrasonic is broken bacterium 10mins, centrifugal (8000rpm, 20mins, 4 ℃) collect supernatant, abandon precipitation.The supernatant of collecting is for next step affinitive layer purification.
3. express the purifying of PsaA albumen:
Supernatant solution adds counter-balanced Glutathione Sepharose 4B gel 3ml room temperature in conjunction with 60min, and loading is collected and penetrated liquid.With 1 × PBS washing pillar of ten times of column volumes, then with GSH elutriant (50 mmol/LTris-HCl pH8.0+10 mmol/L GSH) point three wash-out target proteins of 15ml high density, be the PsaA protein extracellular fragment of purifying.
Result
The albumen of wash-out from Glutathione Sepharose 4B gel column is carried out to SDS-PAGE analysis, and result shows (seeing Fig. 5), obviously gives expression to PsaA/GST fusion rotein through inducing, and expression product is mainly present in supernatant liquor.The about 45kDa of SDS-PAGE Explicit Expression product.
the preparation of protein immunization polyvalent antibody and purifying
the PsaA albumen that purifying obtains can be used for the preparation of polyvalent antibody.We use antigen by with the mode of adjuvant combined immunization New Zealand large ear rabbit, preparation purifying obtain a large amount of rabbit anti-serums, indirect elisa method detects antibody titers.Concrete steps are as follows:
Materials and methods
1. main agents:
DEAE Sepharose tMfast Flow is GE Heathcare company product, and completely not formula adjuvant and incomplete Freund's adjuvant are SIGMA company product.Other reagent is domestic or Import Analysis pure reagent.
2. immunogen and immune programme for children
Use PsaA albumen as immunogen, 5 New Zealand's large ear rabbits of immunity, every each immune 100 μ g antigens of white rabbit, immunization ways is back multiple spot immunity.Immunity is for the first time used completely not formula adjuvant to mix with antigen, and second and third immunity used Freund's incomplete adjuvant.
3. indirect elisa method is surveyed antiserum titre
Latter 2 weeks of each immunity, from rabbit ear edge venous blood sampling, the centrifugal 10min of 4000rpm obtains antiserum(antisera).Coated PsaA antigen, every hole 1 μ g, 4 ℃ are spent the night, 20% calf serum sealing, 37 ℃, 2h, using the antiserum(antisera) gradient dilution of centrifugal acquisition as primary antibodie, 37 ℃, after 2h, add two and resist, 1:5000 dilutes goat anti-rabbit igg, adds A, the colour developing of B liquid reading.
4. sero-fast obtaining
Indirect elisa method is surveyed after antiserum titre reach a certain height, and gets blood from rabbit carotid artery, 4 ℃ spend the night after, 3000rpm, centrifugal 10min, obtains antiserum(antisera), filtration sterilization, 20 ℃ of ﹣ are frozen.
5. sero-fast purifying
Saturated (the NH of equal-volume 4) 2sO 4solution and serum mix, 4 ℃ of standing 1h, and the centrifugal 10min of 3000rpm, abandons supernatant, uses 0.07M Na 3pO 4solution dissolution precipitation, upper prop after dialysis, DEAE Sepharose tMhow anti-Fast Flow anion exchange chromatography purifying is.
Result
After last immunity 2 weeks, adopt rabbit carotid artery blood taking method to obtain blood 60ml, 4 ℃ of placements spend the night after separation of serum, DEAE Sepharose tMhow anti-Fast Flow anion exchange chromatography purifying is as the primary antibodie in ELISA detection, and result shows that 1:200000 is that serum effectively dilutes gradient, i.e. serum titer, as Fig. 6, illustrates that the PsaA albumen of preparation has good immunogenicity and antigenicity.
The gene fragment order table of chemosynthesis streptococcus pneumoniae surface adhesion element A sees appendix document: Nucleotide or the readable carrier of aminoacid sequence list machine.
Figure IDA0000451911390000011
Figure IDA0000451911390000021

Claims (4)

