CN105131094A - Mycobacterium tuberculosis Rv 3248 c recombinant protein, preparation method and application of mycobacterium tuberculosis Rv 3248 c recombinant protein - Google Patents
Mycobacterium tuberculosis Rv 3248 c recombinant protein, preparation method and application of mycobacterium tuberculosis Rv 3248 c recombinant protein Download PDFInfo
- Publication number
- CN105131094A CN105131094A CN201510591293.XA CN201510591293A CN105131094A CN 105131094 A CN105131094 A CN 105131094A CN 201510591293 A CN201510591293 A CN 201510591293A CN 105131094 A CN105131094 A CN 105131094A
- Authority
- CN
- China
- Prior art keywords
- rv3248c
- mycobacterium tuberculosis
- recombinant protein
- tuberculosis
- gene
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a mycobacterium tuberculosis Rv 3248 c recombinant protein, a preparation method and application of the mycobacterium tuberculosis Rv 3248 c recombinant protein. The recombinant protein disclosed by the invention has an amino acid sequence shown as SEQ ID NO. 1. The invention also provides the preparation method of a coded nucleotide sequence of the mycobacterium tuberculosis Rv 3248 c recombinant protein and the recombinant protein. Based on a research result of immunomics, a mycobacterium tuberculosis immunodominant antigen Rv 3248 c is reported for the first time. By applying the mycobacterium tuberculosis Rv 3248 c recombinant protein to a tuberculosis cellular immunology diagnosis, higher sensitivity is achieved, besides the recombinant protein has the advantages of higher speed, safety and reliability in comparison with a conventional skin test.
Description
Technical field
The invention belongs to biological medicine external diagnosis reagent technical field, particularly a kind of novel tuberculosis immunological marker thing Rv3248c recombinant protein, its preparation method and application thereof.
Background technology
Tuberculosis is by pathogenic agent---and caused by m tuberculosis infection body, be still the important transmissible diseases of the mankind so far.Diagnosis of tuberculosis mycobacterium (MTB) infects fast and accurately, significant for control lungy.The diagnostic method that MTB infects has a lot, and clinical method the most conventional still relies on traditional Sputum smears bacteriology checking at present, but recall rate is low; MTB cultivates " gold standard " that can make a definite diagnosis as tuberculosis, but its incubation time is oversize, in general 4-6 week, is difficult to meet clinical needs; Though Bactec technology shortens incubation time, costly, be difficult in the short period of time popularize; X-ray and CT examination only provide iconography possibility to diagnose; Polymerase chain reaction (PCR) technology and other gene diagnosis method remain at complicated operation as clinical diagnosis, to technology and personnel specialty competency profiling higher, and still can not be used for the cloudy quick diagnosis lungy of bacterium.Tuberculosis immunity mainly cellular immunization, the T lymphocyte comprising sensitization and the scavenger cell be activated.The T lymphocyte of sensitization directly can kill the target cell with tubercule bacillus, acts on the phagocytal lymphokine of generation to release is multiple simultaneously, makes macrophage accumulation form the proliferative inflammation based on monocyte at perilesional.The scavenger cell be activated greatly strengthens engulfs digestion, inhibition to tubercule bacillus, stops diffusion, the ability of even destroying, and fully point to wave the effect of cellular immunization.The tuberculosis diagnosis of cell mediated immunity technology of current widespread use still depends on skin test detection reagent---mycobacterium tuberculosis purified protein derivative (PPD).But there is the cross reaction with environment mycobacterium and BCG vaccine strain in PPD, so diagnostic value is on the low side.Thus find new tuberculosis cells immunological markers thing, and to set up new tuberculosis diagnosis of cell mediated immunity method be that diagnosis of tuberculosis needed badly.
Summary of the invention
For overcoming deficiency of the prior art, the object of the present invention is to provide a kind of mycobacterium tuberculosis Rv3248c recombinant protein, preparation method and application thereof.
A kind of mycobacterium tuberculosis Rv3248c recombinant protein provided by the invention, its aminoacid sequence is as shown in SEQIDNO:1.
