CN107043413A - A kind of related urinary biomarkers albumen of pulmonary tuberculosis and its application - Google Patents
A kind of related urinary biomarkers albumen of pulmonary tuberculosis and its application Download PDFInfo
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- CN107043413A CN107043413A CN201610084080.2A CN201610084080A CN107043413A CN 107043413 A CN107043413 A CN 107043413A CN 201610084080 A CN201610084080 A CN 201610084080A CN 107043413 A CN107043413 A CN 107043413A
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- urine
- pulmonary tuberculosis
- protein
- albumen
- rbp4
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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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- 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
Abstract
The invention belongs to biochemical immunity analysis technical field, and in particular to a kind of related urinary biomarkers albumen of pulmonary tuberculosis and its application.The present invention obtains two kinds of pulmonary tuberculosis correlation Urine proteins, i.e. Urine proteins RBP ELISA RBP4, the protein sequence such as sequence table SEQ ID NO of the albumen using protein science screening：Shown in 1.The Urine proteins of the present invention can be used as mark antigen protein application in detection pulmonary tuberculosis Urine in Diagnosis kit is prepared.
Description
Technical field
The invention belongs to biochemical immunity analysis technical field, and in particular to the related urinary biomarkers albumen of a kind of pulmonary tuberculosis and its should
With.
Background technology
Tuberculosis is a kind of zoonosis as caused by M.tuberculosis complex, with higher infection rate and death toll, is arranged
For after HIV the whole world cause death second largest infectious disease.It is largely because not to fail lungy spread of control
(so that effectively treating) cases of infection can fully be detected.Therefore, the one of the global Stop TB plan of the World Health Organization is big
Emphasis is that development is simple, inexpensive and effective new diagnostic methods, to improve existing detection case.In the feelings of resource-constrained
Under condition, active diagnosing tuberculosis.Clinical assessment is relied primarily on, and combines sediments microscope inspection Sputum smears；Separated from clinical sample
It is always diagnosis of tuberculosis goldstandard to cultivate mycobacterium tuberculosis, however, this standard is costly, time-consuming, recall rate is low, and is needed
Bio-safety facility that will be stricter.Therefore, it is to study the key areas of tuberculosis prevention and treatment to find tuberculosis new diagnostic mark.
The development of diagnosis of tuberculosis method is hindered due to lacking reliable and by checking MTB biomarkers at present.Therefore manage
The tuberculosis laboratory diagnostic method thought should possess it is following some：(1) sensitivity and specificity are high；(2) cost is low；(3) can
Early detection and treatment Prognosis scoveillance；(4) detection spectrum is wide, including can detect smear for microscopic examination feminine gender or resistance case；(5)
Sampling is simple, it is therefore desirable to which not damaged is sampled；(6) it is easy to detect, be conducive to carrying out field and domestic medicine service to tuberculosis.Just
In the case of often, protein is few in urine, therefore, in urine kinds of protein and number change have occur with disease, development and
Prognosis is relevant；Meanwhile, urine non-invasively can be obtained largely, and more stable for other biofluids, therefore
Urinary proteomics are turning into an important content during Clinical Proteomics are studied, be it in scientific research and clinically
Using laying a good foundation.
In the present invention, two dimensional electrophoresis and immunoblotting assay show, RBP4 albumen has significance difference in pulmonary tuberculosis and Healthy People urine
Different expression, this show these protein can as pulmonary tuberculosis urine potential source biomolecule marker.RBP4 protein functions are significant,
Because this material may take part in macrophage activation；RBP4 is also considered as being used to resist insulin as Adipocyte Factor, this
Pathophysiological process may be adjusted during bacterium infection.And have been reported that display RBP4 participates in playing important work in immune response
With, but this area does not know its specific influence situation that pulmonary tuberculosis is immunized still, also without it in lunger's urine
Research and development.
The content of the invention
Present invention aims to overcome that the defect of prior art, i.e., detect pulmonary tuberculosis using the disease specific of Urine proteins there is provided one kind
Relevant urinary biomarkers albumen, the marker protein can be used for lunger's Urine proteins biological marker, set up lunger
(retinol-binding protein 4, RBP4, sequence is shown in SEQ ID NO to Urine proteins RBP ELISA：1) express, be lung
Tuberculosis diagnosis in time and treatment provide support.
Present invention firstly discovers that Urine proteins especially RBP ELISA is examined as an important biological indicator in tuberculosis clinic
It is disconnected, there is the effect of marker protein, the research for later tubercular's Urine proteins provides theory in antidiastole, effectively observation
Foundation, and to provide new thought from molecular level diagnosis of tuberculosis, with great theory significance and potential use value.