1. chemosynthesis streptococcus pneumoniae surface adhesion element A(PsaA) gene fragment, this gene fragment coding is containing strong antigen epi-position streptococcus pneumoniae PsaA protein extracellular gene fragment, i.e. 280 amino acid of the 84th amino acid to the, totally 197 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 ecorI restriction enzyme site (underscore part), the gene order total length 606bp of chemosynthesis, sequence is as follows:
GGATCC?CTG?ATC?TTC?TAC?AAT?GGT?ATC?AAT?CTG?GAA?ACG?GGT?GGT?AAC?GCC?TGG?TTC?ACG?AAA?CTG?GTC?GAA?AAT?GCC?AAG?AAA?ACG?GAA?AAC?AAA?GAC?TAT?TTT?GCA?GTC?AGC?GAT?GGC?GTG?GAC?GTT?ATT?TAC?CTG?GAA?GGC?CAG?AAC?GAA?AAA?GGC?AAG?GAA?GAT?CCG?CAT?GCA?TGG?CTG?AAC?CTG?GAA?AAT?GGT?ATT?ATC?TTT?GCG?AAG?AAC?ATC?GCC?AAA?CAA?CTG?TCT?GCT?AAG?GAC?CCG?AAC?AAC?AAG?GAA?TTC?TAC?GAA?AAG?AAC?CTG?AAG?GAA?TAC?ACC?GAT?AAA?CTG?GAC?AAG?CTG?GAT?AAG?GAA?TCA?AAG?GAT?AAG?TTC?AAC?AAG?ATT?CCG?GCG?GAA?AAG?AAA?CTG?ATC?GTG?ACG?TCG?GAA?GGC?GCC?TTT?AAG?TAT?TTC?AGT?AAA?GCA?TAC?GGT?GTT?CCG?TCC?GCT?TAT?ATT?TGG?GAA?ATC?AAT?ACC?GAA?GAA?GAA?GGC?ACG?CCG?GAA?CAG?ATT?AAA?ACC?CTG?GTT?GAA?AAA?CTG?CGT?CAA?ACG?AAA?GTC?CCG?TCC?CTG?TTC?GTC?GAA?AGC?TCT?GTG?GAT?GAC?CGC?CCG?ATG?AAG?ACC?GTG?TCA?CAG?GAT?ACC?AAC?ATT?CCG?ATT?TAT?GCC?CAG?ATT?TTT?ACC?GAC?TGA? CTCGAG?。
2. the gene fragment of chemosynthesis claimed in claim 1, adopts genetic engineering technique to express the streptococcus pneumoniae PsaA protein extracellular gene fragment of this gene order coding, the protein fragments that purifying is expressed, and concrete grammar is as follows:
Express the structure of streptococcus pneumoniae PsaA protein extracellular gene fragment recombinant plasmid:
With bamhI and ecothe streptococcus pneumoniae PsaA protein extracellular gene fragment of R I double digestion plasmid pGEX4T-2 and chemosynthesis, after electrophoresis reclaims, with the connection of T4 DNA ligase enzyme, is inserted in vector pGEX 4T-2 streptococcus pneumoniae PsaA protein extracellular gene fragment bamh I and ecobetween R I site, consistent with the initiator codon translation framework on carrier, express a fusion rotein, 423 amino acid of total length, this fusion rotein N end has merged 226 amino acid on carrier, and C end comprises streptococcus pneumoniae PsaA protein extracellular gene fragment, after electrophoresis reclaims, with the connection of T4 DNA ligase enzyme, make 280 amino acid chains of the 84th amino acid to the in streptococcus pneumoniae PsaA albumen receive vector plasmid, full length amino acid sequence is as follows:
The Screening and Identification of expressed fusion protein engineering bacteria:
By recombinant plasmid transformed e. colibL21 (DE3), coating is containing the LB flat board of 100 μ g/ml penbritins, putting 37 ℃ spends the night, next day, random picking transformed bacterium colony and the contrast bacterium containing plasmid pGEX4T-2, extract plasmid, with restriction enzyme BamHI and XhoI, to plasmid double digestion, 1.0% Agarose gel detection double digestion product should cut the substrate section of about 590bp; 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 3h, add IPTG to final concentration 0.5～1.0 mmol/L, continue shaking culture induction 4～6h, centrifugal collection thalline carries out SDS-PAGE detection, and recon is expressed the streptococcus pneumoniae PsaA albumen that relative molecular weight is about 45kDa, expression amount is about 30%, and contrast bacterium BL21 (DE3) is without this protein band;
Express the purifying of streptococcus pneumoniae PsaA albumen:
By centrifugal the engineering bacteria of abduction delivering fusion rotein receipts bacterium, thalline is resuspended in lysate, lysate is 50 mmol/L Tris-HCl pH8.0,10 mmol/L EDTA, 10 mmol/L DTT, carrying out ultrasonic bacteria breaking 10 min, centrifugal collection supernatant, supernatant solution adds counter-balanced Glutathione Sepharose 4B gel 3ml, and room temperature is in conjunction with 60min, 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) point three wash-out target proteins of 15ml high density, be the streptococcus pneumoniae PsaA protein extracellular fragment of purifying.
3. the gene fragment order of the streptococcus pneumoniae PsaA protein extracellular 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 streptococcus pneumoniae PsaA protein extracellular fragment that described in claim 2 or claim 3 prepared by method, for detection and monoclonal antibody and the how anti-preparation of streptococcus pneumoniae infection antibody.
CN201310752891.1A 2013-12-31 2013-12-31 Gene segment of chemosynthetic streptococcus pneumoniae surface adhesin A as well as expression and application of gene segment Active CN103773779B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201310752891.1A CN103773779B (en) 2013-12-31 2013-12-31 Gene segment of chemosynthetic streptococcus pneumoniae surface adhesin A as well as expression and application of gene segment

