CN105884872A - LppZ antibody identified antigen polypeptide and application thereof - Google Patents
LppZ antibody identified antigen polypeptide and application thereof Download PDFInfo
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- CN105884872A CN105884872A CN201610330711.4A CN201610330711A CN105884872A CN 105884872 A CN105884872 A CN 105884872A CN 201610330711 A CN201610330711 A CN 201610330711A CN 105884872 A CN105884872 A CN 105884872A
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- lppz
- antibody
- polypeptide
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- serum
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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)
-
- 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)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The invention discloses a LppZ antibody identified antigen polypeptide and application thereof. The antigen polypeptide has at least one of the following polypeptide active fragments: (1) SGCARFNDAQSQPFT which is a sequence as shown in SEQ ID NO:1; (2) EPDVVRKDTHAHAWA which is a sequence as shown in SEQ ID NO:2; (3) HAHAWALRMSPDGNV which is a sequence as shown in SEQ ID NO:3; (4) at least two connecting products selected from amino acid sequences as shown in SEQ ID NO:1-3; and (5) amino acid derived sequences formed by modifying, substituting, deleting or adding amino acid residues to the polypeptide active fragment. By utilizing the LppZ antibody identified antigen polypeptide, the LppZ antibody level of a sample (like serum samples), such as blood samples of patients with tuberculosis or suspected tuberculosis can be effectively detected.
Description
Technical field
The present invention relates to biomedical sector.In particular it relates to antigen polypeptide of LppZ antibody recognition and application thereof.
More particularly it relates to the binding site of Serum Antibodies and LppZ albumen, the antigen polypeptide of LppZ antibody recognition,
This antigen polypeptide in preparation detection serum the purposes in the kit of LppZ antibody horizontal, be used for detecting in sample LppZ and resist
The kit of body level.
Background technology
Tuberculosis is one of the whole world three big communicable diseases, and the epidemiological survey report that the World Health Organization (WTO) issues is aobvious
Showing, China's tuberculosis mycobacteria infections pathology accounts for the 12% of the total case load in the whole world, is only second to India, occupies the whole world second.
The lungy occurred frequently and popular social and economic development having had a strong impact on China.The diagnosis of tuberculosis side used clinically at present
Method is mainly using the detection of tulase as goldstandard, and such as Sputum smears, phlegm collection bacterium and the method for Sputum culturing, its diagnostic method is time-consuming
Long, specific or sensitiveness is low.
Detection method based on detection of plasma is one of important supplementary means of present stage diagnosis of tuberculosis, is wherein widely used
Antigentic specificity IFN-r release experiment (IGRA) have commercial reagents box T-spot.TB and QFT-IT.
The antigen that IGRA uses is the holoprotein of Mycobacterium tuberculosis RD (region of difference) district's gene code, produces
Cost is high, expensive, and the World Health Organization does not recommend middle and low income country to be widely used as examination detection means.And
Also there are some researches show, in the country of tuberculosis high prevalence, the diagnosis of IGRA is by the specific reduction of environmental factor interference, false
Positive rising, is not suitable for China's national situation, needs to find more preferable detection of plasma diagnosis marker.
Thus, the current diagnosis of tuberculosis detection of plasma, the research of context of detection still have to be strengthened.
Summary of the invention
The present invention is following discovery based on inventor and completes:
Much's bacillus film surface lipids body protein LppZ can cause Human immune responses, produces corresponding antibodies, and serum resists
The expression of LppZ polyclonal antibody can reflect the m tuberculosis infection state of individuality.Currently with ELISA etc.
The method of Western blotting detection serum anti-LppZ antibody is required to LppZ pure protein as identifying antigen.LppZ
Belong to glycoprotein, its sequence has multiple glycosylation site so that the LppZ protein stability of vivoexpression is poor, and difficult
With renaturation to native conformation, affect the stability of testing result.Additionally, purifying protein preparation process is loaded down with trivial details, time-consuming effort,
Relatively costly.
At least one drawbacks described above that present invention seek to address that prior art, and provide a kind of useful business to select.
Thus, the present invention utilizes the technology screening of polypeptide microarrays to obtain LppZ proteantigen identification high frequency epi-position, and provides
Comprise the core amino acid sequence of serum antibody identification LppZ albumen and protected amino acid sequence coupling carrier albumen such as
The antigen polypeptide of the LPPZ antibody recognition of KLH etc., based on tuberculosis patient serum LppZ antibody expression relatively Healthy People
Notable rising, utilizes these antigen polypeptides can effectively detect the LppZ in person's serum to be measured by immune-blotting method means and resists
Body level, and then can be used in diagnosis of tuberculosis.
