CN109030826A - Albumen Rv3539 for the infection of specific detection mycobacterium tuberculosis - Google Patents
Albumen Rv3539 for the infection of specific detection mycobacterium tuberculosis Download PDFInfo
- Publication number
- CN109030826A CN109030826A CN201710438311.XA CN201710438311A CN109030826A CN 109030826 A CN109030826 A CN 109030826A CN 201710438311 A CN201710438311 A CN 201710438311A CN 109030826 A CN109030826 A CN 109030826A
- Authority
- CN
- China
- Prior art keywords
- albumen
- detection
- mycobacterium tuberculosis
- sequence
- negative control
- 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
-
- 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
-
- 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
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
Abstract
The invention discloses the albumen Rv3539 infected for specific detection mycobacterium tuberculosis.The present invention provides application of the Rv3539 albumen in preparation detection or auxiliary detection mycobacterium tuberculosis product；The Rv3539 is following albumen a) or b): a) protein of the composition of amino acid sequence shown in the sequence 1 in sequence table；B) by the amino acid residue sequence of the sequence 1 in sequence table by one or several amino acid residues substitution and/or deletion and/or addition and with the protein with the same function as derived from a) of albumen shown in sequence 1.The experiment proves that infection due to Mycobacterium tuberculosis recall rate can be improved more effectively.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to the albumen for the infection of specific detection mycobacterium tuberculosis
Rv3539。
Background technique
China's in-vitro diagnosis industry is now in rapid growth period.However the huge market demand and China's diagnostic reagent
The relatively backward independent research and development capacity of industry deposits apparent gap.How to carry out autonomous innovation from source, how to be tried in diagnosis
Without being bound by overseas medical giant individually in agent raw material supply, how by the transformation of scientific findings of existing biomedicine field
It is the hot spot that the Chinese government, scientific research institutions and pharmaceuticals industry are all paid special attention in recent years for product.And it is right on market today
Sensitiveer, efficient diagnostic reagent proposes rigid demand, can fast and accurately make a definite diagnosis tuberculosis patient for medical work
Person and patient are of great significance.
Data survey is the results show that related inspection before having the patient 76.6% of tuberculosis symptoms to receive tuberculosis disease in recent years
It looks into, but only 35.8% is diagnosed as lunger, improves the weight that Patient Detection rate is still current tuberculosis prevention and treatment work
Point is also difficult point.
The in-vitro diagnosis that cause pathogeny imcrobe infection is carried out using the cell immune response of T cells with antigenic specificity, is in recent years
The new detection method of the one kind to grow up.It is gone forward side by side by the peripheral blood mononuclear cells in separation fresh whole blood and assassinates sharp culture,
Then it is capable of the quantity of the cell of secretion of gamma-IFN using ELISPOT detection.This method is mainly used in tuberculosis branch bar at present
The diagnosis of bacterium infection.The Diagnosis of Tuberculosis that clinic generallys use at present depends on clinical symptoms, influences to learn diagnosis and aetology
Diagnosis, it is insensitive to the diagnosis of mycobacterium tuberculosis latent infection.Meanwhile during tuberculosis screening, directly detection is sick
Substance or the sensitivity and specificity for detecting mycobacterium tuberculosis antibody are also undesirable.
T-SPOT.TB is a kind of simpler elisa (ELISPOT) detection method.ELISPOT detection is high
Sensitivity, cell secretion cell factor diffusion dilution before, can capture immediately cell secreted by cell peripheral because
Son.This makes ELISPOT detection more sensitive, tests more than traditional ELISA.T-SPOT.TB is devised for examining
Survey the effector T cell of tuberculosis specific antigen stimulation activation.T-SPOT.TB counts the tuberculosis specific response T cell of each activation.
Summary of the invention
It is an object of the present invention to provide the purposes of Rv3539 albumen.
The present invention provides application of the Rv3539 albumen in preparation detection or auxiliary detection mycobacterium tuberculosis product；
The Rv3539 is following albumen a) or b):
A) protein of the composition of amino acid sequence shown in the sequence 2 in sequence table；
B) by the amino acid residue sequence of the sequence 2 in sequence table by one or several amino acid residues substitution and/
Or deletion and/or addition and with the protein with the same function as derived from a) of albumen shown in sequence 2.
Rv3539 is known albumen, and according to individual difference, which may have different sequences, and there may be one for they
The difference of a or several amino acid, but be within, albumen used in one embodiment of the present of invention
Sequence is sequence 2.