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201310752891.1A CN103773779B (en) 2013-12-31 2013-12-31 Gene segment of chemosynthetic streptococcus pneumoniae surface adhesin A as well as expression and application of gene segment

Publications (2)

Publication Number Publication Date
CN103773779A true CN103773779A (en) 2014-05-07
CN103773779B CN103773779B (en) 2017-02-15

Family

ID=50566544

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201310752891.1A Active CN103773779B (en) 2013-12-31 2013-12-31 Gene segment of chemosynthetic streptococcus pneumoniae surface adhesin A as well as expression and application of gene segment

Country Status (1)

Country Link
CN (1) CN103773779B (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999040200A1 (en) * 1998-02-03 1999-08-12 Center For Disease Control And Prevention RECOMBINANT LIPIDATED PsaA PROTEIN, METHODS OF PREPARATION AND USE
WO1999045121A1 (en) * 1998-03-02 1999-09-10 The Government Of The United States Of America, Represented By The Secretary Of The Department Health And Human Services EPITOPE PEPTIDES IMMUNOGENIC AGAINST $i(STREPTOCOCCUS PNEUMONIAE)
WO2006127020A1 (en) * 2005-05-19 2006-11-30 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, Centers For Disease Control And Prevention FUNCTIONAL EPITOPES OF STREPTOCOCCUS PNEUMONIAE PsaA ANTIGEN AND USES THEREOF
CN102695523A (en) * 2009-09-10 2012-09-26 诺华有限公司 Combination vaccines against respiratory tract diseases

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999040200A1 (en) * 1998-02-03 1999-08-12 Center For Disease Control And Prevention RECOMBINANT LIPIDATED PsaA PROTEIN, METHODS OF PREPARATION AND USE
WO1999045121A1 (en) * 1998-03-02 1999-09-10 The Government Of The United States Of America, Represented By The Secretary Of The Department Health And Human Services EPITOPE PEPTIDES IMMUNOGENIC AGAINST $i(STREPTOCOCCUS PNEUMONIAE)
WO2006127020A1 (en) * 2005-05-19 2006-11-30 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, Centers For Disease Control And Prevention FUNCTIONAL EPITOPES OF STREPTOCOCCUS PNEUMONIAE PsaA ANTIGEN AND USES THEREOF
CN102695523A (en) * 2009-09-10 2012-09-26 诺华有限公司 Combination vaccines against respiratory tract diseases

