CN110244048A - Application of the SERPING1 albumen as marker in exploitation diagnostic activities reagent lungy - Google Patents

Application of the SERPING1 albumen as marker in exploitation diagnostic activities reagent lungy Download PDF

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CN110244048A
CN110244048A CN201910531175.8A CN201910531175A CN110244048A CN 110244048 A CN110244048 A CN 110244048A CN 201910531175 A CN201910531175 A CN 201910531175A CN 110244048 A CN110244048 A CN 110244048A
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serping1
albumen
gene
tuberculosis
peripheral blood
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杨秉芬
翟斐
蒋静
安红娟
苏瑾文
王若
曹志红
刘艳华
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8th Medical Center of PLA General Hospital
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    • 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
    • G01N33/531—Production of immunochemical test materials
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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

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Abstract

Application the invention discloses SERPING1 albumen as marker in exploitation diagnostic activities reagent lungy.Compared with Healthy People and tuberculosis latent infection person, the expression quantity of SERPING1 gene is dramatically increased in the PBMCs of active tuberculosis patient；The expression quantity of SERPING1 gene can be used for distinguishing active tuberculosis patient and tuberculosis latent infection person；The expression quantity of SERPING1 gene can be used for distinguishing active tuberculosis patient and Healthy People.Therefore, SERPING1 albumen and/or SERPING1 gene can be used as marker exploitation diagnostic activities reagent lungy.The present invention has great application value.

Description

SERPING1 albumen is as marker in exploitation diagnostic activities reagent lungy Application
Technical field
The invention belongs to Medical Immunology diagnostic techniques fields, and in particular to SERPING1 albumen is being developed as marker Application in diagnostic activities reagent lungy.
Background technique
Tuberculosis is infectious disease caused by mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB), mainly Pass through respiratory infectious.It may occur in which three kinds of different as a result, first is that immunity of organisms is preferable, MTB directly quilts after MTB infection human body It removes；Second is that MTB is inhibited by immunity of organism, but can not be fully erased, develop as tuberculosis latent infection (latent Tuberculosis infection, LTBI)；Third is that MTB is proliferated rapidly in body, active tuberculosis is developed into.Tuberculosis Disease is the serious infectious diseases that China needs emphasis prevention and control.
Diagnosis of tuberculosis mainly has the methods of imaging diagnosis, tulase diagnosis and immunology diagnosis at present, but has one Fixed disadvantage.Imaging diagnosis is difficult to differentiate between pulmonary tuberculosis and other pulmonary diseases.It is high that tulase diagnoses false negative.Immunology is examined It is disconnected mainly divide antibody test and cellular immunity detect (such as tuberculin skin test (tuberculin skin test, TST) and Interferon release test (interferon gamma release assays, IGRA)).TST and IGRA is to pass through detection The immune main treating tuberculosis of body is cellullar immunologic response to evaluate tuberculosis infection situation.The country is existing mostly by TST as main inspection Survey means, generally by purified protein derivative (PPD) (PPD) strong positive or in a short time from feminine gender switch to it is positive and without clinical tuberculosis evidence Person is judged as tulase latent infection person.Due to TST feature be it is easy to operate, cheap, have become at present clinically most A kind of common and the simplest tubercle bacillus affection diagnostic method.But PPD is the antigen mixture slightly mentioned from mycobacterium tuberculosis, Comprising 200 multiple proteins, wherein being much the common antigen ingredient of non-tuberculous mycobacteria and BCG vaccine, therefore TST is determined Poor specificity is detected, is also easy to produce false positive results in BCG vaccine (BCG) inoculation crowd and non-tuberculous mycobacteria infection population. TST can only only have 70-80% according to the reaction power auxiliary diagnosis of skin, sensitivity.In addition TST there are when check fee, need It wants subject to pay a return visit (72h), skin test operation and result and explains there is the disadvantages of subjective dependence.IGRA is inhaled using enzyme linked immunological Adhesion test (ELISA) or Enzyme linked immunospot (ELISPOT) method quantitatively detect subject's whole blood or the single core of peripheral blood IFN-γ detection release reaction of the cell to Specific Antigen of Mycobacterium Tuberculosis (ESAT6, CFP10 and TB7.7), for The diagnosis of tubercle bacillus affection, but IGRA is difficult to differentiate between active tuberculosis and latent tuberculosis infection.Activity cannot be early diagnosed Property tuberculosis, on the one hand lead to delay treatment, increase medical expense and the death rate；On the other hand it not can be effectively controlled the infection sources, Cause diffusion lungy.Therefore, special, effective active tuberculosis diagnostic reagent is developed to tuberculosis prevention and treatment with important Meaning.