Above-mentioned Rv3539 albumen detects whether patient to be measured infects in mycobacterium tuberculosis product in preparation detection or auxiliary
Application be also the scope of protection of the invention.
It is a further object to provide a kind of detections or auxiliary detection sample to be tested whether to infect tuberculosis branch bar
The product of bacterium.
Product provided by the invention, including Rv3539 albumen.
The said goods further include positive quality control and negative control hole；
The positive quality control is phytohemagglutinin HA；
The negative control hole is nonantigenic cell culture fluid.
In the said goods, the product further includes the readable carrier (can be specification) for being described below content:
If testing result meets following condition: negative control hole spot number is 0-5, and Rv3539 detection hole spot number-yin
Property control wells spot number >=6；If or negative control hole spot number is more than or equal to 6, and (Rv3539 detection hole spot number-feminine gender is right
According to hole spot number) >=2 × (negative control hole spot number)；Then sample to be tested candidate infects mycobacterium tuberculosis, is positive sample；
If testing result meets condition as described below, sample to be tested candidate is uninfected by mycobacterium tuberculosis；
The negative control hole is hole where negative control,
The Rv3539 detection hole is hole where the Rv3539 albumen.
The said goods are kit, and mentioned reagent box is specially T-SPOT kit, or ELISA kit.
Above-mentioned T-SPOT kit further includes well plates in T-SPOT kit, concentration labelled antibody, colour developing bottom
Object solution.Mentioned reagent box is the product of Oxford immunotec (Britain).
Third purpose of the present invention be to provide it is a kind of for identifying, diagnosing, auxiliary diagnosis, screening and/or auxiliary screening knot
The marker of core mycobacteria.
Marker provided by the invention is Rv3539 albumen.
The experiment proves that the present invention has screened the protein fragments in mycobacterium tuberculosis source, a kind of inspection is provided
Mycobacterium tuberculosis marker antigen RV3539 lungy is surveyed, it is anti-come vitro detection Specific T cell immunity by the antigen
It answers, can be used as the reference of diagnosis of tuberculosis patient, for diagnosing whether patient is infected by mycobacterium tuberculosis.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Portion of reagent is as follows:
Lysis buffer:20mM Tris-HCl, 500mM sodium chloride, 10% glycerol, pH 8.0
Wash buffer:20mM Tris-HCl, 20mM imidazoles, 500mM sodium chloride, 10% glycerol, pH 8.0
Elution buffer:20mM Tris-HCl, 250mM imidazoles, 500mM sodium chloride, 10% glycerol, pH8.0
Chromatography buffer: potassium dihydrogen phosphate 13.609g (preparing 1000ml) is taken, adds 0.1mol/l sodium hydroxide solution, adjusts
PH to 7.5 is saved, the PBS buffer solution that pH value is 7.5 is obtained.
Solidify Ni+ chromatographic resin Novagen, Ni-NTA His, (Cat.NO 70691-5, Beijing are magnificent；2-8 degree is protected
It deposits)；Protein binding capacity is greater than 5mg/ml resin chromatographic column (glass or polypropylene).Superdex 75:(GE).
It is the T-SPOT of Oxford immunotec (Britain) that following embodiments, which use tuberculosis infection T cell detection kit,
Kit is 10 pairs of antigenic reagent box of ESAT-6 and CFP.
Embodiment 1, prokaryotic expression destination protein Rv3539
The preparation of destination protein Rv3539: the recombinant vector pET28a-Rv3539 of Rv3539 protein coding gene will be expressed
It imports in e. coli bl21, obtains recombinant bacterium BL21/pET28a-Rv3539；IPTG induces recombinant bacterium BL21/pET28a- again
Rv3539 collects supernatant, as destination protein Rv3539.
It is specific as follows:
1, the building of express express target protein recombinant bacterium
The amino acid sequence of Rv3539 albumen is sequence 2, and the nucleotides sequence of encoding gene is classified as sequence 1.
By the site NdeI and EcoRI of the insertion pET28a carrier of Rv3539 protein coding gene shown in sequence 1, weight is obtained
Group carrier pET28a-Rv3539；
Recombinant vector is imported in e. coli bl21, recombinant bacterium BL21/pET28a-Rv3539 is obtained.
2, protein expression
1) recombinant bacterium BL21/pET28a-Rv3539 stands overnight (16h or so) at 37 DEG C.
