CN106053800A - Tuberculosis serological diagnostic kit - Google Patents
Tuberculosis serological diagnostic kit Download PDFInfo
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- CN106053800A CN106053800A CN201610367677.8A CN201610367677A CN106053800A CN 106053800 A CN106053800 A CN 106053800A CN 201610367677 A CN201610367677 A CN 201610367677A CN 106053800 A CN106053800 A CN 106053800A
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
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- G—PHYSICS
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Abstract
The invention provides a tuberculosis serological diagnostic kit, at least comprising a coating solution, a washing solution, a sealing solution and a stop solution and also comprising the following components: recombinant protein rRv0220, having an amino acid sequence shown as in SEQ ID NO. 1; recombinant protein rRv2958c, having an amino acid sequence shown as in SEQ ID NO. 2. Both the two recombinant proteins provided herein have good immunoreactivity and can specifically bind with mouse-produced antibodies induced by the proteins. Both the two recombinant proteins can specifically bind with serum of a tuberculosis patient; indirect ELISA detection shows that when compared with colloidal gold process (38-16 KDa), rRv0220 has specificity, sensitivity and total accordance respectively of 98.6%, 98.3% and 98.4%, and rRv2985c has specificity, sensitivity and total accordance respectively of 100%, 96.5% and 98.4%, and it is indicated that the two recombinant proteins have high sensitivity and specificity and can function as preferred antigens for tuberculosis serological diagnosis.
Description
Technical field
The present invention relates to a kind of detection kit that can be used in tuberculosis serological diagnosis, particularly relating to two kinds can be with knot
The antigen of mycobacterium tuberculosis of core patient's serum generation specific bond, belongs to biological engineering and medical diagnosis on disease technical field.
Background technology
Tuberculosis (Tuberculosis, TB) is one of topmost infectious disease threatening human health, by infecting
Pathogen mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) infects and causes.MTB be broadly divided into human-like,
Cattle type, Africa type and Mus type, people's paratuberculosis more than 90% is caused by infecting human-like MTB, and minority is caused by cattle type and Africa type.
MTB can invade each tract of whole body, and disease sites is most commonly seen with lungs, it is possible to involves other organs such as liver, bone and brain.
MTB infects and is divided into latency and activeness two states.The source of infection of TB is mainly the lunger of activeness, and it mainly leads to
Cross respiratory infectious, it is also possible to the route infection such as excessively damaged skin, mucosa and digestive tract.After MTB carrier infected by HIV, by
Developing into the probability of active tuberculosis incubation period far above being uninfected by HIV person, its disease is faster.Within 2014, the world defends
Raw tissue is announced the whole world about 9,600,000 and is newly sent out TB case, and 1,100,000 people die from TB, and wherein 400,000 is that HIV is positive.
At present, laboratory diagnostic method lungy mainly has following several: (1) bacteriodiagnosis: Sputum smears Microscopical Method For Detection
Simply, quickly and reliable, but its sensitivity is poor；This cultivation of sputum sample is the goldstandard of diagnosis of tuberculosis, but when conventional solid is cultivated
Between need 60～90d, liquid culture and drug sensitive test about need 20d, and the Sputum smears specimen inspection of the adult pulmonary tuberculosis patient of about 40%
Survey as feminine gender.(2) serodiagnosis: TB serological diagnostic method carries out detecting specificity in serum mainly by immunological technique
The antibody of anti-MTB.(3) tuberculin skin test: the body infecting MTB produces corresponding primed lymphocyte, again notes
After penetrating PPD, body can be in the allergy of 2～3d generation delayeds after skin test is tested, and red and swollen rhabdion occurs in local.This examination
Test the clinical diagnosis being directly applied to TB, but its poor specificity, often with environment mycobacteria and BCG generation cross reaction.(4)
MTB nucleic acid amplification detects: the method sensitivity is good, even if the antibacterial that in specimen, copy number is low also can be detected, but clinical practice
Having deviation, as sample disposal is improper, target sequence or amplified production cross-contamination etc. easily cause false negative.(5) IFN-γ release
Test: the method is the new method of ion vitro immunization detection, and its commercialization diagnostic kit is using EAST6 and CFP10 albumen as thorn
Swash antigen, typically detect body whole blood or peripheral blood mononuclear by enzyme-linked immunosorbent assay or ELISpot method
The T cell quantity of the IFN-γ produced in cell and cytokine variable quantity.
