CN106279383A - A kind of albumen of specific detection m tuberculosis infection - Google Patents
A kind of albumen of specific detection m tuberculosis infection Download PDFInfo
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- CN106279383A CN106279383A CN201510293914.6A CN201510293914A CN106279383A CN 106279383 A CN106279383 A CN 106279383A CN 201510293914 A CN201510293914 A CN 201510293914A CN 106279383 A CN106279383 A CN 106279383A
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- mycobacterium tuberculosis
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Classifications
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- 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)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
-
- 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
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/12—Pulmonary diseases
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Abstract
The present invention relates to the albumen of a kind of specific detection m tuberculosis infection, described albumen source and mycobacterium tuberculosis, aminoacid sequence is as shown in SEQ ID NO.2, can be as detecting mycobacterium tuberculosis marker antigen lungy, carry out vitro detection Specific T cell immunity by this antigen to react, can be used for whether diagnosing patient by m tuberculosis infection as the reference of diagnosis of tuberculosis patient.Meanwhile, further zoopery shows, this proteantigen can induce the immunoreation for mycobacterium tuberculosis, has the function preparing corresponding vaccine.
Description
Technical field
The invention belongs to medical product technical field, in particular to the albumen of a kind of specific detection m tuberculosis infection.
Background technology
The rapid growth period that China's in-vitro diagnosis industry being now in.But the huge market demand is relative with China diagnostic reagent industry falls behind
Independent research and development capacity deposit obvious gap.How from source, to carry out autonomous innovation, how in diagnostic reagent raw material supply without being bound by
Indivedual medicine giants the most overseas, how by the transformation of scientific findings of existing biomedicine field be product be the Chinese government, scientific research institutions and doctor
Medicine industry is at the focus the most all paid special attention to.And on market today, diagnostic reagent sensitiveer, efficient is proposed rigid demand, energy
Enough make a definite diagnosis tuberculosis patient fast and accurately the most significant for medical personnel and patient.
Data survey result shows in recent years, has the patient 76.6% of tuberculosis symptoms to accept coherence check before tuberculosis disease, but only has
35.8% is diagnosed as lunger, and it is also difficult point that raising Patient Detection rate is still the emphasis of current tuberculosis prevention and treatment work.
The cell immune response utilizing T cells with antigenic specificity carries out the in-vitro diagnosis of cause pathogeny imcrobe infection, is the one grown up this year
New detection method.We are gone forward side by side by the peripheral blood lymphocytes in separation fresh whole blood, and assassination is sharp cultivates, and then utilizes ELISPOT to examine
Survey can the quantity of cell of secretion of gamma-IFN.The method is currently mainly applied to the diagnosis of m tuberculosis infection.Clinic is generally adopted at present
Diagnosis of Tuberculosis depend on clinical symptoms, diagnosis and etiological diagnosis are learned in impact, to the diagnosis of mycobacterium tuberculosis latent infection not
Sensitive.Meanwhile, during tuberculosis examination, directly detection pathogen or the sensitivity of detection mycobacterium tuberculosis antibody and specificity are the most not
Preferable.
Summary of the invention
For above-mentioned clinical practice working condition, we have screened the protein fragments in mycobacterium tuberculosis source, it is provided that a kind of detection tuberculosis
Mycobacterium tuberculosis marker antigen, by this antigen come vitro detection Specific T cell immunity react, can be as diagnosis of tuberculosis sufferer
The reference of person, is used for whether diagnosing patient by m tuberculosis infection.Meanwhile, further zoopery shows, this proteantigen can lure
The guide pin immunoreation to mycobacterium tuberculosis, has the function preparing corresponding vaccine.
Specifically, present invention firstly relates to the specific proteins antigen Rv1098c of a kind of mycobacterium tuberculosis, described antigen aminoacid sequence is such as
Shown in SEQ ID NO.2, its amino acid sequence structure is:
The nucleotide sequence of encoding said proteins antigen Rv1098c is as shown in SEQ ID NO.1, and its structural dna sequence is:
The invention still further relates to the preparation method of described proteantigen Rv1098c, the method comprises the steps,
Method one:
Step (1) withLREntry vector (the U.S. Craig Ventor Institute of Enzyme mix catalysis Rv1098c
The PFGRC divided into is provided free) andpDESTTM17 carrier restructuring generate the expression vector of Rv1098c；
The expression vector of step (1) described Rv1098c is converted objective expression host by step (2), expresses target Rv1098c albumen；
Step (3) smudge cells collects albumen renaturation target protein.