2) 37 DEG C of 200rpm of 5ml LB liquid medium (5ul kan is added) are inoculated in, it is (big that culture to OD is greater than 0.6-0.8
About 3h).
3) it is inoculated in 300ml LB liquid medium by inoculum concentration 5ml, 37 DEG C, 200rpm is cultivated to OD600 ≈ 0.6-
0.8 (about 2h).16 DEG C are cooled to, final concentration 0.4mM IPTG is added；
4) 16 DEG C, 200rpm culture culture 16h, 4 DEG C, 4000rpm is centrifuged 10min and collects thallus.
3, protein purification
1) thallus is resuspended: thallus collected by every 1L culture medium is resuspended with 40ml or so lysis buffer and (1% egg is added
White enzyme inhibitor PMSF, then final concentration of 0.5-1mM)
2) bacterial cell disruption: 6s/ times ultrasonic, power 38%, ultrasonic 20min.(bright suspension is standard, is adjusted as one sees fit
Ultrasound works total time).
3) remove bacterial debris: 4 DEG C, 12000g is centrifuged 30 minutes.
4) Ni column handle: 3mlNi column first uses water process 8cv (8 times of column volumes), then use 8cv lysis buffer as put down
Weigh liquid balance, flow velocity 30cm/h.
5) loading: broken bacterium centrifugation supernatant carries out loading with the flow velocity of 30cm/ml, and collection flows through peak.
6) it balances: balancing 5cv with equilibrium liquid after sample loading；
7) it washs: washing 5cv with wash buffer；
8) it elutes: being eluted with elution buffer, be in charge of collection.Eluent is detected with bradford reagent, understands egg
It is white whether complete elution.Such as contain albumen, then continue to elute, until completely, obtaining elution albumen.
9) dialyse: it is slow that elution albumen is fitted into the ion exchange for being put into the bag filter of 7KD and eluting albumen volume equipped with 100 times
Dialysed overnight is carried out in the beaker of fliud flushing A (formula 10mM PB, 50mM NaCl, pH 7.5).Centrifuging and taking supernatant.
10) sieve chromatography
A) chromatographic column: superdex 75 (determines the volume of chromatography media according to the amount of sample)
B) flow velocity；30cm/h
C) chromatography process: water process 5cv is first used, then handles half an hour with 0.6M NaOH aqueous solution, then use chromatography buffer
(PBS, pH 7.5) balance chromatography, until chromatographic column conductance and pH efflux are consistent with chromatography buffer.It 9) is obtained above-mentioned
Supernatant loading elutes (flow velocity 30cm/h) with chromatography buffer, collects corresponding eluting peak, is purpose albumen Rv3539 (size
For 19.516KDa).
The application of embodiment 2, mycobacterium tuberculosis marker antigen Rv3539 in detection mycobacterium tuberculosis
1, T-SPOT kit detects
The specific method is as follows, and some solution of the inside are all from the T-SPOT reagent of Oxford immunotec (Britain)
Box:
1) using heparin vacuum blood collection tube to different patients to be measured (30 clinical detection patients lungy shown in table 1 and
4 clinical detection non-tuberculosis people shown in table 2, and know) peripheric venous blood is directly adopted, obtain anticoagulation sample；
2) anticoagulation sample is mixed by volume 1:1 and RPMI1640 culture solution；Carefully blood sample is added in 3:1 ratio
Ficoll lymphocyte separation medium upper layer (GE Healthcare 17-1440-02) (utilizes Ficoll-PaqueTMplusAccording to
Operating instruction separates PBMCs)；25 DEG C of room temperature, 1000g is centrifuged 22 minutes；Horizontal rotor delays and rises slow drop；
3) by mononuclear cell layer (being located at centrifuge tube middle layer, be the tunica albuginea shape of layer) from Ficoll separating pipe transfer
The sterile 15ml centrifuge tube for having added 10ml AIM-V culture solution (Invitrogen 12055091) is moved on to, is mixed gently, room temperature
600g is centrifuged 7min；
4) supernatant is carefully removed, 1ml AIM-V culture solution is added, AIM-V culture solution is added extremely in light and slow resuspension cell
10ml, 350g are centrifuged 7min；
5) supernatant is carefully abandoned, 1ml AIM-V culture solution is added, cell is resuspended, obtain cell suspension (PBMCs)；
6) it takes 10 μ l cell suspensions that the 1%trypan blue of 40 μ l is added, is diluted with AIM-V culture solution, it is dilute to obtain cell
Release working solution；
7) take out 24 hole pvdf membrane plates from aluminium envelope, be sequentially added into: 50 μ l phytohemagglutinin HA (sigma) are extremely
