CN105223364B - Serum SHBG albumen is as pulmonary tuberculosis blood serum designated object and its application - Google Patents
Serum SHBG albumen is as pulmonary tuberculosis blood serum designated object and its application Download PDFInfo
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- CN105223364B CN105223364B CN201510653780.4A CN201510653780A CN105223364B CN 105223364 B CN105223364 B CN 105223364B CN 201510653780 A CN201510653780 A CN 201510653780A CN 105223364 B CN105223364 B CN 105223364B
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Abstract
A kind of serum SHBG (Sex hormone binding globulin) albumen is as serum of tuberculosis patients mark and its application, the serum SHBG albumen is applying the moon, expression substantially increases in Smear positive tuberculosis and Drug resistant pulmonary tubeculosis patients serum, using iTRAQ combination MALDI TOF/MS technology for detection, 5 peptide fragments of Mass Spectrometer Method SHBG albumen are applying the moon, expression is apparently higher than expression in the serum of pneumonia patient and normal person in Smear positive tuberculosis and Drug resistant pulmonary tubeculosis patients serum, ELISA and immunohistochemistry also verify the high expression in serum of tuberculosis patients and tissue of SHBG albumen.Detected suitable for the auxiliary of pulmonary tuberculosis serum, study of incident mechanism and anti-tuberculosis drugs exploitation.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of application of blood serum designated object, specially a kind of serum SHBG eggs
It is used as serum of tuberculosis patients mark and its application in vain.
Background technology
Tuberculosis is the chronic infectious disease as caused by mycobacterium tuberculosis infection, 4~8 weeks incubation periods, wherein 80% occurs in lung
Portion.China belongs to one of high burden country of 22, whole world tuberculosis, and number of patients is only second to India, occupies second place of the world, removes
Number of patients is more, and the order of severity of China's drug resistance of tuberculosis is only second to Russia in the world, resistance sexploitation will
Tubercufosis control is set to return to no chemotherapy epoch, and the tuberculosis patient that the world today there are about 2/3rds is multi-drug resistant in occurring
Among danger.Therefore, tuberculosis and resistant tuberculosis have turned into the serious public health in China and social concern.Due to facing at present
, the problems such as recall rate is low and/or incubation time is long be present, far in the conventional Sputum smears of bed and the method for Sputum culturing detection tubercle bacillus
Can not meet the needs of clinical detection；Resistance, liver damage, intestines and stomach be present in simultaneously clinical at present anti-tuberculosis drugs treatment
The side effects such as discomfort, allergy, and anti-scarring agent repair focus ability, the problems such as sequelae, therefore, develop new disease mark
Will thing, it is significant to preventing and treating lungy.In recent years, the rapid development with mass-spectrometric technique and proteomics research
Go deep into, generate serum quantitative proteomics new technology, the technology is intended to that various disease is screened and identified using mass-spectrometric technique
The mark in serum is circulated, has been found that and determine many new disease markers using the technology, further to disclose
The mechanism of causing a disease of disease, the discovery of new drug targets is guided to provide important clue, the present invention is exactly serum Quantitative Western
New discovery of the omics technology in tuberculosis research.
The content of the invention
It is an object of the invention to provide a kind of serum SHBG albumen as serum of tuberculosis patients mark and its application, solution
Certainly recall rate existing for prior art is low and/or the problem of incubation time is long, while solves anti-tuberculosis drugs clinical at present
There is the side effect such as resistance, liver damage, gastrointestinal discomfort, allergy in treatment, and anti-scarring agent repairs focus ability, rear to lose
The problem of disease.Aid in detecting suitable for pulmonary tuberculosis serum, study of incident mechanism and anti-tuberculosis drugs exploitation.
The technical scheme is that：A kind of serum SHBG (Sex hormone-binding globulin) albumen conduct
Serum of tuberculosis patients mark, it is to utilize iTRAQ (isobaric tags for relative and absolute
Quantitation, iTRAQ) combine MALDI-TOF/MS technology for detection smear negative tuberculosis patient, Smear positive tuberculosis patient, resistance to
The serum of medicine pulmonary tuberculosis patient, pneumonia patient and normal person, SHBG albumen are applying cloudy, Smear positive tuberculosis and Drug resistant pulmonary tubeculosis disease
Expression is apparently higher than expression in the serum of pneumonia patient and normal person, ELISA and immunohistochemistry in human serum
Also the high expression in serum of tuberculosis patients and tissue of SHBG albumen is verified.