In these diagnostic methods, Serologic detection is the effective ways of in-vitro diagnosis TB, its detection method is simple, quickly,
Efficiently with cheap, become the study hotspot of TB diagnosis in recent years.The method obtains recombinant antigen by genetic engineering, in conjunction with indirectly
The antibody of the anti-MTB in ELISA method detection patients serum, demonstrates superior diagnostic potential in the serodiagnosis of TB,
Although at present existing commercial kit based on recombinant antigen combination, such as colloidal gold method test kit TB38-16KD, but it is sensitive
Property and specificity need to be improved further, and can not diagnose TB incubation period in Clinical Laboratory.Therefore, find high specificity and
The MTB antigen that sensitivity is high is the key improving TB Serologic detection positive rate.
Summary of the invention
The present invention solves the deficiency in background technology, it is provided that a kind of for the serodiagnostic ELISA kit of TB,
Two kinds of recombiant proteins (antigen) in this test kit all can be with tuberculosis patient serum generation specific bond, sensitivity and special
Property high, have a good application prospect.
Realizing the technical scheme that above-mentioned purpose of the present invention used is:
A kind of tuberculosis serological diagnosis test kit, at least includes in this test kit being coated liquid, cleaning mixture, confining liquid and end
Only liquid, also comprises following components: recombiant protein rRv0220, its aminoacid sequence is as shown in SEQ ID NO.1 in test kit；Weight
Histone rRv2958c, its aminoacid sequence is as shown in SEQ ID NO.2.The present invention the most successfully constructs 2 kinds of prokaryotic expression weights
Group carrier pET-28a/Rv0220 and pET-28a/Rv2958c, after it being transformed into respectively in escherichia coli E.coil BL21,
Go out relative molecular weight with IPTG abduction delivering respectively and be about two kinds of recombination fusion proteins of 46kDa and 50kDa (with inclusion body state
Express), after nickel post affinity purification, obtain the recombiant protein of purification.
Described being coated the carbonate buffer solution that liquid is 0.05M, its pH is 9.6；Described cleaning mixture is containing 0.05%
The PBST buffer of Tween-20, its pH is 7.6；Described confining liquid is the PBS containing 5%BSA；Described stop buffer is
The H of 2mol/L2SO4 solution.
Recombiant protein rRv0220 and rRv2958c provided by the present invention all can occur special from different TB patients serums
In conjunction with, detecting through indirect elisa method, compare with colloidal gold method (38-16KDa), the specificity recording rRv0220 is 98.6%,
Sensitivity is 98.3%, and total coincidence rate is 98.4%；The specificity of rRv2985c is 100.0%, and sensitivity is 96.5%, always accords with
Conjunction rate is 98.4%；Illustrate that two kinds of recombiant proteins have good immunoreactivity, it is possible to serodiagnostic preferably as TB
Antigen.
Accompanying drawing explanation
Fig. 1 is PCR primer 1.0% agarose electrophoretic analysis figure in embodiment；
Fig. 2 is two kinds of recombiant plasmid double digestion 1.0% agarose electrophoretic analysis figures in embodiment；
Fig. 3 is two kinds of recombiant plasmid expression figure in E.coliBL21 in embodiment；
Fig. 4 is the purification figure of two kinds of recombiant proteins in embodiment；
Fig. 5 is the immunoreactivity result figure of Western Blot method detection recombiant protein and clinical serum；
Fig. 6 is the immunoreactivity result figure of ELISA method detection recombiant protein and clinical serum.
Detailed description of the invention
Below in conjunction with specific embodiment the present invention done detailed specific description, but protection scope of the present invention not office
It is limited to following example.
One, the structure of prokaryotic expression recombinant vector
1, the PCR amplification of genes of interest
(1) design of genes of interest specific primer and synthesis
DNA sequence according to two kinds of genes of Rv0220 and Rv2958c in GenBank separately designs upstream and downstream primer (lower stroke
Line is restriction enzyme digestion sites), upstream and downstream primer, restriction enzyme site, product length and the annealing temperature of each gene are as follows
Shown in table:
(2) the pcr amplification reaction system of genes of interest and program
With the genomic DNA of TB as template, use PyrbestTMHigh-fidelity DNA polymerase carries out PCR amplifying target genes.