The Rv3539 protein solution that Positive control wells, 50 μ l are prepared (initial concentration 60ug/ml, solvent are AIM V culture solution)
It is added to detection hole, 50 μ l AIM V culture solutions to negative control hole；It is separately added into above-mentioned 3 hole and has prepared cell dilution work
Make liquid 100 μ l (every hole cell quantity 2.5 × 105), and in sample well Rv3539 protein solution final concentration of 20ug/ml；
8) culture plate is put into 37 DEG C, 5%CO2Incubator is incubated for 16 hours；
9) fresh enzyme labelled antibody working solution is prepared by 1:200 with sterile PBS；Culture plate is taken out from incubator, is discarded in hole
Liquid.It is washed 4 times with the sterile PBS of 200 μ l/well；
10) 50 μ l/well enzyme labelled antibody working solutions are added, culture plate is incubated for 1 hour at 2-8 DEG C.It is washed 4 times with PBS
Remove unbonded enzyme labelled antibody；
11) the 50 μ l of substrate working solution that equilibrium at room temperature is crossed is added in every hole, reacts at room temperature 7 minutes.It is terminated with distilled water flushing
Reaction, in 37 DEG C of 2-3hrs or ambient temperature overnight desiccation culture plate, obtains the culture plate containing reaction product.
2, spot counts
1) culture plate containing reaction product is taken out from incubator discard cell culture fluid；
2) 200ul PBS buffer solution (pH 7.5) is added in each reacting hole；
3) PBS buffer solution is discarded；It is washed repeatedly at least 3 times with fresh PBS buffer solution；
4) with the concentration labelled antibody reagent (mouse of alkali phosphatase enzyme mark in 200 times of PBS buffer solution dilution kits
Anti-human gamma interferon monoclonal antibody；
5) 50ul labelled antibody working solution is added in each reacting hole, and 2-8 DEG C is incubated for 1 hour；
6) labelled antibody working solution is discarded, is washed by above-mentioned step 2 and 3；
7) each reacting hole is added 50ul substrate chromophoric solution and is incubated at room temperature 7 minutes；
8) culture plate is thoroughly washed with distilled water or deionized water terminate reaction；
9) in ventilation or 37 DEG C of incubator desiccation culture plates；
10) navy blue that counts in each reacting hole clearly spot.
As a result judge:
If negative control hole spot number is 0-5, and Rv3539 detection hole spot number-negative control hole spot number >=6；
If or negative control hole spot number is more than or equal to 6, and (Rv3539 detection hole spot number-negative control hole spot number)
>=2 × (negative control hole spot number)；
Then sample to be tested candidate infects mycobacterium tuberculosis, is positive sample；
If being unsatisfactory for above-mentioned, sample to be tested and Rv3539 do not generate immune response, and sample to be tested candidate is uninfected by tuberculosis
Mycobacteria.
10 tuberculosis patient samples to be tested in table 1 are detected in aforementioned manners, and the results are shown in Table 1.
The sample to be tested of 4 Healthy Peoples in table 2 is detected in aforementioned manners, and the results are shown in Table 2.
Comparative example:
10 tuberculosis patients shown in T-SPOT kit detection table 1 using Oxford immunotec (Britain) are to be measured
The sample to be tested of 4 Healthy Peoples in sample and table 2, method is same as Example 1, uniquely the difference is that two detection holes are arranged,
And A detection hole is antigen A hole, addition is antigen ESAT-6, and B detection hole is the hole antigen B, and addition is antigens c FP 10.
According to the reaction and judgement result of antigen A and the hole antigen B:
If negative control hole spot number is 0-5, and (antigen A spot number)-(negative control hole spot number) >=6 or (antigen B
Spot number)-(negative control hole spot number) >=6；
Or work as negative control hole spot number >=6, and (antigen A spot number) >=2 × (negative control hole spot number) or (antigen
B spot number) >=2 × (negative control hole spot number).
Then sample to be tested candidate infects mycobacterium tuberculosis, is positive sample；
If being unsatisfactory for above-mentioned, sample to be tested and antigen A or antigen B do not generate immune response, and sample to be tested candidate does not feel
Contaminate mycobacterium tuberculosis.