To this end, in a first aspect of the present invention, the invention provides the antigen polypeptide of a kind of LppZ antibody recognition.According to this
Bright embodiment, its polypeptide active fragment is at least one following:
1) sequence: SGCARFNDAQSQPFT shown in SEQ ID NO:1；
2) sequence: EPDVVRKDTHAHAWA shown in SEQ ID NO:2；
3) sequence: HAHAWALRMSPDGNV shown in SEQ ID NO:3；
4) the connection product of at least two of amino acid sequence shown in SEQ ID NO:1-3 it is selected from；
5) the polypeptide active fragment described in 1)-4) is through the modification of amino acid residue, the ammonia that replaces, lack or add and formed
Base acid derived sequence.
It is surprisingly found by the inventors that, utilize the antigen polypeptide of the above-mentioned LppZ antibody recognition of the present invention, pass through immune-blotting method
Means can effectively detect the LppZ antibody horizontal in person's serum to be measured, and then based on person's serum LppZ antibody expression to be measured
The most relatively Healthy People significantly raises, it is possible to effectively carry out diagnosis of tuberculosis, determines whether person to be measured is Tuberculosis patient.
Wherein, tuberculosis patient serum LppZ antibody expression relatively Healthy People significantly raises.Further, according to the enforcement of the present invention
Example, the above-mentioned antigen polypeptide LppZ antibody recognition of the present invention is the best, and LppZ antibody horizontal detection efficiency is high, and preparation
Simply, low cost, it is easy to carry out industrialized production, beneficially large-scale popularization application.
Further, based on utilizing the antigen polypeptide of above-mentioned LppZ antibody recognition, can effectively be detected by immune-blotting method means
LppZ antibody horizontal in person's serum to be measured, in a second aspect of the present invention, the invention provides foregoing LppZ and resists
The antigen polypeptide of body identification is the purposes in the kit of LppZ antibody horizontal in preparation detection serum.
According to embodiments of the invention, this kit is used for diagnosis of tuberculosis.Wherein, resist based on tuberculosis patient serum LppZ
Body expression relatively Healthy People significantly raises, it is possible to effectively person to be measured is carried out diagnosis of tuberculosis.
In a third aspect of the present invention, the invention provides a kind of for detecting the kit of LppZ antibody horizontal in sample.Root
According to embodiments of the invention, this kit includes: the antigen polypeptide of foregoing LppZ antibody recognition.According to the present invention
Embodiment, utilize this kit can effectively detect LppZ antibody horizontal in sample, and then based on tuberculosis patient serum
LppZ antibody expression relatively Healthy People significantly raises, it is possible to effectively person to be measured is carried out diagnosis of tuberculosis.
Specifically, according to embodiments of the invention, the antigen polypeptide of described LppZ antibody recognition is with at least one following form
There is provided:
1) 96 orifice plate, is coated the antigen polypeptide of described LppZ antibody recognition in the hole of described 96 orifice plates；
2) microballoon, the antigen polypeptide of LppZ antibody recognition described in described microballoon coupling.
Further, according to some concrete examples of the present invention, this kit farther includes: positive control sample, described sun
Property control sample is the antibody of anti-LppZ albumen.Thus, this kit is utilized to carry out LppZ antibody horizontal detection and tuberculosis
During diagnosis, result is relatively reliable.
According to embodiments of the invention, farther including: standard sample, described standard sample is that many cases LppZ antibody expression is
The equal-volume mixture of positive serum.Thus, this kit is utilized to carry out LppZ antibody horizontal detection and diagnosis of tuberculosis
Time result more true and reliable.
According to some concrete examples of the present invention, described standard sample be described many cases LppZ antibody expression be positive serum
The gradient dilution product of equal-volume mixture, the ratio of described gradient dilution is stopped to 1:2000 from 1:50.Thus, utilizing should
It is high that kit carries out reliable results degree when the detection of LppZ antibody horizontal and diagnosis of tuberculosis.
According to embodiments of the invention, the concentration of the antigen polypeptide of described LppZ antibody recognition is 1 μ g/ml.
According to embodiments of the invention, described sample is serum.
The additional aspect of the present invention and advantage will part be given in the following description, and part will become bright from the following description
Aobvious, or recognized by the practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or the additional aspect of the present invention and advantage the accompanying drawings below description to embodiment will be apparent from from combining and
Easy to understand, wherein:
Fig. 1 shows according to one embodiment of the invention, the developing result of polypeptide chip 1；
Fig. 2 shows according to one embodiment of the invention, the developing result of polypeptide chip 2；And
Fig. 3 shows according to one embodiment of the invention, when polypeptide shown in SEQ ID No:1-3 independently uses and is used in combination,
Testing result to the LppZ antibody horizontal in tuberculosis patient serum and healthy population serum.
Detailed description of the invention
Embodiments of the invention are described below in detail, and described embodiment is intended to for explaining the present invention, and it is not intended that to this
The restriction of invention.