PCR reaction system is: PyrbestTMEach 1 μ L, MTB DNA profiling 2 μ L of DNAMix 12.5 μ L, upstream and downstream primer, deionization
Water 8.5 μ L.The Amplification of Rv0220 is: 98 DEG C of 30s, 98 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 30s, totally 35 circulations；72 DEG C are prolonged
Stretch 2min.The Amplification of Rv2958c is: 98 DEG C of 30s；98 DEG C of 10s, 44 DEG C of 30s, 72 DEG C of 30s, totally 35 circulations；72 DEG C are prolonged
Stretch 2min.Amplified production is through 1.0% agarose gel electrophoresis observed result and preserves.
Result shows as it is shown in figure 1, amplify the PCR primer that size is 1212bp and 1287bp respectively, respectively with
The clip size of Rv0220 with Rv2958c gene is consistent.Then use DNA purification kit that PCR primer is purified recovery.
2, carrier is identified and purification recovery with the double digestion of genes of interest
(1) the double digestion system of prokaryotic plasrnid pET-28a and genes of interest Rv0220 and Rv2958c
(2) purification after prokaryotic plasrnid pET-28a and genes of interest double digestion reclaims
Adding each composition by 25 μ L enzyme action systems, 600rpm point, from 10s, puts to 37 DEG C of water baths insulation enzyme action 4h, so
Rear 65 DEG C of water-bath 15min terminate endonuclease reaction.Genes of interest after purification double digestion and prokaryotic plasrnid pET-28a.
3, carrier and the connection of genes of interest
Linked system is as follows:
By above-mentioned system, genes of interest good for purification and plasmid double digestion product and each reactant it is added to 0.1mL EP pipe
In, put after mixing from, 22 DEG C connect 3h
4, convert
(1) take the 10 μ L connection product through 65 DEG C of inactivation 10min, join in competent cell and mix, ice bath 30min；
(2) bacterium solution is placed on heat shock 90s in 42 DEG C of water baths, rapid ice bath 3min；
(3) conversion product will add 800 μ L LB fluid medium mixings, 180rpm, 37 DEG C of shaken cultivation 1h, 4 DEG C,
14000rpm is centrifuged 1min；
(4) suck 800 μ L of supernatant, residual components is blown and beaten gently, resuspended thalline；
(5) resuspended bacterium solution is uniformly laid on LB solid medium (containing kanamycin), is inverted overnight incubation for 37 DEG C.
5, the screening of positive colony and qualification
(1) screening of positive colony
After incubated overnight, observe the single bacterium colony grown on LB solid medium (containing kanamycin), 5 lists of random picking
Bacterium colony, be inoculated in respectively 1mL containing kanamycin LB fluid medium in, 37 DEG C, 220rpm shaken cultivation overnight；
(2) qualification of positive colony
Two kinds of recombiant plasmid double digestion 1.0% agarose electrophoretic analysis figures as in figure 2 it is shown, in Fig. 2, M:DNA Marker；
1:pET-28a/Rv0220PCR product；2:pET-28a/Rv2958c PCR product.
The sequencing result of recombiant plasmid is carried out BLAST analysis, each recombiant plasmid gene Rv0220 that result display builds
It is 100% with the DNA sequence coincidence rate of two kinds of genes of Rv2958c Yu GenBank announcement, shows that construction of recombinant plasmid becomes
Merit.
Two, the expression of two kinds of recombiant proteins, purification and qualification
1, the abduction delivering of RT-PCR expression vector
(1), after construction of recombinant plasmid checks order successfully, take respectively containing recombiant plasmid pET-28a/Rv0220, pET-28a/
On the LB solid medium (containing kanamycin) that the E.coli BL21 of Rv2958c is inoculated in, 37 DEG C of overnight incubation；
(2) respectively picking list colony inoculation in LB fluid medium, 37 DEG C, 220rpm shaken cultivation overnight；
(3) picking recombinant bacterium Rv0220 is after cultivating, and adds IPTG to final concentration of 2mmol/L, abduction delivering at 37 DEG C
3h.Recombinant bacterium Rv2958c, after cultivating, adds IPTG extremely final concentration of 1.5mmol/L, inducing culture 6h at 37 DEG C.