The SHBG albumen high expression in serum of tuberculosis patients and tissue refers to that following 5 peptide fragment expressions increase
It is high：IALGGLLFPASNLR, QAEISASAPTSLR, LPLVPALDGCLR, SCDVESNPGIFLPPGTQAEFNLR, WHQVEVK.
The remarkable result of the present invention is：
, to comprehensive and systematic research lungy, a kind of blood is obtained using based on the quantitative proteomicses that iTRAQ is marked
Clear SHBG albumen passes through the serum ELISA and immunohistochemistry of large sample as tuberculosis patient blood serum designated object, the mark
Checking, it is as a result more reliable, valuable information can be provided for clinical practice.
Brief description of the drawings
Fig. 1 .SHBG iTRAQ relative quantitative assay figure
The iTRAQ of SCDVESNPGIFLPPGTQAEFNLR peptide fragments relative quantitative assay figure, and normal person and pneumonia ratio,
The peptide fragment obvious high expression in pulmonary tuberculosis serum.
Fig. 2 .ELISA detect SHBG expression figure
Healthy control：Normal control, Pneumonia：Pneumonia, SNP-TB：Smear negative tuberculosis, DR-TB：It is resistance to
Medicine pulmonary tuberculosis, SPP-TB：Pulmonary tuberculosis, ELISA detection serum SHBG expression, with normal control and pneumonia phase
Than the expression of SHBG albumen is bright in smear negative tuberculosis patient, drug-resistant pulmonary tuberculosis patients, Smear positive tuberculosis patients serum
Aobvious high expression.
Fig. 3 immunohistochemical assay detects the expression of SHBG albumen
Expression of the Immunohistochemical detection SHBG albumen in tuberculosis and control tissue.With control group
Than the obvious high expression in pulmonary tuberculosis tissue of SHBG albumen.
Embodiment
Technical scheme is further described below by way of specific embodiment.
Embodiment 1
Serum Quantitative Western group technology for detection SHBG five peptide fragments of albumen are expressed in tuberculosis patient serum to be increased
1. detect sample：Multi-drug resistance tuberculosis, apply cloudy tuberculosis, sputum smear positive TB, pneumonia patient and normal control serum
Each 10.Collection 2mL whole bloods, 4 DEG C of standing 1-2h treated that blood clotting separates out serum, 3 000g centrifugation 10min, collected on an empty stomach early morning
Supernatant, on ice dispense after deposit to -80 DEG C it is standby.
2. detection method：(1) high-abundance proteins are removed:Said according to multiple affine removal system human14 chromatogram column operations
It is bright, high-abundant protein from serum is removed, the cut being collected into is concentrated with freeze dryer.(2) desalination and protein content detection:Except Gao Feng
The serum 3000MWCO ultra-filtration centrifuge tubes spent after albumen, the triethylamine bicarbonate buffers of 50mmol/L pH 8.5 are added, repeatedly
3 times, desalination and collect protein fragments；Serum albumin content is determined using BCA methods, every group of low-abundance protein takes 100 μ g/ to manage, and freezes
It is dry.(3) proteolysis and mark iTRAQ:Drying sample is added into trypsase, 37 DEG C of digestion are overnight；Enzymolysis sample vacuum is done
After dry, it is dissolved in iTRAQ solution buffer solutions；ITRAQ reagents 113,117,118,119,120 mark healthy control group, resistance to respectively
Multiple medicine group, apply positive group, apply cloudy group and pneumonia group serum protein antioxidant peptide, the sample after mark removes salt plug by C18spin column
Desalination, freeze.(4) offline two-dimensional HPLC separation and point target:Dry mark sample loading buffer A (10mmol/L
KH2PO4,25%CAN, pH 2.7) redissolve and dilute 10 times, be loaded to SCX prepacked columns, after sample-loading buffer A washings, with containing
There is the buffer B stepwise elution that KCl concentration is respectively 35,50,75,100,125,150,175,200,250 and 300mmol/L,
Collect the polypeptide eluted under the conditions of different gradient concentrations.Anti-phase C18 posts gradient leaching is carried out after each component sample dilution being collected into
Wash and put target.(5) mass spectral analysis and data processing:Mark tandem mass spectrum identification and the relative quantitative assay of peptide fragment public using ABI
The 5800MALDI-TOF/TOF protein analyzers of department, analytical data of mass spectrum is with Protein Pilot 2.0 to SWISSPROT numbers
Retrieval identification albumen is carried out according to storehouse, report confidence level is higher than 95% albumen, while each group detection data and 113 report ions
Integrating peak areas carries out relative quantitative assay, selects the result of P≤0.05 to be reported.(6) statistical procedures：Using
SPSS14.0 softwares carry out statistical analysis, and continuous data is represented with mean ± SD, and the comparison between two sample averages is examined using t
Test, be that difference is statistically significant with P≤0.05.