The antigen polypeptide of LppZ antibody recognition
In a first aspect of the present invention, the present invention proposes the antigen polypeptide of a kind of LppZ antibody recognition.Reality according to the present invention
Executing example, its polypeptide active fragment is at least one following:
1) sequence: SGCARFNDAQSQPFT shown in SEQ ID No:1；
2) sequence: EPDVVRKDTHAHAWA shown in SEQ ID No:2；
3) sequence: HAHAWALRMSPDGNV shown in SEQ ID No:3；
4) the connection product of at least two of amino acid sequence shown in SEQ ID No:1-3 it is selected from,
5) the polypeptide active fragment described in 1)-4) is through the modification of amino acid residue, the ammonia that replaces, lack or add and formed
Base acid derived sequence.
It is surprisingly found by the inventors that, utilize the antigen polypeptide of the above-mentioned LppZ antibody recognition of the present invention, pass through immune-blotting method
Means can effectively detect the LppZ antibody horizontal in person's serum to be measured, and then based on person's serum LppZ antibody expression to be measured
The most relatively Healthy People significantly raises, it is possible to effectively carry out diagnosis of tuberculosis, determines whether person to be measured is Tuberculosis patient.
Wherein, tuberculosis patient serum LppZ antibody expression relatively Healthy People significantly raises.Further, according to the enforcement of the present invention
Example, the above-mentioned antigen polypeptide LppZ antibody recognition of the present invention is the best, and LppZ antibody horizontal detection efficiency is high, and preparation
Simply, low cost, it is easy to carry out industrialized production, beneficially large-scale popularization application.
It should be noted that aforementioned polypeptides (such as derives from protein public resource both from the full length sequence of LppZ albumen
Database uni-prot) corresponding to position.It addition, the term used in this article " polypeptide " refers at least two amino
The oligomer that acid is bonded by peptide and obtains, wherein, the number of the amino acid residue included in polypeptide is limited the most especially
System.According to some embodiments of the present invention, polypeptide can contain 15 amino acid.Certainly, those skilled in the art can manage
Solve be, it is also possible to aforementioned polypeptides is chemically modified, in order to increase polypeptide antigenicity, contribute to polypeptide and be coated
At the bottom of elisa plate.According to embodiments of the invention, utilize the polypeptide with above-mentioned amino acid sequence, it is possible to effectively detect sample
LPPZ antibody horizontal in the blood sample (such as blood serum sample) of product, such as tuberculosis or Tuberculosis patient.Send out
A person of good sense finds that the aforementioned polypeptides obtained from LppZ albumen complete sequence can simulate the epitope of LppZ, it is possible to blood sample
The specific reaction of LPPZ antibody in Ben, therefore, it is possible to be efficiently used for detecting the LPPZ antibody water in object blood sample
Flat, thus, it is possible to be efficiently used for examination tuberculosis suspected patient serum, provide a new inspection for diagnosis lungy
Survey means.
Above-mentioned antigen polypeptide can be obtained by chemical synthesis, it is also possible to is obtained by technique for gene engineering, in this technology is industry
It is familiar with.It will be appreciated by persons skilled in the art that and can effectively pass through conventional synthesis process, synthesize aforementioned polypeptides,
Thus replace recombinant expressed biosynthesis mode.
According to embodiments of the invention, can be not particularly restricted by the type of sample that aforementioned polypeptides carries out detecting.According to
Embodiments of the invention, preferred sample is from tuberculosis or the blood sample of Tuberculosis patient.Thus, it is possible to it is sharp
With the LPPZ antibody horizontal in the blood sample of polypeptide detection tuberculosis or Tuberculosis patient, thus may determine that patient
Whether suffer from tuberculosis, provide supplementary means for diagnosis, or early screening lungy is provided.It addition, also may be used
With by detection object blood in LppZ antibody horizontal, and patient is treated curative effect evaluation monitoring analyze.
Utilize the method for LppZ antibody horizontal in the polypeptide detection sample of the present invention to be not particularly restricted, include but not limited to
ELISA, liquid-phase chip, solid phase chip and other immuning hybridization technology etc..According to embodiments of the invention, enzyme can be passed through
Linked immunosorbent adsorption test (ELISA) method, the polypeptide by the present invention detects the LppZ antibody horizontal in sample.Thus, may be used
Polypeptide is utilized to detect the effect of the LppZ antibody horizontal in the blood sample of tuberculosis or Tuberculosis patient with further raising
Rate.As example, following method can be used to utilize in the blood sample of polypeptide detection tuberculosis or Tuberculosis patient
LppZ antibody horizontal:
First, with the carbonate buffer solution (NaCO of pH9.630.159g, NaHCO30.293g, adds water to 100ml, pH
Value is 9.6) dilution polypeptide to 1 μ g/ml, join in the hole of 96 hole ELISA Plates with the amount in 100 μ l/ holes, shrouding film seal
Rear 4 DEG C overnight.