The present invention, by using pET28a plasmid, has added the recombinant protein that one section of HisTag purification tag obtains mycobacterium tuberculosis Rv3248c before the native protein sequence of mycobacterium tuberculosis Rv3248c; The aminoacid sequence of described HisTag purification tag forms to the 17th amino acids residue sequence from N-terminal the 1st by SEQIDNO:1.From N-terminal the 18th to the native protein aminoacid sequence of the 512nd amino acids residue sequence composition mycobacterium tuberculosis Rv3248c in SEQIDNO:1.
The present invention also provides a kind of gene of above-mentioned mycobacterium tuberculosis Rv3248c recombinant protein of encoding, and it is selected from nucleotide sequence as follows:
1. the nucleotide sequence shown in SEQIDNO:2;
2. SEQIDNO:2 is different from but the aminoacid sequence nucleotide sequence identical with the aminoacid sequence coded by SEQIDNO:2 of coding;
3. under stringent hybridization condition with 1. above-mentioned or 2. in sequence hybridization, and coding has the nucleotide sequence of described mycobacterium tuberculosis Rv3248c recombinant protein activity.
One aspect of the present invention, provides a kind of recombinant plasmid comprising described mycobacterium tuberculosis Rv3248c gene, inserts in commercial expression plasmid sequence by Rv3248c gene order.The Rv3248c gene NCBI number of logging in is: NC_000962.3; Albumen number is: NP_217765.1.
" Rv3248c gene order " of the present invention also comprise to codon one or more in NC_000962.3 replace by the degenerate codon of same amino acid of encoding after produce sequence.Due to the degeneracy of codon, so also to encode identical aminoacid sequence with the degenerate sequence that NC_000962.3 nucleotide sequence homology is low to moderate about 70%.Under this term is also included in the tight condition of moderate, more preferably under highly tight condition with the nucleotide sequence of the nucleotide sequence hybridization of NC_000962.3.This term also comprises the nucleotide sequence homology at least 70% with NC_000962.3, more preferably the nucleotide sequence of at least 90%.
Recombinant plasmid of the present invention, the multiple clone site place usually Rv3248c gene being inserted expression plasmid obtains.
Recombinant plasmid of the present invention, in preparation process, can select various carrier known in the art, then nucleotide sequence of the present invention for coding is connected to expression regulation sequence operably, thus forms protein expression vector.
The present invention also provides a kind of preparation method of above-mentioned mycobacterium tuberculosis Rv3248c recombinant protein, comprises the following steps:
(1) preparation of M. tuberculosis strains genomic dna;
(2) amplification of mycobacterium tuberculosis Rv3248c gene;
(3) mycobacterium tuberculosis Rv3248c gene inserts expression plasmid;
(4) expression plasmid of structure is transformed into host cell;
(5) abduction delivering mycobacterium tuberculosis Rv3248c albumen;
(6) separation and purification abduction delivering product, obtains mycobacterium tuberculosis Rv3248c recombinant protein;
Wherein, in step (2), the primer adopted in amplification mycobacterium tuberculosis Rv3248c gene is primer Rv3248c-F and primer Rv3248c-R, the nucleotide sequence of described primer Rv3248c-F is as shown in SEQIDNo:3, and the nucleotide sequence of described primer Rv3248c-R is as shown in SEQIDNo:4.
In above-mentioned preparation method, various experiment parameter operates selection all routinely, is determined by specific experiment condition.
Rv3248c gene order of the present invention can obtain by pcr amplification method.Can related nucleotide sequences disclosed according to the present invention, especially open reading frame sequence designs primer, and is template with the genomic dna prepared by ordinary method well known by persons skilled in the art, amplification and obtain correlated series.
In the present invention, recombinant protein expression plasmid used can be pET28a etc.
In the present invention, the host cell of expressing Rv3248c albumen used is colibacillary BL21 (DE3) bacterial strain or BL21plysS (DE3) bacterial strain.
In the present invention, abduction delivering Rv3248c albumen can adopt multiple method, as used isopropyl-beta D-thio galactopyranoside (IPTG).