3. testing result：Mass spectrum identifies the unique peptide fragment IALGGLLFPASNLR of 5, SHBG albumen altogether,
QAEISASAPTSLR, LPLVPALDGCLR, SCDVESNPGIFLPPGTQAEFNLR, WHQVEVK, overall coverage 22.4%,
117/113=5.706,118/113=6.0681,119/113=11.1686 P ＜ 0.05；120/113=1.0864 P ＞
0.05.SHBG albumen expression in resistance tuberculosis, painting yin constipation core and sputum smear positive TB human serum substantially increases.Such as Fig. 1 institutes
Show.
Embodiment 2
Preparation contains IALGGLLFPASNLR, QAEISASAPTSLR, LPLVPALDGCLR, SCDVESNPGIFLPPG
5 peptide fragment SHBG protein antibodies such as TQAEFNLR, WHQVEVK, ELISA detections are carried out using the antibody, it is found that SHBG albumen exists
Tuberculosis patient serum expression increases.
1. sample：Collect 24 normal healthy controls, 15 pneumonia patients, 160 tuberculosis patient serum, wherein multi-drug resistant 35
Example, applies cloudy tuberculosis 70, sputum smear positive TB 55.
2. detection method：1. it is loaded：Blank well is set, blank control wells are not added with sample and enzyme marking reagent, remaining each step
Operate identical, gauge orifice, testing sample hole.Each Kong Zhongxian adds 100 μ l sample diluting liquid RD1-75 on enzyme mark coating plate,
Then 50 μ l blank control, titer and testing sample are added toward ELISA Plate bottom hole portion, the final dilution factor of sample is 100 times.To the greatest extent
Amount does not touch hole wall, gently rocks mixing.2. incubate：With 3 hours of the rearmounted incubation at room temperature of shrouding film shrouding.3. match somebody with somebody liquid：By 25
Times concentrated cleaning solution is with standby after 25 times of dilutions of distilled water.4. wash：Shrouding film is thrown off, discards liquid, is dried, per the μ l of hole 400
Cleaning solution, discarded after standing 30 seconds, be so repeated 4 times, pat dry.5. incubate：The μ l of SHBG conjugates 200 are added per hole, use shrouding
1 hour of the rearmounted incubation at room temperature of film shrouding.6. wash：Operation is the same as 4..7. develop the color：The μ l of substrate solution 200 are first added per hole, put room
Temperature incubates half an hour, and gently concussion mixes, 37 DEG C of lucifuge colour developings.8. terminate：Add the μ l of terminate liquid 50 per hole, terminating reaction, this
When blueness is vertical turns yellow.9. determine：With blank air-conditioning zero, 450nm wavelength sequentially measures the absorbance in each hole, i.e. OD values.Measure
Carried out after Ying Jia terminate liquids within 15 minutes.As a result：When the presentation of Quality Control point blank well is colourless, OD values are 0, you can judge detection
As a result.
3. result：SHBG albumen multi-drug resistance tuberculosis people, sputum smear positive TB people, apply cloudy tuberculosis patient, pneumonia patient and
Concentration (nmol/L) in normal control serum is respectively 181.76 ± 200.09,160.43 ± 75.45,159.75 ± 75.47,
60.63 ± 59.38,33.39 ± 34.24, p<0.0001, expression substantially increases in tuberculosis patient serum, such as Fig. 2 institutes
Show.
Embodiment 3
Preparation contains IALGGLLFPASNLR, QAEISASAPTSLR, LPLVPALDGCLR,
The SHBG albumen primary antibodies of 5 peptide fragments such as SCDVESNPGIFLPPGTQAEFNLR, WHQVEVK, immuning tissue is carried out using the primary antibody
Chemical detection, find the high expression in tuberculosis of SHBG albumen.
1. sample：Using US Biomax commercialization organization chip detections SHBG expression, wherein including 40 lung knots
Core tissue, 10 control tissues, control tissue are normal lung tissue and cancerous lung tissue.