It follows that the liquid discarded in hole, 0.1%TBS-T lavation buffer solution (the TBS-T solution containing 0.1%Tween-20
It is formulated as NaCl 8.7g, Tris 1.21g, adds deionized water to 1000ml, pH 7.5, add Tween-20 1ml) washing
ELISA Plate 5 times, each 3min.
Then, in hole, TBS-T Block buffer (its compound method containing 5%BSA and 0.05%Tween-20 is added
For NaCl 0.87g, Tris 0.121g, add deionized water to 100ml, pH 7.5, add Tween-20 0.5ml, BSA
5g), every hole 300 μ l, shrouding film seals latter 37 DEG C and hatches 1h.
Then, standard items and testing sample are added.Close slow with the above-mentioned TBS-T containing 5%BSA and 0.05%Tween-20
Rushing liquid dilution test serum, dilution ratio is 1:100.Gradient dilution is as the serum of standard items, and dilution ratio is from 1:50
Stop to 1:2000.Discarding liquid in hole completely, every hole adds the serum dilution 100 μ l of testing sample, every example testing sample
The multiple hole of serum sample 3, every plate is set up 2 Positive control wells, is added known positive serum, and 2 blank set up by every plate
Control wells, adds 0.1%TBS-T lavation buffer solution.Shrouding film seals latter 37 DEG C and hatches 2h.Discard liquid in hole, washing
ELISA Plate 5 times, each 3min.
Then, SA immune response is carried out.The above-mentioned TBS-T containing 5%BSA and 0.05%Tween-20 is utilized to close
The Goat anti human IgG of buffer solution dilution horseradish peroxidase-labeled, dilution ratio 1:5000, every hole adds 100 μ l, shrouding
Film seals latter 37 DEG C and hatches 1h.Discard liquid in hole, detersive enzyme target 5 times, each 3min.
Then, substrate colour developing is added.According to conventional ELISA kit specification (such as health is century TMB colour reagent box),
Adding chromogenic substrate, every hole adds 100 μ l, and develop the color under the conditions of room temperature lucifuge 10min, and every hole adds 50 μ l 2M H2SO4
Terminate reaction.
Then, optical density OD value is measured under ELIASA 492nm wavelength condition.
Finally, optical density OD value based on gained and the calibration curve of standard concentration matching, it is thus achieved that corresponding detection sample
Relative concentration, then compares with predetermined reference value, determines whether sample to be tested derives from and suffer from tuberculosis patient.
According to embodiments of the invention, it is possible to use the blood sample making a definite diagnosis cancer patient and Healthy People (healthy volunteer) enters
Numerical value obtained by row parallel test is as predetermined reference value.
If when the LppZ antibody relative concentration in sample to be tested is higher than X, then test serum is Tuberculosis patients serum.X
Being that inventor is obtained by experimental calculation, concrete grammar is tubercular and Healthy Human Serum antibody relative concentration in detection crowd
Receiver operating curves's (ROC curve) optimum point under relative concentration.According to embodiments of the present invention, 82 example knots are used
Core patients serum's sample and 38 example Healthy Human Serum samples, calculate the X value of antigen polypeptide shown in sequence SEQ ID NO:1
It is 6.86.
The application of the antigen polypeptide of LPPZ antibody recognition
In a second aspect of the present invention, the invention provides the antigen polypeptide of foregoing LppZ antibody recognition in preparation detection
Purposes in the kit of LppZ antibody horizontal in serum.
As it was previously stated, use the kit of preparation, utilize the antigen polypeptide of its LppZ antibody recognition comprised, pass through Diagnosis of Sghistosomiasis
Mark detection means can effectively detect the LppZ antibody horizontal in person's serum to be measured, and then can effectively carry out diagnosis of tuberculosis.
Thus, according to embodiments of the invention, this kit is used for diagnosis of tuberculosis.Wherein, based on tuberculosis patient serum LppZ
Antibody expression relatively Healthy People significantly raises, it is possible to effectively person to be measured is carried out diagnosis of tuberculosis.
To this end, in a third aspect of the present invention, the invention provides a kind of for detecting the reagent of LppZ antibody horizontal in sample
Box.According to embodiments of the invention, this kit includes: the antigen polypeptide of foregoing LppZ antibody recognition.According to
Embodiments of the invention, utilize this kit can effectively detect LppZ antibody horizontal in sample, and then based on tuberculosis sufferer
Person's serum LppZ antibody expression relatively Healthy People significantly raises, it is possible to effectively person to be measured is carried out diagnosis of tuberculosis.
Specifically, according to embodiments of the invention, the antigen polypeptide of described LppZ antibody recognition is with at least one following form
There is provided:
1) 96 orifice plate, is coated the antigen polypeptide of described LppZ antibody recognition in the hole of described 96 orifice plates；
2) microballoon, the antigen polypeptide of LppZ antibody recognition described in described microballoon coupling.