Table 1 is sample and its testing result
In table ,-it is negative control ,+it is Positive control wells (culture medium for having added PHA), X is Rv3539 Protein Detection hole, A
For antigen A detection hole, B is antigen B detection hole.
Table 2 is Healthy People sample and its testing result
| Serial number | - | A | B | + | X |
| 1 | 1 | 1 | 2 | 862 | 1 |
| 2 | 0 | 3 | 6 | 786 | 2 |
| 3 | 0 | 0 | 0 | 354 | 0 |
| 4 | 0 | 1 | 5 | 891 | 2 |
In table ,-it is negative control (culture medium for being added without any antigen) ,+it is that Positive control wells (have added the culture of PHA
Base), X is Rv3539 Protein Detection hole.
As can be seen that
Rv3539 is detected as antigen, and 9 are in the identified sample to be tested for infecting mycobacterium tuberculosis of 11 clinics
Rv3539 Protein Detection infects the sample of mycobacterium tuberculosis, this is consistent with clinical identification result.But remaining 2 sample does not have
It detected, therefore, the present invention uses Rv3539 to detect whether the recall rate of infection mycobacterium tuberculosis up to 82% as antigen.
Mycobacterium tuberculosis is not detected in 4 Healthy People samples, this is consistent with clinical identification result.
Comparative example detects the identified sample to be tested for infecting mycobacterium tuberculosis of 11 clinics, wherein 7 are comparative example
The sample of detection infection mycobacterium tuberculosis, this is consistent with clinical identification result；But remaining 4 sample is not detected and.
Therefore, comparative example detects whether that the recall rate of infection mycobacterium tuberculosis is only 64%, examines well below Rv3539 as antigen
It surveys.
In comparative example in 4 Healthy People samples 3 mycobacterium tuberculosis is not detected, this with clinical identification result slightly not
Together.
Can be seen that Rv3539 albumen from above-mentioned experiment can detecte or assist whether detection patient to be measured infects tuberculosis point
Branch bacillus, it is specific as follows: using Rv3539 albumen as antigen, to be detected by tuberculosis infection T cell detection kit, trained with cell
It supports base and is used as negative control hole, if negative control hole spot number is 0-5, and Rv3539 detection hole spot number-negative control hole spot
Points >=6；If or negative control hole spot number is more than or equal to 6, and (Rv3539 detection hole spot number-negative control hole spot number)
>=2 × (negative control hole spot number)；Then sample to be tested candidate infects mycobacterium tuberculosis, is positive sample；If being unsatisfactory for
It states, then sample to be tested and Rv3539 do not generate immune response, and sample to be tested candidate is uninfected by mycobacterium tuberculosis.
Sequence table
<110>Guangdong Ti Bikang Biotechnology Co., Ltd
<120>the albumen Rv3539 for the infection of specific detection mycobacterium tuberculosis
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1437
<212> DNA
<213>artificial sequence
<220>
<223>
atggcggatt tcttgacgtt gtcaccagag gtgaattcgg cccggatgta cgcgggtggg 60
gggcccgggt cgctatcggc ggccgcggcg gcctgggatg agttggccgc cgaactgtgg 120
ttggcggcgg cctcgttcga gtcggtgtgc tccggcctgg cggaccgttg gtggcaaggg 180
ccgtcgtctc ggatgatggc ggcgcaggcc gcccgccata cggggtggct ggccgcggcg 240
gccacccagg cagagggagc agccagccag gctcagacga tggcgctggc ctatgaagcg 300
gcgttcgccg caaccgtaca cccggcgctg gtcgcggcga accgcgccct cgtggcctgg 360
ttggcggggt cgaatgtgtt cgggcagaac accccggcga ttgcggccgc cgaggccatc 420
tacgagcaga tgtgggctca ggatgttgtc gcgatgttga actaccatgc ggtggcctcg 480
gcggtcgggg cgcggttgcg gccgtggcag cagttgctgc atgagctgcc caggcggttg 540
ggcggcgaac actccgacag cacaaacacg gaactcgcta acccgagttc aacgacgaca 600