SERPING1 albumen (serine protease inhibitor, clade G, member 1) is serine stretch protein Enzyme C1 inhibitor, encoding gene mutation can lead to hereditary angioedema, related to the morbidity of senile macular degeneration.Most Closely some researches show that HIV infection causes monocyte SERPING1 expression to increase, and the latter is positively correlated with virus titer.Albumen Matter group is the study found that SERPING1 protein concentration increases in the hydrothorax of tuberculous pleurisy.SERPING1 egg is had no at present It is white to be used for diagnostic activities pulmonary tuberculosis.
Summary of the invention
The purpose of the present invention is diagnostic activities tuberculosis.
The present invention protects the substance for detecting SERPING1 albumen preparing the application in product first；The product Function can be following a1) at least one of to a3): a1) diagnostic activities tuberculosis；A2) whether diagnosis person under test is activity Property tuberculosis patient；A3) prevention and control tuberculosis.
The present invention also protects substance and device for detecting SERPING1 albumen preparing the application in product；The production The function of product can be following a1) at least one of to a3): a1) diagnostic activities tuberculosis；A2) diagnosis person under test whether be Active tuberculosis patient；A3) prevention and control tuberculosis；
Described device can be device first and/or device third；
Described device first may include data input device 1, data recordin module 1, data comparison module 1 and conclusion output mould Block 1；
Data input device 1 is used to input the expression numerical quantity of SERPING1 albumen；
Data recordin module 1 is used to store the expression numerical quantity of SERPING1 albumen；
Data comparison module 1 is used for will be in the expression quantity of SERPING1 albumen in person under test's peripheral blood and control peripheral blood The expression quantity of SERPING1 albumen is compared；
Conclusion output module 1 is for showing conclusion, i.e., if the expression quantity of SERPING1 albumen is high in person under test's peripheral blood The expression quantity of SERPING1 albumen in control peripheral blood, then conclusion output module 1 shows that person under test is active tuberculosis sufferer Person；If expression of the expression quantity of SERPING1 albumen lower than SERPING1 albumen in control peripheral blood in person under test's peripheral blood Amount, then conclusion output module 1 shows that person under test is inactive tuberculosis patient；
Described device third may include data input device 3, data recordin module 3, data comparison module 3 and conclusion output mould Block 3；
Data input device 3 is used to input the concentration values of SERPING1 albumen；
Data recordin module 3 is used to store the concentration values of SERPING1 albumen；
Data comparison module 3 is used for will be in the concentration of SERPING1 albumen in person under test's peripheral blood and control peripheral blood The concentration of SERPING1 albumen is compared；
Conclusion output module 3 is for showing conclusion, i.e., if the concentration of SERPING1 albumen is higher than in person under test's peripheral blood The concentration of SERPING1 albumen in peripheral blood is compareed, then conclusion output module 3 shows that person under test is active tuberculosis patient；Such as The concentration of SERPING1 albumen is lower than the concentration of SERPING1 albumen in control peripheral blood in fruit person under test's peripheral blood, then conclusion is defeated Module 3 shows that person under test is inactive tuberculosis patient out；
The control peripheral blood can be tuberculosis latent infection person or the peripheral blood of Healthy People.
Described device first can specifically be exported by data input device 1, data recordin module 1, data comparison module 1 and conclusion Module 1 forms.
Described device third can specifically be exported by data input device 3, data recordin module 3, data comparison module 3 and conclusion Module 3 forms.
The expression quantity of SERPING1 albumen is concretely from the PBMCs separated in peripheral blood in the peripheral blood In the expression quantity of SERPING1 albumen, serum in the expression quantity or blood plasma of SERPING1 albumen SERPING1 albumen expression quantity.
The present invention also protects the substance for detecting SERPING1 gene preparing the application in product；The function of the product Can be following a1) at least one of to a3): a1) diagnostic activities tuberculosis；A2) whether diagnosis person under test is activity Tuberculosis patient；A3) prevention and control tuberculosis.