The concrete grammar of described step (1) is:
The entry vector 1.5 μ L of a. cloning reaction system: Rv1098c,pDESTTM17 carrier 1 μ L,BP
II Enzyme mix 2.5μL；Reaction condition: 25 DEG C, reaction overnight；
B. method for transformation: add 100 μ L bacillus coli DH 5 alpha competence (making by oneself), after ice bath 30min, 42 DEG C of heat shock 90s in reaction system,
System places 10min on ice, after adding 200 μ L LB culture medium renaturation, is coated on the LB solid medium containing ampicillin (100mg/l)
On, cultivate 20h for 37 DEG C；
C. plasmid extraction: extract plasmid with the little extraction reagent kit of N96 high-purity plasmid (DP114), it is thus achieved that the expression vector of Rv1098c；
The concrete grammar of described step (2) is:
A. take the plasmid solution that 1 μ L step (1) obtains and add 100 μ l escherichia coli Rosetta (DE3) competence, after ice bath 30min,
42 DEG C of heat shock 90s；
B. system places 10min on ice, after adding 200 μ L LB culture medium renaturation, is coated on the LB solid containing ampicillin (100mg/l)
In culture medium, cultivate 12h for 37 DEG C；
C. Rv1098c protein expression is induced with 0.75mM IPTG；
The concrete grammar of described step (3) is:
A. with re-suspension liquid: the resuspended antibacterial of 60mM tris PH 9.0,0.15M EDTA, use sonicator ultrasonication thin under the conditions of 4 DEG C
Bacterium；
B.4 DEG C, 10000rpm is centrifuged 15min and collects precipitation and be solid forms inclusion body, and target protein electrophoresis result is as shown in Figure 2.
C. inclusion body is dissolved with lysate (60mM Tris-HCl PH7.0,10M carbamide, 15mM DTT, 1mM EDTA)；Dialysis for the first time:
Albumen is dialysed by the bag filter dialyzed overnight with molecular cut off as 3K, and dialysis solution composition is: 20mM Tris-HCl PH 9.0,1M carbamide,
3mM L-Argine；
Dialysing for the second time, dialysis solution composition is: 20mM Tris-HCl PH 9.0,10mM NaCl the most again, second time dialysis: with molecular cut off
Albumen is dialysed to dialysis solution by the bag filter dialyzed overnight for 3K, can obtain the albumen that can be directly used for immunogen screening.
Method two:
Step (1) is expressed the subject fusion proteins containing polyhistidine label and collects expressive host thalline:
Step (2) collection subject fusion proteins containing polyhistidine label:
Step (3) purification of target fusion protein.
Described step (1) method particularly includes:
1) E.coli BL21 bacterial strain is used to express 6*His-Nus.a-Rv1098cc fusion protein；
2) LB plate streaking activation (adding corresponding antibiotic), 37 DEG C stand overnight (about 12h)；
3) it is inoculated in 37 DEG C of 200rpm of 5ml LB liquid medium (adding corresponding antibiotic), cultivates to OD more than 0.6-0.8 (about 3h)；
4) being inoculated in 300ml LB fluid medium by inoculum concentration 1-3%, 37 DEG C, 200rpm cultivates to OD600 ≈ 0.6-0.8 (about
3h).Cool to 16 DEG C, add IPTG to final concentration 0.1mM；
5) 16 DEG C, 200rpm cultivates 16h, and 4 DEG C, 4000rpm is centrifuged 15min and collects thalline.
Described step (2) method particularly includes:
1) thalline is resuspended: every thalline about 40ml lysis buffer collected by 1L culture medium is resuspended (as added protease suppression
Agent PMSF, the most final concentration of 0.5-1mM；)
2) bacterial cell disruption: ultrasonic 3s, interval 9s, power are 38%, ultrasonic 30min.(suspension is bright for standard, takes the circumstances into consideration to adjust ultrasonic
Work total time) as used high pressure cell cracker to crush 1-2 time, required time about 10min；
3) bacterial debris is removed: 4 DEG C, 15000-18000rpm is centrifuged 20 minutes,
4) loading: above-mentioned supernatant fluid and column material Ni-NTA resin (Novagen Cat.NO 70691-5) 4 DEG C hatch about 1h-1.5h.