(4) being centrifuged collection bacterial sediment and be resuspended in PBS, adding lysozyme to whole content is 4.0g/L；After ice bath 2h
Ultrasonic disruption bacterium solution (each 10s, intermittently 10s；Amount to 5 times).Collect upper cleer and peaceful precipitation after Li Xin respectively, take 32 μ L of supernatant with
5 × SDS sample-loading buffer of 8 μ L uniformly mixes；Bacterial sediment adds 40 μ L PBS and 5 × SDS sample-loading buffer of 10 μ L
Mixing, boil 5min, carry out PAGE gel electrophoresis, coomassie brilliant blue staining with observe recombiant protein rRv0220 and
The existence of rRv2958c.
Albumen optimum condition of the expression is groped by temperature, IPTG concentration and time when changing induction, rRv0220,
RRv2958c all expresses with inclusion bodies.Two kinds of recombiant plasmid expression figure in E.coliBL21 is as shown in Figure 3.37℃、
When 2.0mmol/LIPTG, induction 3h, rRv0220 expression is the highest；37 DEG C, 1.5mmol/LIPTG, induction 6h time rRv2958c
Expression is the highest.
3, the qualification of recombiant protein
(1) by each recombiant protein through SDS-PAGE electrophoresis, condition setting is 90V electrophoresis 30min, treats that sample enters separation gel
After, voltage is adjusted to 120V, after electrophoresis to correct position, cuts glue respectively at the corresponding molecular weight of each recombiant protein；
(2) with half-dried transferring film instrument, the albumen on SDS-PAGE glue is transferred on pvdf membrane through 90mA 2h；
(3) transferring film is complete, film face down is soaked in the confining liquid containing 5%BSA, closes overnight for 4 DEG C；
(4) add TBST buffer and wash film 3 times, each 15min；
(5), during pvdf membrane is soaked in mouse anti-His tag monoclonal antibody (1:15 000 dilutes), incubated at room 2 is little
Time；
(6) add TBST buffer and wash film 5 times, each 15min；
(7) sheep anti-mouse igg antibody (1:20 000 dilutes) incubated at room 45min of horseradish peroxidase-labeled is added；
(8) add TBST buffer and wash film 5 times, each 15min；
(9) use ECL chemiluminescent reagents, use Full-automatic chemiluminescence image analysis system take pictures and stay figure.
4, the extensive preparation of recombiant protein
Express on a large scale according to protein expression condition each in upper step.10 000rpm are centrifuged 15min, collect thalline
Precipitation.Add appropriate PBS washing thalline 2 times, add 8mLPBS buffer by every gram of wet thallus after washing, add lysozyme
To final concentration of 4.0g/L；It is interior in shaking table shaken overnight, ultrasonic disruption (each 10s, intermittently 10s that sample is placed in ice chest；Amount to
60 times).4 DEG C, 12 000rpm are centrifuged 20min and collect inclusion body precipitation.
5, inclusion body washing and dissolving
(1) take the inclusion body that ultrasonic disruption separates, add inclusion body cleaning mixture I (4%TritonX-100,60mmol/L
EDTA, 1.5mol/L NaCl, pH=7.0), vortex oscillation mixes, shaking table vibration 30min in placement ice chest, 4 DEG C, and 10
000rpm is centrifuged 15min, collects precipitation；
(2) inclusion body cleaning mixture II (0.1mol/L Tris-Cl, 20mmol/L EDTA, 2mol/L Urea, pH=are added
7.0), mixing, ice bath shaking table vibration 30min, 4 DEG C, 10 000rpm are centrifuged 15min, collect the inclusion body after washing；
(3) inclusion body after washing is resuspended in lysate (0.1mol/L Tris-Cl, 8mol/L Urea, 5mmOl/L DTT,
PH=8.0), incubated at room overnight, makes inclusion body fully dissolve.
6, the degeneration purification of recombiant protein
(1) obtaining the inclusion body solution after dissolving, 4 DEG C, 10000rpm is centrifuged 15min and collects supernatant；
(2) with Buffer B buffer (8mol/L Urea, 100mmol/L NaH2PO4, 10mmol/L Tris-Cl, pH
=8.0) pre-equilibration Ni-NTA post 2min, discards effluent, with PBS by pillar rinse 2 times；
(3) the solubilization of inclusion bodies liquid supernatant collected is added Ni-NTA post, shaking table vibration 1h, make the abundant hanging column of albumen；
(4) with Buffer C buffer (8mol/L Urea, 100mmol/L NaH2PO4, 10mmol/L Tris-Cl, pH
=6.3) wash post, collect effluent, cyclic washing 3 times；
(5) with Buffer D buffer (8mol/L Urea, 100mmol/LNaH2PO4, 10mmol/L Tris-Cl, pH=
5.9) wash post, collect effluent, cyclic washing 3 times；
(6) with Buffer E buffer (8mol/L Urea, 100mmol/LNaH2PO4, 10mmol/L Tris-Cl, pH=
4.5) carry out eluting, collect effluent, cyclic washing 3 times.