The present invention also protects substance and device second for detecting SERPING1 gene preparing the application in product；It is described The function of product can be following a1) at least one of to a3): a1) diagnostic activities tuberculosis；A2) whether diagnosis person under test For active tuberculosis patient；A3) prevention and control tuberculosis；
Described device second includes data input device 2, data recordin module 2, data comparison module 2 and conclusion output module 2；
Data input device 2 is used to input the expression numerical quantity of SERPING1 gene；
Data recordin module 2 is used to store the expression numerical quantity of SERPING1 gene；
Data comparison module 2 is used for will be in the expression quantity of SERPING1 gene in person under test's peripheral blood and control peripheral blood The expression quantity of SERPING1 gene is compared；
Conclusion output module 2 is for showing conclusion, i.e., if the expression quantity of SERPING1 gene is high in person under test's peripheral blood The expression quantity of SERPING1 gene in control peripheral blood, then conclusion output module 2 shows that person under test is active tuberculosis sufferer Person；If expression of the expression quantity of SERPING1 gene lower than SERPING1 gene in control peripheral blood in person under test's peripheral blood Amount, then conclusion output module 2 shows that person under test is inactive tuberculosis patient；
The control peripheral blood can be tuberculosis latent infection person or the peripheral blood of Healthy People.
Described device second can specifically be exported by data input device 2, data recordin module 2, data comparison module 2 and conclusion Module 2 forms.
The expression quantity of SERPING1 gene is concretely from the PBMCs separated in peripheral blood in the peripheral blood The expression quantity of SERPING1 gene.
The present invention also protects a kind of kit, it may include for detecting the substance of SERPING1 albumen and/or for detecting The substance of SERPING1 gene；The kit can have the function of following a1) at least one of to a3): a1) diagnostic activities knot Core disease；A2) whether diagnosis person under test is active tuberculosis patient；A3) prevention and control tuberculosis.
Any of the above-described " for the detecting the substance of SERPING1 albumen " can be the table for detecting SERPING1 albumen Substance up to the substance of amount and/or for detecting SERPING1 protein concentration.
Any of the above-described " for the detecting the substance of SERPING1 gene " can be the table for detecting SERPING1 gene Up to the substance of amount.
The expression quantity of any of the above-described SERPING1 albumen can be the opposite table of SERPING1 albumen reference internal reference albumen Up to amount.
The expression quantity of any of the above-described SERPING1 gene can be the opposite table of SERPING1 gene reference reference gene Up to amount.
The expression quantity of any of the above-described detection SERPING1 albumen specifically can be used Western Blot experiment and carry out.
Any of the above-described detection SERPING1 protein concentration is particularly used in Elisa experiment and carries out.
Any of the above-described substance or any of the above-described described for detecting for detecting the expression quantity of SERPING1 gene The substance of " relative expression quantity of SERPING1 gene reference reference gene " may include primer pair and internal control primer to composition Primer pair combination；
The primer pair can be made of primer SERPING1-F and primer SERPING1-R；The primer pair Sequence 6 of the target gene containing ordered list DNA fragmentation shown in the 324th to 448 from 5 ' ends；
The internal control primer primers F and primer R to can be made of；The target gene of the internal control primer pair can be people's internal reference base The all or part of cause.
The primer SERPING1-F can be following a1) or a2):
A1) single strand dna shown in the sequence 1 of sequence table；
A2) there is phase by sequence 1 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1 The DNA molecular of congenerous.
The primer SERPING1-R can be following a3) or a4):
A3) single strand dna shown in the sequence 2 of sequence table；
A4) there is phase by sequence 2 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2 The DNA molecular of congenerous.
The primers F can be following b1) or b2):
B1) single strand dna shown in the sequence 3 of sequence table；
B2) there is phase by sequence 3 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 3 The DNA molecular of congenerous.
The primer R can be following b3) or b4):
B3) single strand dna shown in the sequence 4 of sequence table；
B4) there is phase by sequence 4 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 4 The DNA molecular of congenerous.
Any of the above-described substance or any of the above-described described for detecting for detecting the expression quantity of SERPING1 gene The substance of " relative expression quantity of SERPING1 gene reference reference gene " the concretely primer pair combination.
Any of the above-described primer pair also belongs to protection scope of the present invention.
Using the expression quantity or detection SERPING1 gene of any of the above-described primer pair detection SERPING1 gene The relative expression quantity of reference reference gene also belongs to protection scope of the present invention.
Using the expression quantity or detection SERPING1 gene of any of the above-described primer pair combine detection SERPING1 gene The relative expression quantity of reference reference gene also belongs to protection scope of the present invention.
Above, using SERPING1 gene reference internal reference in any of the above-described primer pair combine detection person under test cDNA The method of the relative expression quantity of gene is concretely: using person under test cDNA as template, using any of the above-described primer pair Or then any of the above-described internal control primer uses 2 to real-time fluorescence quantitative PCR is carried out-ΔCtMethod, which calculates, to be obtained.The person under test CDNA can be the cDNA of the PBMCs separated in person under test's peripheral blood.