In the present invention, separation and purification restructuring Rv3248c albumen also can adopt multiple method, as affinity chromatography, ion exchange chromatography etc.
The present invention further provides the application of above-mentioned mycobacterium tuberculosis Rv3248c recombinant protein in the reagent preparing detection or diagnosis of tuberculosis; Above-mentioned mycobacterium tuberculosis Rv3248c recombinant protein is provided to prepare the application in diagnosis test kit; And provide the detectable antigens in a kind of its component of diagnosis test kit to be above-mentioned mycobacterium tuberculosis Rv3248c recombinant protein.
Beneficial effect of the present invention is:
(1) compared with natural mycobacterium tuberculosis Rv3248c albumen, recombinant protein of the present invention more easily adopts the method for affinity chromatography to carry out purifying, purity can reach more than 90%, not only greatly reduce its production cost, and there is higher stability, this recombinant protein when not freeze-drying 4 DEG C can preserve 1 week, can 3 months be preserved for-20 DEG C ,-80 DEG C at least preserve 1 year.
(2) the present invention is based on the achievement in research of immunohistochemistry, reported first Rv3248c specific antigens can be applied to diagnosis of tuberculosis, and be the T cell target antigen that an activity is stronger, diagnose for tuberculosis cellular immunology, there is higher susceptibility high, and compare skin test test, more quick and safe and reliable.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE figure of recombinant protein Rv3248c after being further purified and being concentrated.1 is, Marker, and 2 is thalline before induction, and 3 is thalline after induction, and 4 is albumen after purifying.
Fig. 2 is antigen ESAT-6, CFP-10 and Rv3248c evaluation test.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art, the experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration, but the content of this aspect can not be limited.
The structure of embodiment 1 recombinant plasmid pET28a-Rv3248c
(1) target gene design of primers
Rv3248c-F(SEQIDNo:3)：
GGAATT
CCATATGACCGGAAATTTGGTGACCAAAAATTCAAACAGCGCGCRv3248c-R(SEQIDNo:4)：
CCC
AAGCTTGGTCAGTAGCGGTAGTGGTCCGCGTGCGGTCGAGCCAC
Restriction enzyme site is respectively NdeI, HindIII.
(2) pcr amplification, the clone and sequence of target gene
With mycobacterium tuberculosis H
37rv genomic dna is template, Rv3248c-F and Rv3248c-R is primer, application Taq enzyme (precious biotechnology (Dalian) company limited), directly to be increased Rv3248c protein gene by PCR.PCR reaction conditions: 94 DEG C of denaturation 5min; (94 DEG C, 30s; 58 DEG C, 30s; 72 DEG C, 40s) 35 circulations; 72 DEG C extend 5min; 4 DEG C of preservations.Reaction terminates rear use 1% agarose gel electrophoresis and is separated object fragment, and rear DNA reclaims test kit (Invitrogen) and reclaims.Cut with NdeI and HindIII enzyme, clone is built in pET28a plasmid NdeI and HindIII restriction enzyme site.Order-checking to show in inserted sequence and GeneBank that (the Rv3248c gene NCBI number of logging in is H37Rv tuberculosis full-length genome corresponding gene sequences: NC_000962.3; Albumen number is: NP_217765.1) completely the same.