The present invention is achieved through the following technical solutions：
A kind of detection method of pulmonary tuberculosis Urine proteins, comprises the following steps：
The total protein in lunger and Healthy People control urine is extracted using TCA/ acetone methods, and uses quantification of protein kit
(Bio-Rad, USA) is quantified.Using two dimensional electrophoresis [1], MADEL-TOF-MS and immunoprecipitation method [1] are to pulmonary tuberculosis
The Urine proteins that patient and Healthy People control have been extracted carry out separation identification, analyze expressed differential proteins, and identification can be used for pulmonary tuberculosis to suffer from
The urine markers protein of person's diagnosis.
Present invention firstly discovers that a kind of Urine proteins RBP ELISA (retinol-binding protein 4, RBP4,
P02753-HUMAN) as an important urine biology Testing index, in the diagnosis of pulmonary tuberculosis, antidiastole, effectively
Effect in observation, foundation is provided for the research of pulmonary tuberculosis Urine proteins from now on, and from the horizontal Diagnosis of pulmonary knot of urine protein molecular
Core disease provides new direction, with important theory value and potential Practical significance.
Pulmonary tuberculosis Urine proteins biomarker protein prepared by the present invention can be applied in pulmonary tuberculosis urine detection kit is prepared.
Brief description of the drawings
Sequence table SEQ ID NO：1 is the protein sequence for the Urine proteins RBP ELISA RBP4 that the present invention is separated.
Fig. 1：Differential expression of the Urine proteins in lunger and normal healthy controls person.
Fig. 2：Lunger and the clustering of normal healthy controls person's Urine proteins.
Fig. 3：Immunoprecipitation verifies differential expressions of the RBP4 in lunger and normal healthy controls person.
Embodiment
The examination embodiment in pulmonary tuberculosis patient to RBP4 albumen of embodiment 1
(1) collection and preparation of urine standard
Urine specimen comes from Wuhan City medical treatment center, according to various means such as clinical diagnosis, X images, Phlegm incubations,
It is defined as activity lung pulmonary tuberculosis, and excludes AIDS, the patient newly accepted for medical treatment of disease of urinary tract.76 people successively are have collected,
Everyone collects 30-50mL urina sanguinis mud-stream urines every time.Normal healthy controls urine specimen is collected simultaneously.
(2) urine specimen processing and the optimization of protein extracting method
Carried out the optimization of sample process condition, it is determined that processing routine be：Urine specimen adds protease inhibitors and (is purchased from sieve
Family name company), centrifugation removes cell fragment and impurity, 3kDa ultrafiltration through membranes concentration, with TCA- acetone methods [2] protein precipitation, dialysis
Desalination obtains purifying protein, reaches two dimensional electrophoresis albumen loading standard.
(3) Urine proteins two dimensional electrophoresis
To adhesive tape pH scopes, sample-loading buffer, adhesive tape equilibrium liquid, etc. point focusing program, SDS-PAGE concentration and voltage are carried out
Condition optimizing, finally determines with that pH3-10 is non-linear, the linear adhesive tape of pH4-7 carries out two dimensional electrophoresis；The system of sample-loading buffer
It is standby:8M urea, 2M thiocarbamides, 0.5% (w/v) propane sulfonic acid inner salt, 0.52% (w/v) ampholytes (pH310), the sulphur threoses of 60mM bis-
Alcohol, 40mM trishydroxymethylaminomethane and 0.01% bromophenol blue；Adhesive tape equilibrium liquid be mother liquor (w/v) dithiothreitol (DTT) that adds 2% and
2.5% (w/v) iodacetyl ammonia, respectively balances 15min；Choose 10%SDS-PAGE；SDS-PAGE voltages 50V, 30min, 200V,
6h；Etc. point focusing program such as table 1.
The Urine proteins two dimensional electrophoresis program of table 1
| Program | Voltage | Time |
| Aquation | 50V | 16h |
| S1 | 250V | 3h |
| S2 | 1000V | 6h |
| S3 | 10000V | 3h |
| S4 | 10000V | 60000VH |
| S5 | 500V | 8h |
Lunger and control group crowd's Urine proteins do two dimensional electrophoresis respectively, using PDQuest 8.0 (Bio-Rad) software point
Analysis differential protein carries out MADEL-TOF-MS analyses to it, obtains 19 protein sequences, has carried out bioinformatic analysis and mirror
Fixed, obtaining 1 has the host protein of potential mark meaning and to this difference Urine proteins (retinol-binding protein
4, RBP4, its sequence is shown in SEQ ID NO：1；P02753-HUMAN)
(4) verified using immunoprecipitate
Collect the urine of 25 lungers (5 one group of people, totally 5 groups) and 25 Healthy Peoples (5 one group of people, totally 5 groups)
Liquid, uniformly mixes concentration, carries out Western Blot analyses, experimental verification RBP4 albumen is in pulmonary tuberculosis human urine
Differential expression, and carry out statistical analysis using Image J softwares.As a result Fig. 3 is seen.