Any of the above-described internal reference albumen can be GAPDH albumen.
Any of the above-described reference gene can be GAPDH gene.
The present invention also protects Y1) or Y2) or Y3) or Y4).
Y1) application of the SERPING1 albumen as marker in exploitation diagnostic activities reagent lungy.
Y2) application of the SERPING1 albumen as marker in diagnostic activities tuberculosis.
Y3) application of the SERPING1 gene as marker in exploitation diagnostic activities reagent lungy.
Y4) application of the SERPING1 gene as marker in diagnostic activities tuberculosis.
The amino acid sequence such as sequence table of any of the above-described SERPING1 albumen (No. GeneID are as follows: NP_000053.2) Shown in middle sequence 5.The nucleotide sequence of any of the above-described SERPING1 gene (No. Genebank are as follows: NM_000062.2) is such as In sequence table shown in sequence 6.
Above, described lower than can be being lower than statistically.It is described that be higher than to be being higher than statistically.
It is demonstrated experimentally that compared with Healthy People and tuberculosis latent infection person, in the PBMCs of active tuberculosis patient The expression quantity of SERPING1 gene dramatically increases；The expression quantity of SERPING1 gene can be used for distinguishing active tuberculosis patient With tuberculosis latent infection person；The expression quantity of SERPING1 gene can be used for distinguishing active tuberculosis patient and Healthy People.Cause This, the expression quantity of SERPING1 gene has important application value in terms of diagnostic activities tuberculosis.
Detailed description of the invention
Fig. 1 is that real-time fluorescence quantitative PCR detects active tuberculosis patient, tuberculosis latent infection person and Healthy People The relative expression quantity of SERPING1 gene in PBMCs.
Fig. 2 is SERPING1 gene in ROC curve analytic activity tuberculosis patient and the PBMCs of tuberculosis latent infection person Relative expression quantity.
Fig. 3 is the opposite table of SERPING1 gene in the PBMCs of ROC curve analytic activity tuberculosis patient and Healthy People Up to amount.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even Mean value.
Ficoll-Paque PLUS is the product of U.S. GE company.96 orifice plates are the product of Millipore company.AIM VTMMedium serum free medium is the product of gibco company, catalog number 12055091.RPMI 1640 culture medium is The product of Gibco company, catalog number 11875-093.IFN-γ ELISPOT detection kit is the production for being company up to section Product.IFN-γ monoclonal captures antibody, IFN-γ detection antibody, tubercle bacillus differential mixed polypeptide A, tubercle bacillus differential mixing Polypeptide B and phytohemagglutin phytolectin are the component in IFN-γ ELISPOT detection kit.TRIzolTMReagent is The product of Invitrogen company.PrimeScriptTMRT reagent Kit with gDNA Eraser is TaKaRa The product of Biotechnology company.KAPATM Fast quantification PCR kit is the production of Kapa Biosystems company Product.2 × Green Master Mix is KAPATM Component in fast quantification PCR kit.Nuclease-free water is the U.S. The product of Ambion company.480II fluorescence quantitative PCR instrument is the product of Roche Holding Ag.
Cleaning solution: the PBS buffer solution of pH7.4,0.01M containing 0.05% (v/v) polysorbas20.
Application of the relative expression quantity in diagnostic activities tuberculosis of embodiment 1, SERPING1 gene
One, the acquisition of periphery blood specimen
1, the periphery blood specimen of tuberculosis latent infection person and the periphery blood specimen of Healthy People are distinguished
The sign or symptom of tuberculosis latent infection person and health per capita without tuberculosis morbidity, are distinguished in accordance with the following steps:
A, it is coated with
(1) 96 orifice plates are taken, every hole is added 100 μ L IFN-γ monoclonals and captures antibody, and 4 DEG C of coatings are overnight.
(2) after completing step (1), 96 orifice plate is taken, abandons liquid phase, the PBS buffer solution washing two of pH7.4,0.01M is added Secondary (each 1min), pats dry.
(3) after completing step (2), 96 orifice plate is taken, pH7.4,0.01M that 200 μ L contain 2% (v/v) BSA is added in every hole PBS buffer solution, 37 DEG C of incubation 1h.
(4) after completing step (3), 96 orifice plate is taken, abandons liquid phase, it is primary that the rinse of RPMI 1640 culture medium is added.