Further, according to some concrete examples of the present invention, this kit farther includes: positive control sample, described sun
Property control sample is the antibody (can manually prepare) of anti-LppZ albumen.Thus, this kit is utilized to carry out LppZ
When antibody horizontal detection and diagnosis of tuberculosis, result is relatively reliable.
According to embodiments of the invention, farther including: standard sample, described standard sample is that many cases LppZ antibody expression is
The equal-volume mixture of positive serum.Thus, this kit is utilized to carry out LppZ antibody horizontal detection and diagnosis of tuberculosis
Time result more true and reliable.Wherein, the number of cases of positive serum namely the patient of serum number of originating is not particularly restricted, institute
Stating " many cases " can also be 1 example.According to some concrete examples of the present invention, described standard sample is 10 example LppZ antibody
It is expressed as the serum pond of the serum equal-volume mixing composition of the positive.
According to some concrete examples of the present invention, described standard sample be described many cases LppZ antibody expression be positive serum
The gradient dilution product of equal-volume mixture, the ratio of described gradient dilution is stopped to 1:2000 from 1:50.Wherein, through tonsure
Dilution is follow-up drafting calibration curve.Thus, this kit is utilized to carry out LppZ antibody horizontal detection and tuberculosis
During sick diagnosis, reliable results degree is high.
According to embodiments of the invention, the concentration of the antigen polypeptide of described LppZ antibody recognition is 1 μ g/ml.
According to embodiments of the invention, described sample is serum.
According to some embodiments of the present invention, this kit may include that 96 orifice plates, the hole of described 96 orifice plates is coated before
The antigen polypeptide of described LPPZ antibody recognition.Further, this kit may further include: positive control sample,
Described positive control sample is the antibody of anti-LppZ albumen, or/may further include extraly: standard sample, described
Standard sample be LppZ antibody expression be the combination after gradient dilution of the positive serum.Some according to the present invention are specifically shown
Example, described standard sample be described many cases LppZ antibody expression be that the gradient dilution of the equal-volume mixture of positive serum produces
Thing, the ratio of described gradient dilution is stopped to 1:2000 from 1:50.According to embodiments of the invention, described LppZ antibody is known
The concentration of other antigen polypeptide is 1 μ g/ml.According to embodiments of the invention, described sample is serum.
According to embodiments of the invention, described sample is from tuberculosis or the blood sample of Tuberculosis patient.Thus,
The LppZ antibody horizontal in the blood sample of polypeptide detection tuberculosis or Tuberculosis patient can be utilized, thus can be true
Determine whether patient suffers from tuberculosis, provide supplementary means for diagnosis, or monitor progress lungy.It addition, it is sharp
Can also be by the LppZ antibody horizontal in detection object blood with this kit, and the reaction to patient for treatment's method is entered
Row prognosis.
In still another aspect of the invention, the present invention proposes a kind of for detecting the kit of LPPZ antibody horizontal in sample.
According to embodiments of the invention, this kit includes: the antigen polypeptide of foregoing LppZ antibody recognition.Thus, profit
With according to embodiments of the invention, can effectively by the antigen polypeptide of the LppZ antibody recognition according to the present invention to sample
In LppZ antibody horizontal detect.According to embodiments of the invention, this kit may further include: be suitable to into
The reagent of row ELISA detection.Thus, it is possible to by ELISA detection method, utilize the polypeptide according to the present invention in sample
LppZ antibody detect, utilize polypeptide detection tuberculosis or the blood of Tuberculosis patient such that it is able to improve further
The efficiency of the LppZ antibody horizontal in liquid sample.According to embodiments of the invention, described polypeptide is arranged in 96 orifice plates, example
As the hole of 96 orifice plates can be coated, such as can by conventional treatment as by be coated liquid and retardance liquid carry out.Thus, it is possible to
Detect the most with high throughput.Can improve further and utilize polypeptide detection tuberculosis or the blood of Tuberculosis patient
The efficiency of the LppZ antibody horizontal in sample.Kit can also comprise other reagent, such as lavation buffer solution, sample
Dilution buffer, tmb substrate solution, reaction terminating liquid, the two of horseradish peroxidase-labeled resist, positive control sample,
Standard sample.Wherein, positive control sample is the antibody of anti-LppZ albumen, and standard sample is that LppZ antibody expression is for positive
Serum combination after gradient dilution.Or, described standard sample be described many cases LppZ antibody expression be positive blood
The gradient dilution product of clear equal-volume mixture.
According to embodiments of the invention, described sample is from tuberculosis or Tuberculosis patient and the blood of healthy volunteer
Liquid sample.
It addition, utilize this kit can also be by the LppZ antibody horizontal in detection object blood, and to patient for treatment side
The reaction of method carries out prognosis.
Below by specific embodiment, the present invention is explained.It should be noted that the following example is the most illustrative
, and limit the present invention never in any form.It addition, all appts employed in the following example, material etc. are city
Sell available, such as the operation the most not explicitly pointed out, the operation that those skilled in the art are conventional can be passed through
Method is carried out.