cgcattaccg tccccggcgc atctccggtg catgcagcga cgttactgcc gttcatcgga 660
aggctactgg cggcgcgtta tgccgagctg aacaccgcga tcggcacgaa ctggtttccg 720
ggcaccacgc cagaagtggt gagctatccg gccaccatcg gggtccttag cggctctctt 780
ggcgccgtcg atgccaacca gtccatcgct atcggtcagc agatgttgca caacgagatc 840
ctggccgcca cggcctccgg tcagccggtg acggtggccg gactgtcgat gggcagcatg 900
gtcatcgacc gcgaacttgc ctatctggcc atcgacccca acgcgccacc ctcgagcgcg 960
ctcacattcg tcgagctcgc cggcccggaa cgcggtcttg cccagaccta cctgcccgtt 1020
ggcaccacca ttccaatcgc ggggtacacc gtggggaatg cgcccgagag ccagtacaac 1080
accagcgtgg tttatagcca gtacgatatc tgggccgatc cgcccgaccg tccgtggaac 1140
ctgttggccg gcgccaacgc actgatgggc gcggcttact ttcacgatct gaccgcctac 1200
gccgcaccac aacaggggat agagatcgcc gctgtcacga gttcactggg cggaaccacg 1260
acaacgtaca tgattccgtc gcccacgctg ccgttgctgt tgccactgaa gcagatcggt 1320
gtcccagact ggatcgtcgg cgggctgaac aacgtgctga agccgctcgt cgacgcgggc 1380
tactcacagt acgcccccac cgccggccct tatttcagcc acggcaacct ggtgtgg 1437
<210> 2
<211> 479
<212> PRT
<213>artificial sequence
<220>
<223>
<400> 2
Met Ala Asp Phe Leu Thr Leu Ser Pro Glu Val Asn Ser Ala Arg Met
1 5 10 15
Tyr Ala Gly Gly Gly Pro Gly Ser Leu Ser Ala Ala Ala Ala Ala Trp
20 25 30
Asp Glu Leu Ala Ala Glu Leu Trp Leu Ala Ala Ala Ser Phe Glu Ser
35 40 45
Val Cys Ser Gly Leu Ala Asp Arg Trp Trp Gln Gly Pro Ser Ser Arg
50 55 60
Met Met Ala Ala Gln Ala Ala Arg His Thr Gly Trp Leu Ala Ala Ala
65 70 75 80
Ala Thr Gln Ala Glu Gly Ala Ala Ser Gln Ala Gln Thr Met Ala Leu
85 90 95
Ala Tyr Glu Ala Ala Phe Ala Ala Thr Val His Pro Ala Leu Val Ala
100 105 110
Ala Asn Arg Ala Leu Val Ala Trp Leu Ala Gly Ser Asn Val Phe Gly
115 120 125
Gln Asn Thr Pro Ala Ile Ala Ala Ala Glu Ala Ile Tyr Glu Gln Met
130 135 140
Trp Ala Gln Asp Val Val Ala Met Leu Asn Tyr His Ala Val Ala Ser
145 150 155 160
Ala Val Gly Ala Arg Leu Arg Pro Trp Gln Gln Leu Leu His Glu Leu
165 170 175
Pro Arg Arg Leu Gly Gly Glu His Ser Asp Ser Thr Asn Thr Glu Leu
180 185 190
Ala Asn Pro Ser Ser Thr Thr Thr Arg Ile Thr Val Pro Gly Ala Ser
195 200 205
Pro Val His Ala Ala Thr Leu Leu Pro Phe Ile Gly Arg Leu Leu Ala
210 215 220
Ala Arg Tyr Ala Glu Leu Asn Thr Ala Ile Gly Thr Asn Trp Phe Pro
225 230 235 240
Gly Thr Thr Pro Glu Val Val Ser Tyr Pro Ala Thr Ile Gly Val Leu
245 250 255
Ser Gly Ser Leu Gly Ala Val Asp Ala Asn Gln Ser Ile Ala Ile Gly
260 265 270
Gln Gln Met Leu His Asn Glu Ile Leu Ala Ala Thr Ala Ser Gly Gln
275 280 285
Pro Val Thr Val Ala Gly Leu Ser Met Gly Ser Met Val Ile Asp Arg
290 295 300
Glu Leu Ala Tyr Leu Ala Ile Asp Pro Asn Ala Pro Pro Ser Ser Ala
305 310 315 320
Leu Thr Phe Val Glu Leu Ala Gly Pro Glu Arg Gly Leu Ala Gln Thr
325 330 335
Tyr Leu Pro Val Gly Thr Thr Ile Pro Ile Ala Gly Tyr Thr Val Gly
340 345 350
Asn Ala Pro Glu Ser Gln Tyr Asn Thr Ser Val Val Tyr Ser Gln Tyr
355 360 365
Asp Ile Trp Ala Asp Pro Pro Asp Arg Pro Trp Asn Leu Leu Ala Gly
370 375 380
Ala Asn Ala Leu Met Gly Ala Ala Tyr Phe His Asp Leu Thr Ala Tyr
385 390 395 400
Ala Ala Pro Gln Gln Gly Ile Glu Ile Ala Ala Val Thr Ser Ser Leu
405 410 415
Gly Gly Thr Thr Thr Thr Tyr Met Ile Pro Ser Pro Thr Leu Pro Leu
420 425 430
Leu Leu Pro Leu Lys Gln Ile Gly Val Pro Asp Trp Ile Val Gly Gly
435 440 445
Leu Asn Asn Val Leu Lys Pro Leu Val Asp Ala Gly Tyr Ser Gln Tyr
450 455 460
Ala Pro Thr Ala Gly Pro Tyr Phe Ser His Gly Asn Leu Val Trp
465 470 475
Claims (7)
- Application of the 1.Rv3539 albumen in preparation detection or auxiliary detection mycobacterium tuberculosis product；The Rv3539 is following albumen a) or b):A) protein of the composition of amino acid sequence shown in the sequence 2 in sequence table；B) amino acid residue sequence of the sequence 2 in sequence table by the substitution of one or several amino acid residues and/or is lacked Lose and/or addition and with the protein with the same function as derived from a) of albumen shown in sequence 2.