Collect a large amount of abduction delivering rRv0220、rRv2958c, ultrasonication thalline obtains inclusion body, scrubbed molten with degeneration
After solution, upper Ni-NTA post, uses Buffer B、BufferC, Buffer D and Buffer E carries out eluting.Result shows and all can obtain
The rR that purity is higherv0220、rRv2958c, as shown in Figure 4.RR after purificationv0220, rRv2958c is through bag filter dialysis renaturation,
PEG800 concentrates, and is respectively 1100 μ g/mL, 2200 μ g/mL by BCA protein quantification kit measurement concentration.
7, recombiant protein dialysis renaturation
(1) respectively rRv0220, rRv2958c after purification is added in bag filter, with dialysis solution (50mmol/L NaCl,
0.5mmol/L EDTA, 50mmol/L Tris, 1mmol/L GSH, 0.1mmol/L GSSG, 50% glycerol, pH=8.0) in 4
DEG C dialysed overnight；
(2) dialysis solution is replaced by (4mol/L Urea, 2mmol/L GSH, 0.2mmol/L GSSG, PBS pH=7.4), and 4
DEG C dialysed overnight；
(3) in dialysis cup, it is dividedly in some parts the PBS of common 1L, slowly reduces Urea concentration and make recombiant protein naturally multiple
Property；
(4) finally the albumen after renaturation is concentrated with Polyethylene Glycol PEG800.
8, sum up
The present embodiment successfully constructs two kinds of prokaryotic expression carrier pET-containing MTB Rv0220 and Rv2958c gene
28a/Rv0220 and pET-28a/Rv2958c.Respectively the expression condition of two kinds of recombiant proteins rRv0220, rRv2958c is carried out
Grope and optimize, content include derivant IPTG concentration (0.2mmol/L, 0.5mmol/L, 1.0mmol/L, 1.5mmol/L and
2.0mmol/L), inducing temperature (16 DEG C, 28 DEG C, 30 DEG C and 37 DEG C) and induction time (3h, 6h, 8h, 12 and 24h).Result shows
Show: rRv0220 is 2.0mmol/L in IPTG concentration, when 37 DEG C, induce 3h expression in precipitation the highest, without table in supernatant
Reach；RRv2958c is 1.5mmol/L in IPTG concentration, induces 6h expression in precipitation the highest, without table in supernatant when 37 DEG C
Reach.Illustrating that two kinds of recombiant proteins are all expressed with inclusion bodies, the possible cause that restructuring destination protein forms inclusion body is as follows:
(1) when destination protein rRv0220, rRv2958c of a large amount of synthesis, during protein folding, in E. coli BL21
The factor that participation albumen correctly folds can not meet demand, therefore produces inclusion body；(2) express as E. coli BL21
During to its virose albumen, the inclusion body that rRv0220, rRv2958c are formed is a kind of autoprotection of expressive host bacterium
Behavior.The present invention improves by the following method: (1) reduces IPTG inducer concentration；(2) inducing temperature is reduced；(3) again
Extract the successful positive bacteria recombiant plasmid that checks order, change prokaryotic expression bacterium E.coliRosetta.But recombiant protein is still with inclusion body
Form is expressed.Therefore, the present invention uses the lysate containing 8M carbamide to dissolve inclusion body rRv0220, rRv2958c, through Ni
The recombiant protein of column purification is Denatured protein, and Denatured protein is inserted bag filter, and bag filter is placed in containing oxidisability and reproducibility paddy
The dialysis solution of the sweet peptide of Guang so that it is be folded into correct space structure to recover the biologic activity of recombiant protein.
Three, the immunocompetence research of recombiant protein
The female BAl BIc of 30 8 week old/c mice is randomly divided into PBS group, rRv0220 group and rRv2958c group.Through experiment
Research, rRv0220 and rRv2958c is respectively provided with good immunogenicity, can produce specific antibody by inducing mouse.Mice serum
Middle specific IgG level increases in rising trend with immune time.After final immunization mice two weeks, rRv0220 and rRv2958c
The titer of group immune serum IgG antibody is respectively 1: 12 800 and 1: 12 800；Detect through Westernblot, two kinds of weights
Histone all can be with the mice serum generation specific bond of final immunization.