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
GOWRISANKAR RAJAM ET AL.: "A 28-aa Pneumococcal Surface Adhesin A–Derived Peptide, P4, Augments Passive Immunotherapy and Rescues Mice from Fatal Pneumococcal Infection", 《THE JOURNAL OF INFECTIOUS DISEASES》 *
GOWRISANKAR RAJAM ET AL.: "A functional epitope of the pneumococcal surface adhesin A activates nasopharyngeal cells and increases bacterial internalization", 《MICROBIAL PATHOGENESIS》 *
N. SRIVASTAVA ET AL.: "Selection of an Immunogenic and Protective Epitope of the PsaA Protein of Streptococcus pneumoniae Using a Phage Display Library", 《HYBRIDOMA》 *
SANDRA ROMERO-STEINER ET AL.: "Adherence of recombinant pneumococcal surface adhesin A (rPsaA)-coated particles to human nasopharyngeal epithelial cells for the evaluation of anti-PsaA functional antibodies", 《VACCINE》 *
SHIFENG WANG ET AL.: "Immune Responses to Recombinant Pneumococcal PsaA Antigen Delivered by a Live Attenuated Salmonella Vaccine", 《INFECTION AND IMMUNITY》 *
吴艳丽: "肺炎链球菌表面粘附素A的原核表达和免疫保护性的初步研究", 《中国优秀硕士学位论文全文数据库·医药卫生科技辑》 *
唐玉龙: "肺炎球菌表面黏附素A和表面膜蛋白A的研究进展", 《中国生物制品学杂志》 *

Also Published As

Publication number Publication date
CN103773779B (en) 2017-02-15

Similar Documents

Publication Publication Date Title
CN102636641B (en) Detection kit of helicobacter pylori emulsion method and preparation process thereof
CN105884902A (en) Fusion protein of mycoplasma pneumonia protein epitope and preparation and application thereof
CN104198741A (en) Tilapia streptococcus agalactiae IgM antibody capture ELISA detection kit
CN101659975B (en) Preparation method of HRPII protein monoclonal antibody of plasmodium falciparum
CN105925597B (en) Concatenated recombination of a kind of PEDV S gene Main Antigenic and its preparation method and application
CN111487417B (en) MCR-1 drug-resistant protein double-antibody sandwich ELISA detection kit and detection method
CN101402960B (en) Recombinant rubella virus E1 protein and uses thereof
CN105348391A (en) Enteric cytopathogenic human orphan virus 6-type VP1 protein specific epitope, fusion protein of enteric cytopathogenic human orphan virus 6-type VP1 protein specific epitope and preparation method and use of fusion protein
WO2008119358A2 (en) Surface-located streptococcus pneumoniae polypeptides for use in vaccine compositions
CN103941020A (en) Indirect ELISA (enzyme-linked immuno sorbent assay) kit for detecting haemophilus parasuis antibody
CN114276445B (en) Rotavirus recombinant protein specific antibody, plasmid vector and method
CN103834667A (en) Chemosynthetic extracellular region gene fragment of streptococcus pneumonia PspA protein, and expression and application thereof
CN104610443B (en) A kind of high stability restructuring Procalcitonin, Preparation method and use
CN111378628B (en) Hybridoma cell strain secreting mycobacterium tuberculosis ESAT6 protein specific antibody, antibody and application thereof
CN109975552A (en) A kind of recombination cystatin C albumen and its application in detection kit
CN105949320A (en) Preparation and application of echovirus type 1 VP1 protein specific antigen epitope and fusion protein thereof
CN102690351A (en) Preparation method of plasmodium vivax aldolase protein monoclonal antibody
CN109384834A (en) Recombinate Protein A albumen and its high efficient expression and application
CN103773779A (en) Gene segment of chemosynthetic streptococcus pneumoniae surface adhesin A as well as expression and application of gene segment
CN105906716B (en) Preparation and application of echovirus 9 type VP1 protein specific epitope and fusion protein thereof
CN103725698A (en) Surface protein ClfA gene segment of chemically-synthesized staphylococcus aureus as well as expression and application of surface protein ClfA gene segment
JPH022357A (en) Production of haemopilus influenza b-type main crust membrane protein antigen and composition
CN110540597B (en) Preparation method of latex microsphere immunochromatographic test paper based on haemophilus influenzae surface protein
CN110540599B (en) Klebsiella pneumoniae Elisa detection kit based on Klebsiella pneumoniae surface protein antibody and preparation method thereof
CN102827292B (en) Fusion protein TgMEP as well as preparation method and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
TR01 Transfer of patent right

Effective date of registration: 20230918

Address after: 210002 No. 293 East Zhongshan Road, Jiangsu, Nanjing

Patentee after: EASTERN THEATER DISEASE PREVENTION AND CONTROL CENTER OF PLA

Address before: 210002 Military Medical Research Institute, Nanjing military area command, 293 East Zhongshan Road, Nanjing, Jiangsu

Patentee before: Li Yuexi

TR01 Transfer of patent right