B, the preparation of PBMCs suspension
(1) the RPMI 1640 culture medium of 2mL peripheral blood to be measured and 2mL is uniformly mixed；Then it is slowly added into being equipped with In the sterile centrifugation tube of 3mL Ficoll-Paque PLUS, room temperature, 2000rcf are centrifuged 20min, are from top to bottom divided into three layers.
(2) it after completing step (1), draws middle layer and is transferred in the centrifuge tube of the RPMI 1640 culture medium equipped with 10mL, Mixing is gently blown and beaten with dropper, room temperature, 1400rpm are centrifuged 7min.
(3) after completing step (2), the centrifuge tube is taken, abandons supernatant, the culture of RPMI 1640 that 6mL is preheated to 37 DEG C is added Liquid is resuspended, and room temperature, 1400rpm are centrifuged 7min.
(4) after completing step (3), the centrifuge tube is taken, abandons supernatant, addition is preheated to 37 DEG C of AIM VTMMedium without Blood serum medium is resuspended, and obtaining concentration is 2.5 × 106The PBMCs suspension of a/mL.
C, immunodotting detects
With reference to the specification of IFN-γ ELISPOT detection kit, immunodotting detection is carried out using the kit.Reagent The equal by specification of dosage carries out.Specific step is as follows:
(1) take into 96 orifice plate of step a, every hole be added 100 μ L step b preparation PBMCs suspension (about 2.5 × 105A PBMC).
(2) after completing step (1), tubercle bacillus differential mixed polypeptide A is added in each detection hole or tubercle bacillus differential is mixed Close polypeptide B；Serum free medium is added in each negative control hole；Phytohemagglutin phytolectin is added in each Positive control wells.
(3) after completing step (2), 96 orifice plate is placed in incubator, 37 DEG C, 5%CO2Cultivate 20h.
(4) after completing step (3), 96 orifice plate is taken, abandons supernatant, the ice water of 200 μ L pre-cooling, 4 DEG C of placement 10min are added (purpose is lytic cell).
(5) after completing step (4), 96 orifice plate is taken, abandons supernatant, 5 times is washed with cleaning solution and (200 μ L is added every time to wash Liquid is washed, washs 1min every time), it pats dry.
(6) after completing step (5), 96 orifice plate is taken, every hole is added 100 μ L IFN-γ and detects antibody (Avidin mark Note) dilution (being mixed by the PBS buffer solution of 99 parts by volume pH7.4,0.01M and 1 parts by volume IFN-γ detection antibody), 37 DEG C be incubated for 1h.
(7) after completing step (6), 96 orifice plate is taken, abandons supernatant, 5 times is washed with cleaning solution and (200 μ L is added every time to wash Liquid is washed, washs 1min every time), it pats dry.
(8) after completing step (7), take 96 orifice plate, every hole be added the streptomysin of 100 μ LHRP label dilution (by The streptomysin of the PBS buffer solution of 99 parts by volume pH7.4,0.01M and 1 parts by volume HRP label mixes), 37 DEG C of incubation 1h.
(9) after completing step (8), 96 orifice plate is taken, abandons supernatant, 5 times is washed with cleaning solution and (200 μ L is added every time to wash Liquid is washed, washs 1min every time), it pats dry.
(10) after completing step (9), 96 orifice plate is taken, zymolyte is added in every hole, at room temperature black out colour developing 15-45min.
(11) after completing step (10), 96 orifice plate is taken, with distilled water flushing 3 times (purpose is stopped reaction), then 96 orifice plate is stored at room temperature, naturally dry.
(12) after completing step (11), 96 orifice plate is taken, immunodotting calculating instrument (Cellular is used Technology Ltd, USA) image and spot count are carried out, then make the following judgment: the number of spots of negative control hole is small When 6, if the number of spots that the number of spots of detection hole subtracts negative control hole be 6 or more, if detection hole be positive, if The number of spots that the number of spots of detection hole subtracts negative control hole is less than 6, then detection hole is negative；The spot of negative control hole Point number be 6 or more when, if the number of spots of detection hole be negative control hole number of spots 2 times or more, if detect Hole be the positive, if the number of spots of detection hole than negative control hole number of spots less than 2 times, if detection hole be feminine gender.Inspection Gaging hole is the positive, then peripheral blood to be measured provides (i.e. the periphery blood specimen of tuberculosis latent infection person) by tuberculosis latent infection person；Inspection Gaging hole is feminine gender, then peripheral blood to be measured provides (i.e. the periphery blood specimen of Healthy People) by Healthy People.