Described step (3) method particularly includes:
1) the lysis buffer about 10 times of column volumes flows through gravity post, is repeated 2 times and (the most just can must enter by liquid stream in gravity post every time
Row adds liquid next time)；
2) the lysis buffer of the 20mM imidazoles about 10 times of column volumes flows through gravity post, is repeated 3 times (ibid)；
3) collect eluting mixing sample and wash miscellaneous effect for detection；
4) the Elution buffer about 1-2 times of column volume flows through gravity post, is repeated 3 times (ibid), is in charge of collection, uses bradford
Reagent detection eluent, understands albumen eluting the most completely.As containing albumen, then continue eluting, until completely；
5) detection: whether SDS-PAGE testing goal albumen exists；
6) dialysis bag filter dialyzed overnight eluent at 4 DEG C with molecular cut off as 3K, peripheral dialysis solution is PBS, is simultaneously introduced HRV3c
Protease restricted enzyme action fusion protein, produces 6*His-Nus.a fragment and purpose fragment Rv1098c；
7) digestion products and Ni-NTA resin 4 DEG C are fostered 30min, collect and penetrate sample acquisition purpose fragment Rv1098c, simultaneously
6*His-Nus.a, HRV3c and non-enzyme action fusion protein completely all will with Ni-NTA resin-bonded thus be eliminated；
8) more than HiTrap Q HP (GE Cat.NO 17-1154-01) purification to obtain albumen, it is thus achieved that high-purity sample.
The invention still further relates to the immunogenic application as detection mycobacterium tuberculosis of the described Rv1098c proteantigen, described application includes,
(1) it is individually used for this Rv1098c proteantigen detecting mycobacterium tuberculosis；
Or (2) are by this Rv1098c antigen and the detection antigen combination of existing mycobacterium tuberculosis, are used for detecting mycobacterium tuberculosis.
The invention still further relates to the application in preparing Mycobacterium tuberculosis detection kit of the described Rv1098c proteantigen, described application is,
(1) described Rv1098c proteantigen is prepared detection kit separately as detection antigen；
Or (2) are by described Rv1098c proteantigen and the collaborative combination of existing mycobacterium tuberculosis detection antigen, increase existing antigen immune detection standard
Exactness.
Described existing mycobacterium tuberculosis detection antigen is the ESAT-6 antigen in mycobacterium tuberculosis source, CFP10 antigen, PPD antigen, BCG
Antigen, LAM antigen, ES-31 antigen.
The invention still further relates to the single antigenic type Mycobacterium tuberculosis detection kit prepared by described Rv1098c proteantigen, described test kit bag
Contain,
(1) the Rv1098c proteantigen of detection effective dose concentration (preferred concentration is 75ug/ml)；
(2) necessary detectable and colour reagent.
The invention still further relates to the many antigen combination type Mycobacterium tuberculosis detection kit comprising described Rv1098c proteantigen, described test kit bag
Contain,
(1) the Rv1098c proteantigen of detection effective dose concentration (preferred concentration is 75ug/ml), and other antigens of detection effective dose；
(2) necessary detectable and colour reagent.
The invention still further relates to described unitary type or the application of combination type Mycobacterium tuberculosis detection kit, it is characterised in that described detection method
For,
(1) detection sample is the anticoagulated whole blood prepared by blood sample of patient to be measured or PBMC cell；
(2) Rv1098c proteantigen is as immunogen and anticoagulated whole blood or PBMC cell incubation 20-30 hour, individual hour of preferably 20-24
(3) detection is by the secretomotor cytokine of Rv1098c proteantigen, determines testing result, described cell with positive with reference to contrast
The factor is IFN-gamma, IFN-alpha, TNF-alpha, IL-2, IL-13, IP-10, IL-1ra, GM-CSF, MIP-1betta, IL-6,
IL-8, MCP-1, IL-10 and IL-12, preferably IFN-gamma, TNF-alpha, IL-2, IL-13, IP-10, IL-1ra, GM-CSF, MIP-1betta,
Further preferably IFN-gamma, TNF-alpha, IL-2, IL-13, most preferably IFN-gamma.