Sum up: by rRv0220, rRv2958c recombiant protein through subcutaneous multi-point injection method immunization. Female BALB/c mouse, send out
Existing rRv0220 and rRv2958c albumen is respectively provided with good immunogenicity, can effectively induce immune mouse to produce corresponding antibody.
After immune 6th week, IgG antibody titer is respectively 1: 12800 and 1: 12800, hence it is evident that higher than matched group (P < 0.01).Western
The result of blot detection shows, two kinds of recombiant proteins all can be with the mice serum generation specific bond of immunity.
Four, the immunoreactive clinical evaluation of recombiant protein
1, human serum and source employed in the present embodiment are as follows:
(1) TB positive serum (Sputum smears acid-fast stain is positive, and detects its anti-MTB antibody positive through colloidal gold method) 70
Part, from clinical laboratory of the People's Hospital of Hengyang City the 3rd；
(2) mycoplasma pneumoniae positive serum (detecting its anti-MTB antibody for feminine gender through colloidal gold method) 32 parts, by University Of Nanhua
Attached First Hospital clinical laboratory provides；
(3) (Sputum smears acid-fast stain is negative to TB negative serum, and confirms that MTB antibody and pneumonia are propped up former through colloidal gold method
Body antibody is feminine gender) 57 parts, from clinical laboratory of the People's Hospital of Hengyang City the 3rd.
2, Western Blot detection recombiant protein and the immunoreactivity of clinical serum
By 8 parts of TB positive serum mixing, the dilution proportion using 1: 200 resists as one, and Western Blot identifies restructuring egg
White rRv0220, rRv2958c and the reactivity of tubercular's serum.
(1) each albumen is carried out on PAGE gel electrophoresis, 90V electrophoresis 30min, after sample enters separation gel,
Adjustment voltage, to 120V electrophoresis to invention required time, cuts glue respectively at the corresponding molecular weight of destination protein；
(2) taking pvdf membrane slightly larger than the size of glue, pvdf membrane soaks 10s before use in methanol, then pvdf membrane, turn
Film filter paper and the gel cut all are soaked in half an hour in transferring film buffer.With half-dried transferring film instrument by the albumen on SDS-PAGE glue
It is transferred on pvdf membrane through 90mA 2h；
(3) transferring film is complete, closes overnight with the confining liquid containing 5%BSA 4 DEG C；
(4) add TBST buffer and wash film 3 times, each 15min；
(5) pvdf membrane combines, incubated at room 2h with clinical serum (1: 200 dilution)；
(6) add TBST buffer and wash film 5 times, each 15min；
(7) goat anti-human igg's (1: 10 000 dilution) of HRP labelling, incubated at room 45min are added；
(8) add TBST buffer and wash film 5 times, each 15min；
(9) the chemiluminescence agent of enhancement mode ECL is uniformly added on pvdf membrane, uses Full-automatic chemiluminescence graphical analysis system
System is taken pictures and preserves.
3, indirect elisa method evaluates the immunoreactivity of recombiant protein and clinical serum
With purification, identify and measure after rRv0220, rRv2958c be antigen coated ELISA microwell plate, with clinic collect
Tuberculosis positive serum, tuberculosis negative serum and mycoplasma pneumoniae positive serum be one resist, indirect elisa method detection clinical serum
Middle specific IgG, specifically comprises the following steps that
(1) it is coated: respectively the carbonate buffer solution of rRv0220, rRv2958c pH 9.6 of purification is diluted to respectively
Concentration is 10 μ g/mL, 100 μ L/ holes, is coated overnight in 4 DEG C；
(2) washing: fully get rid of liquid in ELISA microwell plate, adds PBST buffer (containing 0.05%Tween-20's
PBS, pH 7.6), 100 μ L/ holes are washed 3 times, pat dry；
(3) close: add the PBS containing 5%BSA according to 200 μ L/ holes, close 2h for 37 DEG C；
(4) washing: repeat step (2)；
(5) hatch one to resist: added afterwards in microwell plate by clinical serum dilution (1: 500), 100 μ L/ holes, hatch 2h for 37 DEG C；
(6) washing: repeat step (2)；
(7) hatch two to resist: the goat anti-human igg antibody of HRP labelling is diluted (1: 10 000), add in microwell plate, 50 μ L/
Hole, hatches 1h for 37 DEG C；
(8) washing: repeat step (2)；
(9) colour developing: add TMB developer A liquid and each 50 μ L of B liquid, add stop buffer after lucifuge colour developing 10min and terminate reaction；
(10) measure: measure the A respectively treating gaging hole by microplate reader450Value；Negative control is done, with it with tuberculosis negative serum
Average A450+ 2S is positive marginal value, measures hole A450Value is positive more than positive dividing value, is otherwise judged to feminine gender；According to each restructuring
The response situation of each clinical serum of albumen, evaluates the immunoreactivity of recombiant protein.