2, the acquisition of periphery blood specimen
(1) active tuberculosis group: 40 periphery blood specimens.
40 periphery blood specimens: 40 patient's (all trouble for being clinically diagnosed as active tuberculosis disease are extracted respectively The equal informed consent of person) peripheral blood 2-3mL, be placed in the pipe of anticoagulant blood-collecting containing EDTA, turn upside down 5-6 times (purpose be anti-freezing liquid and Peripheral blood mixes), obtain 40 periphery blood specimens.
(2) tuberculosis latent infection group: 25 periphery blood specimens.
25 periphery blood specimens: 25 tuberculosis latent infection persons for being clinically diagnosed as tuberculosis latent infection are extracted respectively The peripheral blood 2-3mL of (equal informed consent) is placed in the pipe of anticoagulant blood-collecting containing EDTA, and turning upside down 5-6 times, (purpose is for anti-freezing liquid and outside All blood mixes), obtain 25 periphery blood specimens.
(3) healthy control group: 46 periphery blood specimens.
46 periphery blood specimens: the peripheral blood 2-3mL of 46 Healthy Peoples (equal informed consent) is extracted respectively, is placed in containing EDTA Anticoagulant blood-collecting pipe turns upside down 5-6 times (purpose is that anti-freezing liquid and peripheral blood mix), obtains 46 periphery blood specimens.
111 periphery blood specimens need to be placed in room temperature (do not freeze or refrigerate), and standing time is less than 6h.
Two, application of the relative expression quantity of SERPING1 gene in diagnostic activities tuberculosis
The amino acid sequence of SERPING1 albumen (No. GeneID are as follows: NP_000053.2) is as shown in sequence 5 in sequence table. Encode the nucleotide sequence of the gene (abbreviation SERPING1 gene, No. Genebank are as follows: NM_000062.2) of SERPING1 albumen As shown in sequence 6 in sequence table.
1, the acquisition of the cDNA of 111 periphery blood specimens
(1) preparation of PBMCs suspension
The peripheral blood to be measured of b in step 11 is replaced in step 12 111 periphery blood specimens, other steps respectively It is constant, obtain the PBMCs suspension of 111 periphery blood specimens.
(2) RNA is extracted
1. taking PBMCs suspension (each sample about 1 × 10 of 111 periphery blood specimens respectively6A cell), 400rcf centrifugation 5min collects precipitating.
2. the precipitating is taken, using TRIzol after completing step 1.TMReagent extracts RNA.
(3) synthesis of cDNA
The RNA for taking the PBMCs suspension of 111 periphery blood specimens respectively, using PrimeScriptTM RT reagent Kit With gDNA Eraser carries out reverse transcription, obtains the cDNA of 111 periphery blood specimens.
2, the preparation of primer pair combination
According to the nucleotide sequence of SERPING1 gene, primer pair shown in table 1 is designed and synthesized.
According to the nucleotide sequence of GAPDH gene, internal control primer pair shown in table 1 is designed and synthesized.
Primer pair combination is by primer pair and internal control primer to forming.
Each primer (HPLC purifying) is synthesized by Shanghai Sheng Gong Biotechnology Co., Ltd.
Table 1
3, the relative expression quantity of real-time fluorescence quantitative PCR detection SERPING1 gene
Respectively using the cDNA of 111 periphery blood specimens as template, the primer pair or internal control primer that are prepared using step 2 To progress real-time quantitative PCR, and then obtain the relative expression quantity of SERPING1 gene in each template.Specific step is as follows:
(1) reaction system 1 and reaction system 2 are prepared
Reaction system 1 is 20 μ L, by 10 μ L2 × Green Master Mix, 0.4 μ LSERPING1-F aqueous solution (concentration Be 10 μM), 0.4 μ L SERPING1-R aqueous solution (concentration is 10 μM), 2 μ L templates (5-20ng) and 7.2 μ L nuclease-free water groups At.
Reaction system 2 is 20 μ L, and by 10 μ L2 × Green Master Mix, 0.4 μ L GAPDH-F aqueous solution, (concentration is 10 μM), 0.4 μ L GAPDH-R aqueous solution (concentration be 10 μM), 2 μ L templates (5-20ng) and 7.2 μ L nuclease-free waters form.
(2) real-time quantitative PCR detects
Each reaction system that step (1) is prepared is existedIt is carried out on 480II fluorescence quantitative PCR instrument real When quantitative PCR detection.Use 2-ΔCtMethod calculates the relative expression quantity of SERPING1 gene in each template.