The invention still further relates to the application in preparing against mycobacterium tuberculosis vaccine of the described Rv1098c antigen.
Accompanying drawing explanation
Fig. 1 .Pdest17-Rv1098c expression vector digestion verification result, swimming lane A2 is the band after enzyme action.
Fig. 2 .Rv1098c protein S DS-PAGE detection figure, left side be Marker (unit K d),
Fig. 3. pulmonary's photo of mycobacterium tuberculosis counteracting toxic substances infection model Cavia porcellus
Fig. 4. the SABC photo of mycobacterium tuberculosis counteracting toxic substances infection model each tissue slice of Cavia porcellus
Detailed description of the invention
Embodiment 1.Rv1098c-pDEST17 expression vector establishment and the expression of target protein Rv1098c and renaturation (method one)
WithLREnzyme mix is catalyzedRv1098cEntry vector (U.S. Craig Ventor Institute divides into
PFGRC provided free) andpDESTTM17 carrier restructuring generate the expression vector of Rv1098c.
Concrete grammar:
The entry vector 1.5 μ L of A. cloning reaction system: Rv1098c,pDESTTM17 carrier 1 μ L,BP
II Enzyme mix 2.5μL；Reaction condition: 25 DEG C, reaction overnight.
B. method for transformation: add 100 μ L bacillus coli DH 5 alpha competence (making by oneself), after ice bath 30min, 42 DEG C of heat shock 90s in reaction system,
System places 10min on ice, after adding 200 μ L LB culture medium renaturation, is coated on the LB solid medium containing ampicillin (100mg/l)
On, cultivate 20h for 37 DEG C.
C. plasmid extraction: extract plasmid with the little extraction reagent kit of N96 high-purity plasmid (DP114), it is thus achieved that the expression vector of Rv1098c.
D. digestion verification: whether checking clone completes with BsrG1 cleavage reagent box (R0575S NEB) digested plasmid, and result is as shown in Figure 1
Result shows, endonuclease bamhi size is about 700bp, plasmid construction success.
The expression vector of the Rv1098c obtained is imported in rosetta (DE3) expression vector, method particularly includes:
A. take 1 μ L plasmid solution and add 100 μ rosetta (DE3) competence (making by oneself), after ice bath 30min, 42 DEG C of heat shock 90s,
B. system places 10min on ice, after adding 200 μ L LB culture medium renaturation, is coated on the LB solid containing ampicillin (100mg/l)
In culture medium, cultivate 12h for 37 DEG C；
C. Rv1098c protein expression is induced with 0.75mM IPTG.
2. ultracentrifugation after ultrasonication antibacterial, obtains inclusion body solid precipitation and collects albumen and renaturation step method particularly includes:
A. with re-suspension liquid: the resuspended antibacterial of PBS, under the conditions of 4 DEG C, sonicator ultrasonication antibacterial is used；
DEG C b.4,10000rpm is centrifuged 20min and collects precipitation and be solid forms inclusion body, to the electrophoresis result of the albumen collected as shown in Figure 2.
C. inclusion body is dissolved with lysate (PBS+10M carbamide)；Dialysis for the first time: the bag filter dialyzed overnight with molecular cut off as 3.5K
Being dialysed by albumen, dialysis solution composition is: PBS, 1M carbamide, 3mM L-Argine；
Dialysing for the second time, dialysis solution is PBS the most again, second time dialysis: albumen is dialysed by the bag filter dialyzed overnight with molecular cut off as 3.5K
To dialysis solution, the albumen that can be directly used for immunogen screening can be obtained.