- 2.Rv3539 albumen detects whether patient to be measured infects the application in mycobacterium tuberculosis product in preparation detection or auxiliary.
- 3. whether a kind of detection or auxiliary detection sample to be tested infect the product of mycobacterium tuberculosis, including Rv3539 albumen.
- 4. product according to claim 3, it is characterised in that: the product further includes positive quality control and negative control hole；The positive quality control is phytohemagglutinin HA；The negative control hole is nonantigenic cell culture fluid.
- 5. product according to claim 3 or 4, it is characterised in that: the product further includes be described below content readable Carrier:If testing result meets following condition: negative control hole spot number is 0-5, and Rv3539 detection hole spot number-feminine gender is right According to hole spot number >=6；If or negative control hole spot number is more than or equal to 6, and (Rv3539 detection hole spot number-negative control hole Spot number) >=2 × (negative control hole spot number)；Then sample to be tested candidate infects mycobacterium tuberculosis, is positive sample；If inspection It surveys result and meets condition as described below, then sample to be tested candidate is uninfected by mycobacterium tuberculosis；The negative control hole is hole where negative control,The Rv3539 detection hole is hole where the Rv3539 albumen.
- 6. according to the product any in claim 3-5, it is characterised in that: the product is kit.
- 7. it is a kind of for identifying, diagnosing, auxiliary diagnosis, screening and/or assist screening mycobacterium tuberculosis marker, be Rv3539 albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710438311.XA CN109030826A (en) | 2017-06-12 | 2017-06-12 | Albumen Rv3539 for the infection of specific detection mycobacterium tuberculosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710438311.XA CN109030826A (en) | 2017-06-12 | 2017-06-12 | Albumen Rv3539 for the infection of specific detection mycobacterium tuberculosis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109030826A true CN109030826A (en) | 2018-12-18 |
Family
ID=64629134
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710438311.XA Pending CN109030826A (en) | 2017-06-12 | 2017-06-12 | Albumen Rv3539 for the infection of specific detection mycobacterium tuberculosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109030826A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012057904A1 (en) * | 2010-10-27 | 2012-05-03 | Infectious Disease Research Institute | Mycobacterium tuberculosis antigens and combinations thereof having high seroreactivity |
| CN102713629A (en) * | 2009-11-20 | 2012-10-03 | 俄勒冈健康科学大学 | Methods for detecting a mycobacterium tuberculosis infection |
| WO2016141347A2 (en) * | 2015-03-04 | 2016-09-09 | Wayne State University | Systems and methods to diagnose sarcoidosis and identify markers of the condition |
| CN106405107A (en) * | 2016-08-31 | 2017-02-15 | 中国疾病预防控制中心传染病预防控制所 | Use of mycobacterium tuberculosis antigen protein Rv2941 and its T cell epitope peptide |
| CN106645733A (en) * | 2015-07-16 | 2017-05-10 | 广东体必康生物科技有限公司 | Protein for specific detection of mycobacterium tuberculosis infection |
-
2017
- 2017-06-12 CN CN201710438311.XA patent/CN109030826A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102713629A (en) * | 2009-11-20 | 2012-10-03 | 俄勒冈健康科学大学 | Methods for detecting a mycobacterium tuberculosis infection |
| WO2012057904A1 (en) * | 2010-10-27 | 2012-05-03 | Infectious Disease Research Institute | Mycobacterium tuberculosis antigens and combinations thereof having high seroreactivity |
| WO2016141347A2 (en) * | 2015-03-04 | 2016-09-09 | Wayne State University | Systems and methods to diagnose sarcoidosis and identify markers of the condition |