Reaction condition: 95 DEG C of initial denaturation 3min；95 DEG C of 5s, 60 DEG C of 30sec, 40 circulations, fluorescence signal are extending the stage Acquisition.
Experimental result is shown in Fig. 1 (TB is active tuberculosis group, and LI is tuberculosis latent infection group, and Nor is healthy control group). The result shows that compared with healthy control group and tuberculosis latent infection group, SERPING1 gene in the PBMCs of active tuberculosis group Relative expression quantity dramatically increase.
(3) it statisticallys analyze
It is for statistical analysis using result of the GraphPad Prism 5 to step (2).
According to the relative expression quantity of SERPING1 gene in the PBMCs of active tuberculosis group and tuberculosis latent infection group, Receiver operating curve's analysis is carried out using GraphPad Prism 5.As a result see Fig. 2.The result shows that below ROC curve Product is 0.849 (p < 0.0001), illustrates that the relative expression quantity of SERPING1 gene can be used for distinguishing active tuberculosis patient With tuberculosis latent infection person.When the transcriptional level cut-off value of SERPING1 gene takes 0.01349, youden index (Youden ' s index, YI) is maximum, and sensitivity at this time is 77.50%, and specificity is 88.00%.
According to the relative expression quantity of SERPING1 gene in the PBMCs of active tuberculosis group and healthy control group, use GraphPad Prism 5 carries out Receiver operating curve's analysis.As a result see Fig. 3.The result shows that area is under ROC curve 0.856 (p < 0.0001) illustrates that the relative expression quantity of SERPING1 gene can be used for distinguishing active tuberculosis patient and be good for Health people.When the transcriptional level cut-off value of SERPING1 gene takes 0.01789, youden index is maximum, and sensitivity at this time is 70.00%, specificity is 89.13%.
It is important that the above results show that the relative expression quantity of SERPING1 gene has in terms of diagnostic activities tuberculosis Application value.
<110>the 8th medical center of Chinese People's Liberation Army General Hospital
<120>application of the SERPING1 albumen as marker in exploitation diagnostic activities reagent lungy
<160> 6
<170> PatentIn version 3.5
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305 310 315 320
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340 345 350
Leu Ser Leu Val Ile Leu Val Pro Gln Asn Leu Lys His Arg Leu Glu
355 360 365
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370 375 380
Lys Leu Glu Met Ser Lys Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg
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Glu Phe Phe Asp Phe Ser Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu
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Asp Pro Asp Leu Gln Val Ser Ala Met Gln His Gln Thr Val Leu Glu
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Leu Thr Glu Thr Gly Val Glu Ala Ala Ala Ala Ser Ala Ile Ser Val
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tccttaccca ggtcctgctc ggggctgggg agaacaccaa aacaaacctg gagagcatcc 780
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gtgtcacctc agtctctcag atcttccaca gcccagacct ggccataagg gacacctttg 900
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Claims (10)

1. the substance for detecting SERPING1 albumen is preparing the application in product；The function of the product is following a1) extremely At least one of a3): a1) diagnostic activities tuberculosis；A2) whether diagnosis person under test is active tuberculosis patient；A3) prevent Control tuberculosis.
2. the substance and device for detecting SERPING1 albumen are preparing the application in product；The function of the product is as follows A1) at least one of to a3): a1) diagnostic activities tuberculosis；A2) whether diagnosis person under test is active tuberculosis patient； A3) prevention and control tuberculosis；
Described device is device first and/or device third；
Described device first includes data input device 1, data recordin module 1, data comparison module 1 and conclusion output module 1；
Data input device 1 is used to input the expression numerical quantity of SERPING1 albumen；
Data recordin module 1 is used to store the expression numerical quantity of SERPING1 albumen；
Data comparison module 1 is used for will be in the expression quantity of SERPING1 albumen in person under test's peripheral blood and control peripheral blood The expression quantity of SERPING1 albumen is compared；
Conclusion output module 1 is for showing conclusion, i.e., if the expression quantity of SERPING1 albumen is higher than pair in person under test's peripheral blood According to the expression quantity of SERPING1 albumen in peripheral blood, then conclusion output module 1 shows that person under test is active tuberculosis patient；Such as The expression quantity of SERPING1 albumen is then tied lower than the expression quantity of SERPING1 albumen in control peripheral blood in fruit person under test's peripheral blood Show that person under test is inactive tuberculosis patient by output module 1；
Described device third includes data input device 3, data recordin module 3, data comparison module 3 and conclusion output module 3；
Data input device 3 is used to input the concentration values of SERPING1 albumen；
Data recordin module 3 is used to store the concentration values of SERPING1 albumen；
Data comparison module 3 is used for SERPING1 in the concentration of SERPING1 albumen in person under test's peripheral blood and control peripheral blood The concentration of albumen is compared；
Conclusion output module 3 is for showing conclusion, i.e., if the concentration of SERPING1 albumen is higher than control in person under test's peripheral blood The concentration of SERPING1 albumen in peripheral blood, then conclusion output module 3 shows that person under test is active tuberculosis patient；If to The concentration of SERPING1 albumen is lower than the concentration of SERPING1 albumen in control peripheral blood in survey person's peripheral blood, then conclusion exports mould Block 3 shows that person under test is inactive tuberculosis patient；
The control peripheral blood is the peripheral blood of tuberculosis latent infection person or Healthy People.