Rv1098c expression vector establishment containing polyhistidine label of embodiment 2. and the expression of target protein Rv1098c and renaturation (method two)
1. express the subject fusion proteins containing polyhistidine label and collect host bacterial:
1) E.coli BL21 bacterial strain is used to express 6*His-Nus.a-Rv1098c fusion protein；
2) LB plate streaking activation (adding corresponding antibiotic), 37 DEG C stand overnight (about 12h)；
3) it is inoculated in 37 DEG C of 200rpm of 5ml LB liquid medium (adding corresponding antibiotic), cultivates to OD more than 0.6-0.8 (about 3h)；
4) being inoculated in 300ml LB fluid medium by inoculum concentration 1-3%, 37 DEG C, 200rpm cultivates to OD600 ≈ 0.6-0.8 (about
3h).Cool to 16 DEG C, add IPTG to final concentration 0.1mM；
5) 16 DEG C, 200rpm cultivates 16h, and 4 DEG C, 4000rpm is centrifuged 15min and collects thalline；
2. the collection subject fusion proteins containing polyhistidine label:
1) thalline is resuspended: every thalline about 40ml lysis buffer collected by 1L culture medium is resuspended (as added protease suppression
Agent PMSF, the most final concentration of 0.5-1mM；)
2) bacterial cell disruption: ultrasonic 3s, interval 9s, power are 38%, ultrasonic 30min.(suspension is bright for standard, takes the circumstances into consideration to adjust ultrasonic
Work total time) as used high pressure cell cracker to crush 1-2 time, required time about 10min；
3) bacterial debris is removed: 4 DEG C, 15000-18000rpm is centrifuged 20 minutes,
4) loading: above-mentioned supernatant fluid and column material Ni-NTA resin (Novagen Cat.NO 70691-5) 4 DEG C hatch about 1h-1.5h；
3. purification of target fusion protein:
3.1 wash foreign protein:
1) the lysis buffer about 10 times of column volumes flows through gravity post, is repeated 2 times and (the most just can must enter by liquid stream in gravity post every time
Row adds liquid next time)；
2) the lysis buffer of the 20mM imidazoles about 10 times of column volumes flows through gravity post, is repeated 3 times (ibid)；
3) collect eluting mixing sample and wash miscellaneous effect for detection；
3.2 destination protein eluting:
1) the Elution buffer about 1-2 times of column volume flows through gravity post, is repeated 3 times (ibid), is in charge of collection.Use bradford
Reagent detection eluent, understands albumen eluting the most completely.As containing albumen, then continue eluting, until completely；
2) detection: whether SDS-PAGE testing goal albumen exists；
3) dialysis bag filter dialyzed overnight eluent at 4 DEG C with molecular cut off as 3K, peripheral dialysis solution is PBS, is simultaneously introduced HRV3c
Protease restricted enzyme action fusion protein, produces 6*His-Nus.a fragment and purpose fragment Rv1098c；
4) digestion products and Ni-NTA resin 4 DEG C are fostered 30min, collect and penetrate sample acquisition purpose fragment Rv1098c, simultaneously
6*His-Nus.a, HRV3c and non-enzyme action fusion protein completely all will with Ni-NTA resin-bonded thus be eliminated；
5) more than HiTrap Q HP (GE Cat.NO 17-1154-01) purification to obtain albumen, it is thus achieved that high-purity sample.
The immunogenicity test of embodiment 3. target protein Rv1098c
In order to detect the immunogenicity of Rv1098c and for the reinforced effects of existing antigen detection kit, for clinical from BJ Chest Science Hospital
The multiple cases made a definite diagnosis have carried out the screening of further Detection of antigen.