| CN106645733A (en) * | 2015-07-16 | 2017-05-10 | 广东体必康生物科技有限公司 | Protein for specific detection of mycobacterium tuberculosis infection |
| CN106405107A (en) * | 2016-08-31 | 2017-02-15 | 中国疾病预防控制中心传染病预防控制所 | Use of mycobacterium tuberculosis antigen protein Rv2941 and its T cell epitope peptide |
Non-Patent Citations (1)
| Title |
|---|
| 乐军 等: "酶联免疫斑点试验快速诊断结核分枝杆菌感染的临床应用价值", 《中华检验医学杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110146697B (en) | Immune test strip for rapidly detecting malignant malaria, vivax malaria, triumphaniae and oval malaria | |
| CN101294964A (en) | Reagent and method for detecting active tuberculosis and tuberculosis dormant infection | |
| CN101419237B (en) | ELISA fleck diagnosis kit for tubercle bacillus infect and method for preparing specific antigen | |
| CN105542014A (en) | TP recombinant antigen and preparing method and application thereof | |
| CN107076744A (en) | For the composition for the mycobacterium tuberculosis for diagnosing latent infection | |
| Dey et al. | Recombinant antigen-based latex agglutination test for rapid serodiagnosis of leptospirosis | |
| CN106939035B (en) | Mycobacterium tuberculosis T cell epitope polypeptide and application thereof | |
| CN105861520A (en) | Application of Rv3121 protein for detecting mycobacterium tuberculosis infection | |
| CN111443208B (en) | Composition for distinguishing active tuberculosis from latent tuberculosis | |
| CN103063837B (en) | Reagent, method and kit for detecting mycobacterial infection | |
| CN106349350A (en) | Protein capable of specifically detecting mycobacterium tuberculosis infection | |
| CN109991417B (en) | Immune marker for tuberculosis and application | |
| CN106645733A (en) | Protein for specific detection of mycobacterium tuberculosis infection | |
| CN106279381B (en) | A kind of albumen of specific detection mycobacterium tuberculosis infection | |
| CN109030826A (en) | Albumen Rv3539 for the infection of specific detection mycobacterium tuberculosis | |
| CN109187987B (en) | Application of MS4A3 protein as marker in diagnosis of active tuberculosis | |
| CN108254553A (en) | For the albumen Rv1791 of specific detection mycobacterium tuberculosis infection | |
| CN109030825A (en) | Albumen Rv2031c for the infection of specific detection mycobacterium tuberculosis | |
| CN109406776B (en) | Protein Rv2818c for specific detection of mycobacterium tuberculosis infection | |
| CN109406777B (en) | Protein Rv2824c for specific detection of mycobacterium tuberculosis infection | |
| CN101979406B (en) | Multi-epitope protein for South Africa type II foot-and-mouth disease, and preparation method and application thereof | |
| CN108267589B (en) | Application of mycobacterium tuberculosis protein in preparing product for diagnosing latent tuberculosis infected person and/or active tuberculosis | |
| CN112646007B (en) | Combined protein for detecting mycobacterium tuberculosis and detection reagent | |
| CN103525829A (en) | Preparation method of recombinant antigen of mycobacterium tuberculosis capable of being used for diagnosing tuberculosis infection | |
| CN116589576B (en) | Preparation method of anti-HBP monoclonal antibody, monoclonal antibody and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20200819 Address after: 4 / F, building a, China Europe center, No.2 South Lingnan Avenue, Lecong Town, Shunde District, Foshan City, Guangdong Province 528300 Applicant after: GUANGZHOU BCBIO BIOTECHNOLOGY Co.,Ltd. Address before: 528000 6th Floor, Block A, Central Europe Center, South 2 Lingnan Avenue, Lecong Town, Shunde District, Foshan City, Guangdong Province Applicant before: TB Healthcare Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181218 |
|
| RJ01 | Rejection of invention patent application after publication |