The preparation of embodiment 1 polypeptide
1. the preparation in peptide storehouse
The amino acid sequence of LppZ (Rv3006) obtains from protein common source databases uni-prot, and its sequence is from N end
As follows to C end:
MWTTRLVRSGLAALCAAVLVSSGCARFNDAQSQPFTTEPELRPQPSSTPPPPPPLPPVP
FPKECPAPGVMQGCLESTSGLIMGIDSKTALVAERITGAVEEISISAEPKVKTVIPVDPAGD
GGLMDIVLSPTYSQDRLMYAYISTPTDNRVVRVADGDIPKDILTGIPKGAAGNTGALIFTSP
TTLVVMTGDAGDPALAADPQSLAGKVLRIEQPTTIGQTPPTTALSGIGSGGGLCIDPVDGS
LYVADRTPTADRLQRITKNSEVSTVWTWPDKPGVAGCAAMDGTVLVNLINTKLTVAVRL
APSTGAVTGEPDVVRKDTHAHAWALRMSPDGNVWGATVNKTAGDAEKLDDVVFPLFP
QGGGFPRNNDDKT (SEQ ID NO:4).
LppZ full length protein sequence splits into the polypeptide of one group of 15 amino acid length, overlapping 12 of order between adjacent polypeptide
Amino acid residue, composition LppZ peptide storehouse, 121 altogether.
2. polypeptide chip (peptide array) is prepared in fabricated in situ LppZ protein peptides storehouse
Peptide synthesizer ASP SL (Germany, intavis is carried out according to albumen and polypeptide amino acid residues in length, overlap length
Company) MutilPep synthesis control program setting, adds a kind of specific amino acids according to programme-control at each synthesis cycle and arrives
The synthesis site, nitrocellulose filter surface of activation, and between catalytic amino acid, peptide bond is formed.Synthetic peptide chain length is 15 ammonia
Base acid residue, need to carry out the reaction in 15 cycles, according to the operating process of ASP SL Peptide synthesizer, by 20 kinds of FMOC-
The native amino acid solution of radical protection is put on machine the position of correspondence.Between each cycle amino acid will through capping,
The a series of processes such as FMOC-group deprotection could be combined with next amino acid, during last end cycle, and be by
Amino acid whose side chain protecting group is taken off.Save backup in-20 DEG C of sealings after polypeptide chip synthesis.
The polypeptide (screening of epitope) that embodiment 2 screening is relevant to tuberculosis
1. the aquation of polypeptide chip and Western blotting
The polypeptide chip (referred to herein as " polypeptide chip 1 ") embodiment 1 prepared immerses in 40ml 100% ethanol, shaking table
On shake 5 minutes.Then, this polypeptide chip is immersed in 40ml 75% ethanol, shaking table shakes 5 minutes.It follows that will be many
Peptide chips immerses in 40ml 50% ethanol, and shaking table shakes 5 minutes.Then, add 150ml PBS to soak 30 minutes.?
After, during just polypeptide chip immerses 40ml 5% skimmed milk power/PBS-T solution, incubated at room temperature is closed for 3 hours.
The serum sample 5% skimmed milk power/PBS-T solution 1:1000 related in following primary dcreening operation and identification experiment is diluted preparation
Become immune response liquid, polypeptide chip is put into hybridization bag, add reactant liquor by 0.1ml/cm2, remove all bubbles in bag, envelope
Film machine seals, 4 DEG C of shaken overnight gently.
After sero-immunity reaction terminates, cutting off hybridization bag, discard reactant liquor, PBS-T 40ml washes polypeptide chip 3 times, every time
15 minutes, polypeptide chip is put into hybridization bag, add Goat anti human's IgG solution of 1:2000 dilution, by 0.1ml/cm2
The anti-reactant liquor that adds two, seals hybridization bag, and room temperature with gentle vibrates 2 hours, and 40ml PBS-T washes film 3 times, each 15 points
Clock.
It follows that carry out ECL development, exposure, exposure results AlphaView software system analysis image.
2. the primary dcreening operation of epitope
10 example tuberculosis patient peripheral blood serum mixed in equal amounts are prepared as tuberculosis pooled serum pond, 10 example PPD detection feminine genders
Volunteer's peripheral blood serum mixed in equal amounts is prepared as healthy pooled serum pond.Above-mentioned serum pond is with polypeptide chip according to the method described above
Carry out immuning hybridization, according to immune response result, select and healthy volunteer serum reverse positive with the reflection of tuberculosis patient serum
Reflect the differential peptides site of feminine gender, 16 altogether, be LppZ albumen antigen recognizing epi-position in antitubercle sera.Polypeptide
Chip developing result is as shown in Figure 1.