2. experimental procedure：1. dewax：Successively by slide be put into dimethylbenzene-alcohol of the alcohol of dimethylbenzene -100% -100% -
The alcohol of -80% alcohol of -90% alcohol of 95% alcohol -70%.Put 10min in above two reagents, behind 6 reagents be
5min.2. antigen retrieval：A period of time is rinsed after dewaxing in clear water, adds 3%H2O210min is soaked, then outwells H2O2,
Washed in clear water twice, add citrate buffer solution, be put into boiling 3min in microwave ingle, it is general just to arrive boiling,
Room temperature is cooled to, then boiling once, is cooled to room temperature again.3. serum is closed：After being cooled to room temperature, citrate buffer solution is fallen
Fall, wash 2 times, and slide is placed in 5min in PBS, wash 2 times, dry the PBS liquid around tissue, at once plus serum, so
After be put into half an hour in room temperature or 37 DEG C of incubators.4. plus primary antibody：Slide in incubator is taken out, slide is dried with blotting paper
Serum around reverse side and face weave, add primary antibody, control experiment is just in the tissue plus PBS of control.Primary antibody is added after 4 DEG C
Preserved overnight in refrigerator.5. plus secondary antibody：Slide is taken out from refrigerator, is put into PBS and washes 3 times, each 5min, dry tissue
Around PBS after add secondary antibody, after be placed in half an hour in room temperature or 37 DEG C of incubators.6. slice, thin piece is taken out from incubator, it is put into
Washed in PBS 3 times, each 5min, reagent solution C is added dropwise, is subsequently placed in 10-15min in room temperature or 37 DEG C of incubators.7. plus developer：
Slice, thin piece is taken out from incubator, is put into PBS and washes 3 times, each 5min, developer is added after drying the PBS around tissue.Colour developing
The configuration of agent：In 1ml water plus 1 drips developer A, shakes up, and then plus 1 drips developer B, shakes up, then adds 1 drop developer C, shakes
It is even.8. redye：After the slice, thin piece after colour developing is rinsed into a period of time with clear water, it is soaked in haematine and dyes.9. mounting：With neutrality
Natural gum is dropped in beside tissue, then is covered with cover glass, to be first laid flat side, then gently be put down opposite side, in order to avoid bubble is produced,
Seal to be placed in vent cabinet after slice, thin piece and dry.
3. result：SHBG protein expression total positiveses in 40 tuberculosis, SHBG expression is complete in 10 control tissues
Portion is negative, as shown in Figure 3.
Claims (1)
- A kind of 1. application of SHBG albumen in preparing for lunger's blood serum designated object detection reagent, it is characterised in that Cloudy lunger, Smear positive tuberculosis patient are applied using iTRAQ combination MALDI-TOF/MS technology for detection, Drug resistant pulmonary tubeculosis is suffered from The serum of person, patients with pneumonia and normal person, five peptide fragment IALGGLLFPASNLR, QAEISASAPTSLR contained by SHBG albumen, LPLVPALDGCLR, SCDVESNPGI FLPPGTQAEFNLR and WHQVEVK, applying cloudy, painting sun and Drug resistant pulmonary tubeculosis patient Expression is applying apparently higher than expression in patients with pneumonia and the serum of normal person, ELISA detection SHBG albumen in serum High expression, Immunohistochemical detection SHBG albumen are high in tuberculosis in the moon, Tu Yang, Drug resistant pulmonary tubeculosis patients serum Expression.
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| CN108226515A (en) * | 2017-11-28 | 2018-06-29 | 泰州泽成生物技术有限公司 | A kind of kit and its test method for measuring sex hormone binding globulin content |
| TWI682175B (en) * | 2018-04-16 | 2020-01-11 | 臺北醫學大學 | Method of gastric cancer diagnosis |
| CN109030834A (en) * | 2018-08-11 | 2018-12-18 | 南京市儿童医院 | A method of the aortic coaractation marker detection based on proteomics |
| CN109852629A (en) * | 2019-02-22 | 2019-06-07 | 广西医科大学 | A kind of expression and purification method of recombined human sex hormone binding globulin N-terminal 51-218aa |
| CN110437322B (en) * | 2019-08-30 | 2021-11-30 | 上海市肺科医院 | Marker for tuberculosis diagnosis and application thereof |
| CN114778656B (en) * | 2022-03-29 | 2023-02-14 | 浙江苏可安药业有限公司 | Serum metabolic marker for detecting drug-resistant tuberculosis and kit thereof |
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