5. result of the test
1) general features of research object, the results are shown in Table 2.
The case-control crowd characteristic of table 2 is distributed
2) expression of the RBP4 albumen in lunger and control group and its diagnostic value to lung pulmonary tuberculosis：
Such as Fig. 1：The present invention is altogether to using two dimensional electrophoresis and MADEL-TOF-MS in 29 consumptives and 7 normal healthy controls persons
Verify this Urine proteins.MADEL-TOF-MS is carried out to it using (Bio-Rad) the software analysis differential proteins of PDQuest 8.0
Analysis, obtains 19 protein sequences, has further carried out bioinformatic analysis and immunoprecipitate identification, and the present invention is obtained
RBP4 has the host protein of potential mark meaning, while having carried out variance analysis to RBP4 albumen.As a result show that this RBP4 urinates
There is significant difference between the two groups in the expression of albumen.
In the checking of Image J gray analysis immunoprecipitation, expression of the RBP4 Urine proteins in consumptive is right higher than health
According to 3.82 times of person.The testing result that the mark albumen for relying on the present invention is obtained further illustrates the index of this Urine proteins to pulmonary tuberculosis
The examination of disease has certain diagnostic value, and albumen of the invention can detect the diagnostic kit of pulmonary tuberculosis for preparation
The middle application as antigen protein.
The RBP4 albumen of the present invention of embodiment 2 application of the sandwich ELISA in the diagnostic kit for preparing detection pulmonary tuberculosis
The clonal expression of 1.RBP4 albumen
According to SEQ ID NO disclosed by the invention：Sequence shown in 1, clones RBP4 genes, and in expression in escherichia coli,
Obtain target antigen albumen；Recombinant protein further is extracted using affinity chromatography (according to a conventional method), the restructuring target for obtaining purifying resists
Former rRBP4 (or recombinant protein rRBP4)；
2. it is prepared by recombinant protein rRBP4 monoclonal antibody and polyclonal antibody
Antibody is prepared (referring to document 3,4) according to a conventional method, including：RRBP4 immunizing rabbits will be purified, rabbit-anti RBP4 is obtained
Antibody；Immune mouse, obtains the anti-RBP4 monoclonal antibodies of mouse (monoclonal antibody and the reference of the preparation method of polyclonal antibody simultaneously：
Zhu Li equalitys,《Immunology common experimental method》, People's Medical Officer Press, the method reported in 2000 editions and Xue Qingshan,《Body
The philosophy and technique of outer culture》, Science Press, the method reported in version for 2001)；
3. set up detection RBP4 sandwich ELISA
The anti-rRBP4 of mouse monoclonal antibody is coated with ELISA, closed with 5% defatted milk, the urine containing RBP4 or other body fluid
For sample to be tested, rabbit-anti rRBP4 is detection antibody, and HRP- goat-antis rabbit (commercialization reagent) is secondary antibody, and TMB/H2O2 is substrate,
HF terminating reactions, OD value (OD is detected with ELIASA at wavelength 630nm630).Make standard with the rRBP4 of above-mentioned purifying bent
Line, using healthy negative human urine mean protein values ± 2~3SD as threshold value, sets up urine RBP4 detection sandwich ELISAs, further group
Dress up kit.Kit component is as follows：
(1) one piece of elisa plate (being coated with the anti-rRBP4 polyclonal antibodies of mouse)
(2) rRBP4 positive criteria products (dried frozen aquatic products)
(3) sample diluting liquid (conventional formulation) and cleaning solution (conventional formulation)
(4) rabbit-anti rRBP4 polyclonal antibodies
(5) HRP- goat-antis rabbit secondary antibody
(6) nitrite ion (conventional formulation) and terminate liquid (conventional formulation)
Bibliography：
1.Rembert Pieper,Christine L.Gatlin,et al(2004)Characterization of the human
urinary proteome:A method for high-resolution display of urinary proteins on
two-dimensional electrophoresis gels with a yield of nearly 1400distinct protein
spots.Proteomics,4:1159–1174.
2.Chih-Ming Lu1,Yu-Jen Wu et al(2011)Identification of low-abundance proteins via
fractionation of the urine proteome with weak anion exchange chromatography.Proteome
Science,9:17.
3. Zhu Li equalitys,《Immunology common experimental method》, People's Medical Officer Press, 2000 editions
4. Xue Qing is apt to,《The philosophy and technique of in vitro culture》, Science Press, version in 2001.
Claims (2)
1. a kind of Urine proteins RBP ELISA RBP4, its protein sequence such as SEQ ID NO：Shown in 1.
2. the Urine proteins RBP ELISA described in claim 1 is in detection pulmonary tuberculosis urine reagent box is prepared as mark
The application of will antigen protein.
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Application publication date: 20170815 |