Fig. 1 shows the developing result of polypeptide chip 1, and wherein black box show region, LppZ protein peptides storehouse.
3. there is the antigen polypeptide screening that diagnosis of tuberculosis is worth
According to the preparation method described in embodiment 1, preparation comprises above-mentioned 16 differential peptides and to impinging upon interior polypeptide chip (this
In be referred to as " polypeptide chip 2 "), by carrying out immune response with 100 example tuberculosis patient serum and 90 example Healthy Human Serums,
Filtering out 3, LppZ epitope high frequency site, they have differentiation tuberculosis patient and healthy people integration, its sequence
With the positive rate in antitubercle sera as shown in table 1, polypeptide chip 2 developing result is as shown in Figure 2:
Table 1
| Serial number | Polypeptide title | Sequence | Positive rate % |
| 1 | SEQ ID NO:1 | SGCARFNDAQSQPFT | 20.6% |
| 2 | SEQ ID NO:2 | EPDVVRKDTHAHAWA | 5.9% |
| 3 | SEQ ID NO:3 | HAHAWALRMSPDGNV | 15.3% |
Fig. 2 shows the developing result of polypeptide chip 2, wherein show LppZ polypeptide region in black box.
Embodiment 3 ELISA checks
It is that antigen is coated 96 orifice plates respectively so that embodiment 2 to be screened three peptide species (shown in table 1) of acquisition, uses ELISA
Method carries out the detection of LppZ antibody to following sample: 82 example tubercular's serum and 38 example normal healthy controls serum.
Concrete detection method is as follows:
First, described antigen polypeptide is synthesized with Peptide synthesizer MultiPep RS according to amino acid residue order in sequence table.
Then, with the carbonate buffer solution (NaCO of pH9.630.159g, NaHCO30.293g, adds water to 100ml, pH
Value is 9.6) dilution polypeptide to 1 μ g/ml, join in the hole of 96 hole ELISA Plates with the amount in 100 μ l/ holes, shrouding film seal
Rear 4 DEG C overnight.
It follows that the liquid discarded in hole, 0.1%TBS-T lavation buffer solution (the TBS-T solution containing 0.1%Tween-20
It is formulated as NaCl 8.7g, Tris 1.21g, adds deionized water to 1000ml, pH 7.5, add Tween-20 1ml) washing
ELISA Plate 5 times, each 3min.
Then, in hole, TBS-T Block buffer (its compound method containing 5%BSA and 0.05%Tween-20 is added
For NaCl 0.87g, Tris 0.121g, add deionized water to 100ml, pH 7.5, add Tween-20 0.5ml, BSA
5g), every hole 300 μ l, shrouding film seals latter 37 DEG C and hatches 1h.
Then, standard items and testing sample are added.Described standard items are the periphery of the wherein 10 example tuberculosis patients randomly selected
The equal-volume mixture of blood serum.Treat with the dilution of the above-mentioned TBS-T Block buffer containing 5%BSA and 0.05%Tween-20
Surveying serum, dilution ratio is 1:100.Gradient dilution as the serum mixture of standard items, dilution ratio from 1:50 to 1:2000
Only.Discarding liquid in hole completely, every hole adds the serum dilution 100 μ l of testing sample, the serum sample of every example testing sample
3 multiple holes, every plate is set up 2 Positive control wells, is added known positive serum, and 2 blank control wells set up by every plate,
Add 0.1%TBS-T lavation buffer solution.Shrouding film seals latter 37 DEG C and hatches 2h.Discard liquid in hole, detersive enzyme target 5
Secondary, each 3min.
Then, SA immune response is carried out.The above-mentioned TBS-T containing 5%BSA and 0.05%Tween-20 is utilized to close
The Goat anti human IgG of buffer solution dilution horseradish peroxidase-labeled, dilution ratio 1:5000, every hole adds 100 μ l, shrouding
Film seals latter 37 DEG C and hatches 1h.Discard liquid in hole, detersive enzyme target 5 times, each 3min.
Then, substrate colour developing is added.According to conventional ELISA kit specification (health is century TMB colour reagent box), add
Entering chromogenic substrate, every hole adds 100 μ l, and develop the color under the conditions of room temperature lucifuge 10min, and every hole adds 50 μ l 2M H2SO4Eventually
Only reaction.
Then, optical density OD value is measured under ELIASA 492nm wavelength condition.
Finally, optical density OD value based on gained and the calibration curve of standard concentration matching, it is thus achieved that corresponding detection sample
Relative concentration, then compares with predetermined reference value, determines whether sample to be tested derives from and suffer from tuberculosis patient.
If when the LppZ antibody relative concentration in sample to be tested is higher than X, then test serum is Tuberculosis patients serum.X
Receiver operating curves's (ROC curve) optimum point for tubercular in detection crowd and Healthy Human Serum antibody relative concentration
Under relative concentration.Based on using 82 example tubercular's serum samples and 38 example Healthy Human Serum samples, it is calculated each anti-
The X value that former polypeptide is corresponding, wherein the X value of antigen polypeptide shown in sequence SEQ ID NO:1 is 6.86.