The abduction delivering of embodiment 2 recombinant protein Rv3248c and purifying
The eppdorf pipe that 100 μ lBL21 (DE3) physS competent cell (TIANGEN) are housed is put on ice immediately from-80 DEG C of refrigerators.After waiting liquid in pipe to melt after 3-5 minute, the restructuring pET28a-Rv3248c plasmid checking order correct by 0.5 μ l puts into competent cell, place 45min on ice, in 42 DEG C of water-baths, thermal shock 90s, leaves standstill 3min on ice, add the LB substratum not containing antibiotic preheating of 500 μ l, 37 DEG C of shaking tables, 220rmp, cultivates 45-60min.Getting a certain amount of being coated with containing on the solid LB media plate of kantlex, after drying at room temperature, being inverted in overnight incubation in 37 DEG C of incubators.Picked clones, put into the LB liquid nutrient medium of the resistance containing 50 μ g/ml kantlex, 220rmp, cultivate about OD to 0.6 for 37 DEG C, adding final concentration is 10mMIPTG, 37 DEG C of 3h, collect the thalline after IPTG induction, in the resuspended rear ultrasonication of the ratio of the wet bacterium of 10mlWashingBufferI every gram.Result target protein is contained in supernatant.The centrifugal 30min of 10000g, collects supernatant.Affinity purification (2CV deionized water rinsing is carried out with IMAC affinity chromatographic column (Bio-Rad), 5CV balances containing the washingBufferI of 5mM imidazoles, 6CV is containing target protein supernatant loading, 6CV rinses containing the washingBufferI of 5mM imidazoles, 6CV rinses containing the washingBuffer2 of 10mM imidazoles, 10CV is containing the ElutionBuffer wash-out of 500mM imidazoles), collected the target protein (as shown in Figure 1) of column purification, with 20mMTris-HCl, the solution dialysis of pH8.0, 10kDa super filter tube concentrates the target protein liquid after dialysis, by the concentration of target protein after BCA method mensuration purifying, SDS-PAGE electrophoretic analysis target protein purity.
After the recombinant protein nickel post affinity purification of the present embodiment, purity can reach more than 90%, not only greatly reduces its production cost, and there is higher stability, this recombinant protein when not freeze-drying 4 DEG C can preserve 1 week, can 3 months be preserved for-20 DEG C ,-80 DEG C at least preserve 1 year.
Embodiment 3 recombinates Rv3248c antigen as detection reagent to clinical suspicious tuberculosis patient detection.
The present embodiment detects using recombinant protein Rv3248c antigen as detection reagent clinical suspicious tuberculosis patient, control group is done with Healthy People, simultaneously to commonly use diagnosis of tuberculosis antigen ESAT-6 at present, CFP-10 synchronously divides into groups to test, the ELISPOT test design of concrete antigen is in table 1, and operation steps is as follows:
1, sample collection: aseptic collection human peripheric venous blood is about 5ml in anticoagulant heparin pipe, can preserve after sample collection under room temperature, is not placed on refrigeration or refrigeration chamber; And carry out mark;
2, the lymphocytic separation of peripheral blood single core, collection and counting:
A, get the whole blood of 5ml, add isopyknic RTPMI-1640 serum-free medium, and mix;
B, get the lymphocyte separation medium that a 15ml centrifuge tube adds 4ml, and the blood sample of mixing is slowly joined the surface of lymphocyte separation medium, boundary is between the two wanted obviously, not mix, cover tightly pipe lid, handle with care;
C, centrifuge tube is put into horizontal centrifuge, 1000g, room temperature, centrifugal 22min, careful take out after visible blood ingredient layering;
D, with Dispette, single core buffy coat is transferred in new 15ml centrifuge tube, and add 10mlRTPMI-1640 serum-free medium and mix, put into whizzer 600g centrifuge washing 10min, adding serum-free medium, the centrifugal 10min of 350g;
E, taking-up centrifuge tube abandon supernatant, add the RPMI-1640 of 400 μ l containing serum free culture system liquid, and the mixing of piping and druming cell, wants during piping and druming softly, avoid cell to be subject to the damage of external force as far as possible;
F, get 120ul carry out lymphocyte count on automatic counter for counting (Sysmex, XT-2000i);
G, adjustment cell concn to 2.5 × 10
6/ ml, prepares 500 μ l;
3, the activation of plate: 200 μ l/wellRPMI-1640 are containing serum free culture system liquid, and 5-10min, topples over.