Test blood sample: go to a doctor patient from BJ Chest Science Hospital
Positive control agent and consumptive material: T-SPOT test kit (10 pairs of antigenic reagent box of ESAT-6 and CFP), Oxford immunotec (Britain)
Product,
Negative control: Rv2327 proteantigen, another section randomly selected derives from the known protein fragments Rv2327 of mycobacterium tuberculosis, its core
Nucleotide sequence and aminoacid sequence are respectively as shown in SEQ ID NO.3 and SEQ ID NO.4
SEQ ID No.4
SEQ ID No.3
Experimental technique and evaluation methodology are as follows:
1) heparin anti-coagulating, anticoagulation sample by volume 1:1 Yu RPMI1640 mixes；Blood sample is carefully added in Ficoll in 2-3:1 ratio drench
Bar cell separation liquid upper strata；
2) 1000g is centrifuged 22 minutes；Horizontal rotor, slow liter delays fall；
3) transfer to add 10ml AIM-V from Ficoll separation pipe by mononuclear cell layer (being positioned at centrifuge tube intermediate layer, for the tunica albuginea shape of layer)
The aseptic 15ml centrifuge tube of culture fluid, mixes gently, and room temperature 600g is centrifuged 7min；
4) carefully remove supernatant, add 1ml AIM-V, light and slow re-suspended cell, add AIM-V culture fluid and be centrifuged 7min to 10ml, 350g；
5) carefully abandon supernatant, add 1ml AIM-V culture fluid re-suspended cell；
6) cell counting and dilution, takes the 1%trypan blue that 10 μ l cell suspension add 40 μ l, prepares 1:5 cell diluent, carries out
Viable count, calculates volume that cell dilution needs and adds corresponding AIM-V serum-free medium and prepare cell diluent, and test kit detection is wanted
Seek addition every hole (24 hole pvdf membrane plate) cell quantity 2.5 × 105；
7) culture plate is taken out from aluminum envelope, is sequentially added into:
50 μ l AIM V are to negative control hole；50 μ l antigen ESAT-6 are to antigen A hole；50 μ l antigens c FP 10 to antigen B holes；50 μ l are positive
Comparison is to Positive control wells；Above-mentioned 4 holes are separately added into and prepare cell dilution working solution 100 μ l.The Sample that 50 μ l embodiments 2 prepare
Protein (initial concentration is 60ug/ml) joins in sample well, is subsequently added culture medium and cell, Sample protein in sample well
The 1/3 of final concentration of initial concentration.
8) culture plate being put into 37 DEG C, 5%CO2 incubator hatches 16-20hrs.
9) fresh enzyme labelled antibody working solution is prepared with aseptic PBS by 1:200.Take out culture plate from incubator, discard liquid in hole.With 200 μ
The aseptic PBS of l/well washs 4 times.
10) add 50 μ l/well enzyme labelled antibody working solutions, culture plate is hatched 1 hour at 2-8 DEG C.Wash 4 times with PBS and remove uncombined enzyme
Labeling antibody.
11) every hole adds the substrate working solution 50 μ l that equilibrium at room temperature is crossed, room temperature reaction 7 minutes.Reaction is terminated, at 37 DEG C with distilled water flushing
2-3hrs or ambient temperature overnight desiccation culture plate.
Immunogen test result and analysis:
Immunogenicity test is carried out in Molecular Biology Lab of chest hospital, and the tuberculosis patient of a number of clinical definite is selected in test, logical
Cross the blood sample using various proteantigen to detect above-mentioned patient diagnosed, and the thus immunogenicity of interpretation of result Rv1098c proteantigen and application side
To, concrete testing result statistics see table shown in 1,
Table 1.Rv1098c antigen has made a definite diagnosis the mycobacterium tuberculosis testing result of tuberculosis patient to being in hospital
* note: reactionless response patient is different from T-spot test kit detection nonreply object.
Having 12 examples is that the detection of T-spot test kit is come to nothing, but Rv1098c can filter out.The result of Rv1098c is better than the inspection of T-spot
Surveying result, during Rv1908c and T-spot combination, Detection results is optimal.
Compared to the testing result with commercially available T-spot test kit, Rv1098c proteantigen high response rate higher, nonreply response rate is lower,
Therefore, Rv1098c immunogen can more effectively detect mycobacterium tuberculosis compared to commercially available double antigen detection kits, if positive anti-with other
Former combination, it is possible to more effectively improve recall rate.
The immunogenicity detection of embodiment 4. target protein Rv1098c
1. the foundation of guinea pigs model
Taking Cavia porcellus, use the hypodermic mode of groin to inject m tuberculosis infection source, injection dosage is 0.2ml, counteracting toxic substances dosage 4x106~
4x107CFU。
Counteracting toxic substances puts to death Cavia porcellus, lung organ's infection conditions of Cavia porcellus in detection infectable infection source after completing, and takes linked groups's section and carry out SABC
Analyze.
Fig. 3 is shown in by pulmonary's photo of infection model Cavia porcellus, it is seen then that obvious inflammatory reaction occurs in the Cavia porcellus pulmonary after counteracting toxic substances.
The immunohistochemical analysis photo of infection model each tissue slice of Cavia porcellus is shown in that Fig. 4, internal organs section result show,
There is interstitial pneumonia (30-40%), interstitial lung congestion (40-50%) in Cavia porcellus pulmonary after counteracting toxic substances；
Visible epithelioid cell tuberosity in the red pulp of spleen local, a small amount of lymphocyte infiltration of periphery, caseous necrosis stove seen from center；
There is hepatic congestion (20-30%) in liver, and partially visible little granulation forms (1-5%).