Testing result, as it is shown on figure 3, the polypeptide shown in SEQ ID NO:1-3 the most independently uses or is used in combination, all detects
In tuberculosis patient serum, LppZ antibody horizontal is all remarkably higher than healthy population serum.
In sum, inventor finds, the LppZ antibody horizontal of tuberculosis patient is all remarkably higher than healthy population serum.Thus,
Demonstrate the antigen polypeptide of the LppZ antibody recognition of the present invention, can be effectively used for auxiliary diagnosis lungy.
In the description of this specification, reference term " embodiment ", " some embodiments ", " example ", " specifically show
Example " or the description of " some examples " etc. means to combine this embodiment or example describes specific features, structure, material or
Feature is contained at least one embodiment or the example of the present invention.In this manual, the schematic representation to above-mentioned term
It is not necessarily referring to identical embodiment or example.And, the specific features of description, structure, material or feature can be
Any one or more embodiments or example combine in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: without departing from
These embodiments can be carried out multiple change in the case of the principle of the present invention and objective, revise, replace and modification, this
Bright scope is limited by claim and equivalent thereof.
Claims (10)
1. the antigen polypeptide of a LppZ antibody recognition, it is characterised in that its polypeptide active fragment is at least one following:
1) sequence: SGCARFNDAQSQPFT shown in SEQ ID NO:1；
2) sequence: EPDVVRKDTHAHAWA shown in SEQ ID NO:2；
3) sequence: HAHAWALRMSPDGNV shown in SEQ ID NO:3；
4) the connection product of at least two of amino acid sequence shown in SEQ ID NO:1-3 it is selected from,
5) the polypeptide active fragment described in 1)-4) is through the modification of amino acid residue, the ammonia that replaces, lack or add and formed
Base acid derived sequence.
2. the antigen polypeptide of the LppZ antibody recognition described in claim 1 LppZ antibody horizontal in preparation detection serum
Purposes in kit.
Purposes the most according to claim 2, it is characterised in that described kit is used for diagnosis of tuberculosis.
4. one kind is used for detecting the kit of LppZ antibody horizontal in sample, it is characterised in that including:
The antigen polypeptide of the LppZ antibody recognition described in claim 1.
Kit the most according to claim 4, it is characterised in that below the antigen polypeptide of described LppZ antibody recognition
At least one form of row provides:
1) 96 orifice plate, is coated the antigen polypeptide of described LppZ antibody recognition in the hole of described 96 orifice plates；
2) microballoon, the antigen polypeptide of LppZ antibody recognition described in described microballoon coupling.
Kit the most according to claim 5, it is characterised in that farther include: positive control sample, described sun
Property control sample is the antibody of anti-LppZ albumen.
Kit the most according to claim 5, it is characterised in that farther include: standard sample, described standard sample
Product be many cases LppZ antibody expression be the equal-volume mixture of positive serum.
Kit the most according to claim 7, it is characterised in that described standard sample is described many cases LppZ antibody
Be expressed as the gradient dilution product of equal-volume mixture of the serum of the positive, the ratio of described gradient dilution from 1:50 to
1:2000 is only.
Kit the most according to claim 5, it is characterised in that the antigen polypeptide of described LppZ antibody recognition dense
Degree is 1 μ g/ml.
Kit the most according to claim 4, it is characterised in that described sample is serum.
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|---|---|---|---|---|
| US20080171345A1 (en) * | 2006-08-10 | 2008-07-17 | Colorado State University Research Foundation | Biomarkers of tuberculosis that distinguish disease categories: use as serodiagnostic antigens |
| CN104004069A (en) * | 2014-06-06 | 2014-08-27 | 上海交通大学医学院 | Reagent for mycobacterium tuberculosis infection detection, clinical treatment effect tracking and antituberculous vaccine development and application thereof |
| CN104597239A (en) * | 2014-12-17 | 2015-05-06 | 广州一代医药科技有限公司 | Antigen stimulant and kit for detecting mycobacterium tuberculosis infection, and application of antigen stimulant |
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| US20080171345A1 (en) * | 2006-08-10 | 2008-07-17 | Colorado State University Research Foundation | Biomarkers of tuberculosis that distinguish disease categories: use as serodiagnostic antigens |
| CN104004069A (en) * | 2014-06-06 | 2014-08-27 | 上海交通大学医学院 | Reagent for mycobacterium tuberculosis infection detection, clinical treatment effect tracking and antituberculous vaccine development and application thereof |
| CN104597239A (en) * | 2014-12-17 | 2015-05-06 | 广州一代医药科技有限公司 | Antigen stimulant and kit for detecting mycobacterium tuberculosis infection, and application of antigen stimulant |
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