4, according to experimental design arrangement: each detection sample needs 2 holes, adds different sample cell 100 μ l/well;
Negative control: every hole adds 10 μ lRPMI-1640 containing serum free culture system liquid
Positive control: every hole adds 10 μ lPHA
Test hole a: every hole adds 10 μ l Specific Antigen of Mycobacterium Tuberculosis ESAT-65 μ g/ml
Test hole b: every hole adds 10 μ l Specific Antigen of Mycobacterium Tuberculosis CFP-105 μ g/ml
Stimulator antigen: every hole adds 10 μ l mycobacterium tuberculosis recombinant antigen Rv3248c10 μ g/ml
Remarks:
Self-control recombinant antigen ESAT-6 and CFP-10 albumen: with M. tuberculosis genes group DNA for template, with Auele Specific Primer gene engineering method structure pET21a-esat6 or the pET28a-cfp10 recombinant plasmid routinely of albumen, proceed to e. coli bl21 (DE3) physS expression strain, 1mMIPTG37 DEG C of induction 3h can obtain a large amount of recombinant proteins, its purity of nickel post affinity purification reaches more than 90%, by recombinant protein 20mMTris-HCl, ultrafiltration and concentration after pH8.0 dialysis, BCA method detects protein concentration, by the packing of 1mg/ pipe, freeze-drying is preserved.
5, after adding all samples, cover plate lid, put into 37 DEG C, 5%CO
216-20hr (17h) cultivated by incubator;
6, half hour before taking-up culture plate, be ready to clap water paper, loading slot (washing lotion), wash bottle, 50 × washing lotion placed equilibrate at room temperature for a moment, preparation 1 × Washingbuffer, experimentally scheme and determining;
7, wash plate: liquid in pouring aperture, the 1 × Washingbuffer of 200 μ l/well, wash 5 times, each 30sec, pats dry, and firmly clap, and pats dry clean; PBS washing lotion;
8, add the enzyme labelled antibody diluted, 50 μ l/well, 4 DEG C of incubators, hatch 1hr; Single stage method is reacted;
9, wash plate: liquid in pouring aperture, the 1 × Washingbuffer of 200 μ l/well, wash 5 times, each 30sec, pats dry, and firmly clap, and pats dry clean;
10, develop the color: add the nitrite ion prepared, 100 μ l/well, room temperature keeps in Dark Place 7min;
11, grow into after applicable size until spot, with deionized water wash 2 times, color development stopping process.Take off protective layer after patting dry, naturally dry.Plate is not placed in baking box, prevent film embrittlement from breaking; Also directly can use directly flushing with tap water, but by hole directly facing to current, and endways for plank water can not should be rinsed, current will ease up, and lath can be taken off, and dry after rinsing bottom surface gently, place and treat that nature dries;
12, ELISPOT plate spot count, and the various parameters recording spot, do statistical study (as shown in Figure 2).
Result judges: be then judged to be positive findings when test hole A or B reaches following standard:
(1) if negative control hole spot number is 0-5, negative control hole spot number >=6 are deducted with the spot number of test hole A or B;
(2) if negative control hole spot number >=6, the spot number of test hole A or B must be greater than the negative control hole spot number of 2 times.
Result is explained: positive findings prompting detects in sample and contains for the special effector T cell of mycobacterium tuberculosis; Negative findings prompting detects in sample and does not contain for the special effector T cell of mycobacterium tuberculosis.Table 2 is that recombinant protein Rv3248c and existing ESAT-6, CFP-10 stimulate clinical samples to produce sensitized T lymphocyte experimental result respectively.
Table 1ELISPOT test design
Table 2
Recombinant protein Rv3248c and ESAT-6, CFP-10 stimulate the lymphocytic ELISPOT experimental result of clinical samples T respectively
The result of table 2 shows, Rv3248c recombinant protein antigen produces responsiveness T lymphocyte at stimulation clinical samples and detects, and its susceptibility detected is 74.1% (20/27), and specificity is 88.5% (24/26).Its susceptibility and specificity are close to ESAT-6 antigen and CFP10 antigen.
Rv3248c recombinant protein of the present invention separately as diagnostic reagent, also can combinationally use with existing known diagnostic antigen, thus improves the detection sensitivity to latent tuberculosis patient.
In addition to the implementation, the present invention can also have other embodiment.All technical schemes adopting modification, replacing, equivalent replacement or equivalent transformation to be formed, all fall within the scope of protection of the present invention.