The above results shows, the Cavia porcellus after mycobacterium tuberculosis counteracting toxic substances occurs in that obvious infection symptoms, model construction success
2. immunogenicity detection
Take successfully counteracting toxic substances modeling after Cavia porcellus two, the antigen protein that the embodiment 2 respectively at subcutaneous injection variable concentrations prepares, little in 72
Observing the immunoreation inflammatory plaques size that antigen causes time after, result is as shown in the table, and result shows, similar with positive control, and Rv1098c resists
The former immunoreation that also can cause the Cavia porcellus infecting mycobacterium tuberculosis, illustrates that this antigen has the ability as mtb vaccine.
| Sample | 72h transverse diameter X indulges footpath (mm) |
| PBS (negative control) | - |
| BCG-PPD 20ug/ml (positive control) | 13x18 |
| Rv1098c 25ug/ml | 9x10 |
Last it should be noted that above example is intended for skilled artisan understands that the essence of the present invention, it is not used as this law protection domain
Limit.
Claims (10)
1. a specific proteins antigen for mycobacterium tuberculosis, described antigen sequence is as shown in SEQ ID NO.2.
2. shown in a specific proteins antigen for mycobacterium tuberculosis, described antigen sequence and SEQ ID NO.2 between sequence, have more than 95% same
Source property.
3. the nucleotide fragments of antigen described in coding claim 1 or 2.
4. the preparation method of the antigen described in claim 1 or 2, it is characterised in that described method is:
Method one:
Step (1) withThe entry vector of catalysis Rv1098c is (under U.S. Craig Ventor Institute
If PFGRC provided free) andCarrier restructuring generates the expression vector of Rv1098c；
The expression vector of step (1) described Rv1098c is converted objective expression host by step (2), expresses target Rv1098c albumen；
Step (3) smudge cells collects albumen renaturation target protein；
Method two:
Step (1) is expressed the subject fusion proteins containing polyhistidine label and collects expressive host thalline:
Step (2) collection subject fusion proteins containing polyhistidine label:
Step (3) purification of target fusion protein.
5. the antigen described in claim 1 or 2 is as the immunogenic application of detection mycobacterium tuberculosis, and described application includes,
(1) it is individually used for this proteantigen detecting mycobacterium tuberculosis；
Or (2) are by this antigen and the detection antigen combination of existing mycobacterium tuberculosis, are used for detecting mycobacterium tuberculosis.
6. the single antigenic type Mycobacterium tuberculosis detection kit prepared by the antigen described in claim 1 or 2, described test kit comprises,
(1) antigen of detection effective dose concentration (preferred concentration is 75ug/ml)；
(2) necessary detectable and colour reagent.
7. comprising many antigen combination type Mycobacterium tuberculosis detection kit of antigen described in claim 1 or 2, described test kit comprises,
(1) antigen described in strong request 1 or 2 of detection effective dose concentration (preferred concentration is 75ug/ml), and the existing tuberculosis of detection effective dose
Mycobacteria detection antigen；
(2) necessary detectable and colour reagent.
Application the most according to claim 5, it is characterised in that the described ESAT-6 that existing mycobacterium tuberculosis detection antigen is mycobacterium tuberculosis source
Antigen, CFP10 antigen, PPD antigen, BCG antigen, LAM antigen, ES-31 antigen.
Test kit the most according to claim 7, it is characterised in that described existing mycobacterium tuberculosis detection antigen is mycobacterium tuberculosis source
ESAT-6 antigen, CFP10 antigen, PPD antigen, BCG antigen, LAM antigen, ES-31 antigen.
10. the application in preparing against mycobacterium tuberculosis vaccine of the antigen described in claim 1 or 2.
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| CN111574600A (en) * | 2020-05-08 | 2020-08-25 | 华南农业大学 | A kind of Mycobacterium tuberculosis PPE10 protein and its preparation method and application |
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2015
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| CN111574600A (en) * | 2020-05-08 | 2020-08-25 | 华南农业大学 | A kind of Mycobacterium tuberculosis PPE10 protein and its preparation method and application |
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