4, recombiant protein immunoreactive clinical evaluation result
It is respectively adopted the immunoreation of Western Blot and ELISA method detection rRv0220, rRv2958c and clinical serum
Property, as shown in Figure 5 and Figure 6, result shows that rRv0220, rRv2958c have higher for detecting clinical serum to testing result
Specificity and sensitivity: compare with colloidal gold method (38-16KDa), the specificity of rRv0220 is 98.6%, and sensitivity is
98.3%, total coincidence rate is 98.4%；The specificity of rRv2985c is 100%, and sensitivity is 96.5%, and total coincidence rate is
98.4%.Testing result is as shown in the table:
The Sensitivity and Specificity of indirect elisa method detection clinical serum
Claims (5)
1. a tuberculosis serological diagnosis test kit, at least includes in this test kit being coated liquid, cleaning mixture, confining liquid and termination
Liquid, it is characterised in that also comprise following components in test kit: recombiant protein rRv0220, its aminoacid sequence such as SEQ ID NO.1
Shown in；Recombiant protein rRv2958c, its aminoacid sequence is as shown in SEQ ID NO.2.
Tuberculosis serological diagnosis test kit the most according to claim 1, it is characterised in that being coated liquid described in: is 0.05M
Carbonate buffer solution, its pH is 9.6；Described cleaning mixture is the PBST buffer containing 0.05%Tween-20, and its pH is
7.6；Described confining liquid is the PBS containing 5%BSA；Described stop buffer is the H of 2mol/L2SO4 solution.
3. the prokaryotic expression recombinant vector pET-28a/ being used for expressing recombiant protein rRv0220 described in claim 1
Rv0220。
4. the prokaryotic expression recombinant vector pET-28a/ being used for expressing recombiant protein rRv2958c described in claim 1
Rv2958c。
5. the tuberculosis branch in preparation detection tuberculosis patients serum of the tuberculosis serological diagnosis test kit described in claim 1
Application in bacillus antibody reagent.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610367677.8A CN106053800A (en) | 2016-05-30 | 2016-05-30 | Tuberculosis serological diagnostic kit |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610367677.8A CN106053800A (en) | 2016-05-30 | 2016-05-30 | Tuberculosis serological diagnostic kit |
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| CN106053800A true CN106053800A (en) | 2016-10-26 |
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|---|---|---|---|---|
| US20070161562A1 (en) * | 2005-12-07 | 2007-07-12 | Kolattukudy Pappachan E | Targeting of long chain triacylglycerol hydrolase gene for tuberculosis treatment |
| CN101661044A (en) * | 2008-08-27 | 2010-03-03 | 复旦大学附属华山医院 | Diagnostic reagent of tuberculosis and kit |
| WO2012164088A1 (en) * | 2011-06-03 | 2012-12-06 | Universite Montpellier 2 Sciences Et Techniques | Diagnosis method of active tuberculosis |
| CN102993283A (en) * | 2010-07-15 | 2013-03-27 | 华中农业大学 | Antigen protein for mycobacterium tuberculosis and application |
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| US20070161562A1 (en) * | 2005-12-07 | 2007-07-12 | Kolattukudy Pappachan E | Targeting of long chain triacylglycerol hydrolase gene for tuberculosis treatment |
| CN101661044A (en) * | 2008-08-27 | 2010-03-03 | 复旦大学附属华山医院 | Diagnostic reagent of tuberculosis and kit |
| CN102993283A (en) * | 2010-07-15 | 2013-03-27 | 华中农业大学 | Antigen protein for mycobacterium tuberculosis and application |
| WO2012164088A1 (en) * | 2011-06-03 | 2012-12-06 | Universite Montpellier 2 Sciences Et Techniques | Diagnosis method of active tuberculosis |
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Application publication date: 20161026 |