Claims (8)
1. a mycobacterium tuberculosis Rv3248c recombinant protein, is characterized in that, its aminoacid sequence is as shown in SEQIDNO:1.
2. the gene of mycobacterium tuberculosis Rv3248c recombinant protein described in claim 1 of encoding.
3. gene according to claim 2, is characterized in that, its nucleotide sequence is selected from any one of following sequence:
1. the nucleotide sequence shown in SEQIDNO:2;
2. SEQIDNO:2 is different from but the aminoacid sequence nucleotide sequence identical with the aminoacid sequence coded by SEQIDNO:2 of coding;
3. under stringent hybridization condition with 1. above-mentioned or 2. in sequence hybridization, and coding has the nucleotide sequence of described mycobacterium tuberculosis Rv3248c recombinant protein activity.
4. one kind comprises the recombinant plasmid of gene described in claim 2.
5. the preparation method of mycobacterium tuberculosis Rv3248c recombinant protein according to claim 1, is characterised in that, comprises the following steps:
(1) M. tuberculosis strains genomic dna is prepared;
(2) increase mycobacterium tuberculosis Rv3248c gene;
(3) mycobacterium tuberculosis Rv3248c gene inserts expression plasmid;
(4) expression plasmid of structure is transformed into host cell;
(5) abduction delivering mycobacterium tuberculosis Rv3248c albumen;
(6) separation and purification abduction delivering product, obtains mycobacterium tuberculosis Rv3248c recombinant protein;
Wherein, in step (2), the primer adopted in amplification mycobacterium tuberculosis Rv3248c gene is primer Rv3248c-F and primer Rv3248c-R, the nucleotide sequence of described primer Rv3248c-F is as shown in SEQIDNo:3, and the nucleotide sequence of described primer Rv3248c-R is as shown in SEQIDNo:4.
6. the application of mycobacterium tuberculosis Rv3248c recombinant protein according to claim 1 in the reagent preparing detection or diagnosis of tuberculosis.
7. mycobacterium tuberculosis Rv3248c recombinant protein according to claim 1 is preparing the application in diagnosis test kit.
8. a diagnosis test kit, is characterized in that, the detectable antigens in its component is mycobacterium tuberculosis Rv3248c recombinant protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510591293.XA CN105131094A (en) | 2015-09-17 | 2015-09-17 | Mycobacterium tuberculosis Rv 3248 c recombinant protein, preparation method and application of mycobacterium tuberculosis Rv 3248 c recombinant protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510591293.XA CN105131094A (en) | 2015-09-17 | 2015-09-17 | Mycobacterium tuberculosis Rv 3248 c recombinant protein, preparation method and application of mycobacterium tuberculosis Rv 3248 c recombinant protein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105131094A true CN105131094A (en) | 2015-12-09 |
Family
ID=54716713
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510591293.XA Pending CN105131094A (en) | 2015-09-17 | 2015-09-17 | Mycobacterium tuberculosis Rv 3248 c recombinant protein, preparation method and application of mycobacterium tuberculosis Rv 3248 c recombinant protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105131094A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724182A (en) * | 2019-11-15 | 2020-01-24 | 上海交通大学 | Marker combination for improving detection sensitivity of bacterial scrotum infection and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1807456A (en) * | 2005-01-19 | 2006-07-26 | 上海赛金生物医药有限公司 | Recombinant human parathormone PTH1-34 preparation method |
| CN1888069A (en) * | 2006-07-13 | 2007-01-03 | 复旦大学 | Recombinant protein Rv3425 and its application of in preparing tuberculosis kit |
| CN101235090A (en) * | 2008-01-31 | 2008-08-06 | 复旦大学 | Specificity fusion protein applied to tuberculosis rapid diagnosis |
-
2015
- 2015-09-17 CN CN201510591293.XA patent/CN105131094A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1807456A (en) * | 2005-01-19 | 2006-07-26 | 上海赛金生物医药有限公司 | Recombinant human parathormone PTH1-34 preparation method |