3. the substance for detecting SERPING1 gene is preparing the application in product；The function of the product is following a1) extremely At least one of a3): a1) diagnostic activities tuberculosis；A2) whether diagnosis person under test is active tuberculosis patient；A3) prevent Control tuberculosis.
4. the substance and device second for detecting SERPING1 gene are preparing the application in product；The function of the product is such as Lower a1) at least one of to a3): a1) diagnostic activities tuberculosis；A2) whether diagnosis person under test is active tuberculosis sufferer Person；A3) prevention and control tuberculosis；
Described device second includes data input device 2, data recordin module 2, data comparison module 2 and conclusion output module 2；
Data input device 2 is used to input the expression numerical quantity of SERPING1 gene；
Data recordin module 2 is used to store the expression numerical quantity of SERPING1 gene；
Data comparison module 2 is used for will be in the expression quantity of SERPING1 gene in person under test's peripheral blood and control peripheral blood The expression quantity of SERPING1 gene is compared；
Conclusion output module 2 is for showing conclusion, i.e., if the expression quantity of SERPING1 gene is higher than pair in person under test's peripheral blood According to the expression quantity of SERPING1 gene in peripheral blood, then conclusion output module 2 shows that person under test is active tuberculosis patient；Such as The expression quantity of SERPING1 gene is then tied lower than the expression quantity of SERPING1 gene in control peripheral blood in fruit person under test's peripheral blood Show that person under test is inactive tuberculosis patient by output module 2；
The control peripheral blood is the peripheral blood of tuberculosis latent infection person or Healthy People.
5. a kind of kit, including the substance for detecting SERPING1 albumen and/or the object for detecting SERPING1 gene Matter；The kit has the function of following a1) at least one of to a3): a1) diagnostic activities tuberculosis；A2 person under test) is diagnosed It whether is active tuberculosis patient；A3) prevention and control tuberculosis.
6. the application as described in Claims 1-4 is any or the kit as described in claim 5, it is characterised in that:
" for the detecting the substance of SERPING1 albumen " be for detect the expression quantity of SERPING1 albumen substance and/or For detecting the substance of SERPING1 protein concentration；
" for the detecting the substance of SERPING1 gene " is the substance for detecting the expression quantity of SERPING1 gene.
7. application as claimed in claim 6 or kit, it is characterised in that:
The expression quantity of the SERPING1 albumen is the relative expression quantity of SERPING1 albumen reference internal reference albumen；
The expression quantity of the SERPING1 gene is the relative expression quantity of SERPING1 gene reference reference gene.
8. application or kit as claimed in claims 6 or 7, it is characterised in that:
The substance or described for detecting " SERPING1 gene reference internal reference for detecting the expression quantity of SERPING1 gene The substance of the relative expression quantity of gene " includes that primer pair and internal control primer combine the primer pair of composition；
The primer pair is made of primer SERPING1-F and primer SERPING1-R；The target gene of the primer pair Sequence 6 containing ordered list DNA fragmentation shown in the 324th to 448 from 5 ' ends；
The internal control primer is formed to by primers F and primer R；The target gene of the internal control primer pair is the whole of people's reference gene Or part.
9. primer pair described in claim 8.
10.Y1) or Y2) Y3) or Y4):
Y1) application of the SERPING1 albumen as marker in exploitation diagnostic activities reagent lungy；
Y2) application of the SERPING1 albumen as marker in diagnostic activities tuberculosis；
Y3) application of the SERPING1 gene as marker in exploitation diagnostic activities reagent lungy；
Y4) application of the SERPING1 gene as marker in diagnostic activities tuberculosis.
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Application publication date: 20190917