| CN1888069A (en) * | 2006-07-13 | 2007-01-03 | 复旦大学 | Recombinant protein Rv3425 and its application of in preparing tuberculosis kit |
| CN101235090A (en) * | 2008-01-31 | 2008-08-06 | 复旦大学 | Specificity fusion protein applied to tuberculosis rapid diagnosis |
Non-Patent Citations (1)
| Title |
|---|
| LEW J M等: "adenosylhomocysteinase[Mycobacterium tuberculosis H37Rv]，Genbank accession No. NP_217765.1", 《GENBANK数据库》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724182A (en) * | 2019-11-15 | 2020-01-24 | 上海交通大学 | Marker combination for improving detection sensitivity of bacterial scrotum infection and application thereof |
| CN110724182B (en) * | 2019-11-15 | 2022-04-08 | 上海交通大学 | Marker combination for improving detection sensitivity of bacterial scrotum infection and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | The haemagglutinin of Avibacterium paragallinarum is a trimeric autotransporter adhesin that confers haemagglutination, cell adherence and biofilm formation activities | |
| Ahmad et al. | Mammalian cell‐entry proteins encoded by the mce3 operon of Mycobacterium tuberculosis are expressed during natural infection in humans | |
| CN106771250B (en) | A kind of luciferase co-immunoprecipitation kit for detecting mammal Lyme disease | |
| CN103059109B (en) | Mycoplasma pneumonia antigen, preparation method and immunodetection kit | |
| CN102964435B (en) | Truncated-form streptococcus hemolyticus bacteriolysin O and detection kit using same | |
| EP0922056A1 (en) | New fibrinogen binding protein originating from coagulase-negative staphylococcus | |
| CN108314710B (en) | Mycoplasma pneumoniae recombinant antigen and application thereof | |
| CN102718848A (en) | Recombinant protein of mycobacterium tuberculosis Rv 3120, preparation method and application in cellular immunological diagnosis thereof | |
| CN101294154B (en) | Method for producing fasciola gigantica cathepsin L1 | |
| CN105131094A (en) | Mycobacterium tuberculosis Rv 3248 c recombinant protein, preparation method and application of mycobacterium tuberculosis Rv 3248 c recombinant protein | |
| CN105294844A (en) | Mycobacterium tuberculosis Rv3457c recombinant protein as well as preparation method and application thereof | |
| CN102676562B (en) | Preparation method and application of fusion protein with broad spectrum adsorption capacity to antibodies | |
| CN101619313B (en) | Oligonucleotides aptamer of targeted mycobacterium tuberculosis Ag85B, preparation method and application thereof | |
| CN103063843A (en) | Specific marker of mycobacterium tuberculosis and application thereof | |
| CN104151411B (en) | Albumen and pharmaceutical composition for diagnosis of tuberculosis | |
| CN105131095A (en) | Mycobacterium tuberculosis Rv 2991 recombinant protein, preparation method and application of mycobacterium tuberculosis Rv 2991 recombinant protein | |
| CN102718871A (en) | Recombinant protein of mycobacterium tuberculosis specific marker Rv 1284, preparation method and application thereof | |
| CN105524177A (en) | Mycobacterium tuberculosis specific fusion protein, and encoding gene and application thereof | |
| CN102993283B (en) | Antigen protein for mycobacterium tuberculosis and application | |
| CN102584962A (en) | Preparation method and application of mycobacterium tuberculosis Rv3117 recombinant protein | |
| KR101270662B1 (en) | Diagnostic kits of Brucella abortus using 26kDa recombinant antigenic protein and Lipopolysaccaride | |
| CN110724182A (en) | Marker combination for improving detection sensitivity of bacterial scrotum infection and application thereof | |
| Chanana et al. | Coordinated expression and immunogenicity of an outer membrane protein from Salmonella enterica serovar Typhi under iron limitation, oxidative stress and anaerobic conditions | |
| CN108084256A (en) | Procalcitonin mutant and its preparation method and application | |
| CN106405113A (en) | Specific marker for detecting potential tuberculosis and application of specific marker |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151209 |