CN107267659A - Detect the purposes of the product of TRIM genes and/or protein level - Google Patents
Detect the purposes of the product of TRIM genes and/or protein level Download PDFInfo
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention provides the purposes of detection TRIM genes and/or the product of protein level, the products application is in the product for preparing discriminating, diagnosis and/or examination active tuberculosis.When the product of present invention detection TRIM genes and/or protein level is used to prepare the product of discriminating, diagnosis and/or examination active tuberculosis, enable the product distinguish latent infection person and active tuberculosis patient, and sample is readily available, with quick, efficiently, high sensitive, high accuracy, operate relative ease, it is easy to the characteristics of promoting.
Description
Technical field
The present invention relates to biological technical field, the purposes of the product of TRIM genes and/or protein level is more particularly to detected.
Background technology
Tuberculosis is a kind of chronic infectious disease caused by mycobacterium tuberculosis infection.Although the mankind with it is lungy
Struggle never stopped, but it still seriously threatens the publilc health in the whole world.According to the newest report of the World Health Organization, 2015
Year, the new hair cases of tuberculosis in the whole world is about 10,400,000, and about 1,800,000 people die from tuberculosis, and Chinese neopathy number is 91.8
Ten thousand, death toll is 3.8 ten thousand, and morbidity total number of persons occupies the 4th, the world, popular situation very severe.In recent years, with resistance
The coinfection of the extensive appearance, in addition HIV of tuberculosis and multi-drug resistance tuberculosis, makes control lungy face a severe challenge.
One of important measures of TB endemic are controlled to be turned off the infection sources, and active tuberculosis patient is the main infection sources, because
This early, be accurately diagnosed to be active tuberculosis patient, and give and timely treat, just can effectively reduce lungy climing
Prolong.
At present, clinically there is notable defect, such as Sputum smears acid-fast stain inspection-sensitivity in diagnosis of tuberculosis method
It is relatively low；Traditional mycobacterium tuberculosis cultivation-cumbersome, positive rate is low and time-consuming；Automate shell vial method-needs
Expensive instrument and equipment；Chest x-ray detection-pulmonary tuberculosis feature is sexually revised not clear aobvious and can not timely found early stage；Tuberculosis
Rhzomorph skin test-cannot distinguish between natural infection and BCG vaccination；IFN-r release tests (IGRAs)-cannot distinguish between latent infection
Person and active tuberculosis patient；GeneXpert MTB/RIF methods-directly from the genetic test of sputum specimen progress, in sensitiveness side
Face is not significantly improved, and there is certain false negative rate and false positive rate.Serology antibody test-specificity and sensitiveness
It is undesirable.Therefore, in the urgent need to developing new diagnostic marker, diagnostic method quick, efficiently, sensitive is set up.
After people's infection mycobacterium tuberculosis, about only 5%~10% people eventually develops into active tuberculosis, and
Most people are in a kind of latent infection state without clinical symptoms.However, about 5%~10% latent infection person, when it
During hypoimmunity, mycobacterium tuberculosis can again activate and develop into active tuberculosis.Mycobacterium tuberculosis infects body
Afterwards, the immune state of host cell plays vital effect in pathogenic process lungy, wherein, in immune response
Regulator, such as cell factor, chemotactic factor (CF), cell surface receptor etc. always are the emphasis of research.And Recent study is sent out
Existing three domain proteins (TRIM) family can as host restriction factor, participate in innate immunity reaction, therefore and standby
It is concerned.
TRIM protein families are made up of numerous members, are almost present in all multicellular animals bodies, and it can be as immune
Regulatory protein and E3 ubiquitin ligases, participate in the innate immune response of body.With deepening continuously for research, it has been found that TRIM
Protein family can not only maintain the normal physiological function of body, such as cell growth, cell differentiation, film reparation, signal path and
Apoptosis etc., moreover it is possible to participate in the regulation process of a variety of diseases.In addition, TRIM albumen has mark sheet in mankind's various disease
Up to spectrum, all there is important suggesting effect for advancing of disease, diagnosis or prognosis.Such as Kosaka has found TRIM29 in stomach cancer
Up-regulated expression in patient, and the transfer of lymph node can be used to refer to as a mark.Yamada etc. is had found in testis life
TRIM44 up-regulated expressions in cell colonization tumour (TGCT) patient, are expected to turn into a new prognostic factor.In addition, Nenasheva
Deng the versatile stem cell by inducing Parkinson (PD) patient and Healthy People, compare transcript profile and find TRIM6 and TRIM24 bases
Because the expression quantity in two groups of crowds has notable difference, these genes are pointed out to develop as disease and immune system disorder
Ews gene.However, the research of current expression and adjustment effect on TRIM genes in tuberculosis also has much not
Part is known, if the feature TRIM gene related to tuberculosis can be found, it is possible to prepare prevention lungy, diagnosing and control
The product for the treatment of provides new target spot and thinking.
It would therefore be highly desirable to which a pair TRIM gene related to tuberculosis infection is studied, new diagnosis marker is found, is set up
A kind of discriminating more rapidly, efficiently, sensitive, diagnosis, the method for examination active tuberculosis.
The content of the invention
One aspect of the present invention be for lack in the prior art more rapidly, efficiently, sensitively differentiate, diagnose and/or
There is provided the purposes of a kind of detection TRIM genes and/or the product of protein level for examination method lungy.
In order to solve the above-mentioned technical problem, the technical scheme that provides of the present invention is：
Detect the purposes of the product of TRIM genes and/or protein level, the said goods be applied to prepare differentiate, diagnosis and/
Or the product of examination active tuberculosis.
Preferably, in an embodiment of the invention, above-mentioned TRIM genes be selected from TRIM35, TRIM47,
One or more in TRIM65 or TRIM68.
Preferably, in an embodiment of the invention, above-mentioned TRIM albumen be selected from TRIM35, TRIM47,
The albumen expressed by one or more of genes in TRIM65 or TRIM68.
In the present invention, the product of any appropriate detection TRIM genes and/or protein level can realize the present invention
Purpose, include but is not limited to quantitatively and/or qualitatively product.But it is above-mentioned preferably, in an embodiment of the invention
Detect product of the product of TRIM genes and/or protein level in sample for quantitatively detection TRIM genes and/or protein level.
It is highly preferred that in above-mentioned detection sample TRIM genes and/or protein level product be selected from gene magnification primer,
One or more in probe, genetic chip, antibody, protein-chip or antibody.
It is highly preferred that above-mentioned is for expanding one or more of genes in TRIM35, TRIM47, TRIM65 or TRIM68
PCR primer.
It is further preferred that PCR primer is a pair or several to PCR primer in SEQ ID No.1-8.
Above-mentioned PCR primer is preferably real-time fluorescence quantitative PCR (qRT-PCR) primer.
Preferably, in an embodiment of the invention, inventor screens target using following methods：
1. one group of target TRIM gene is provided, including TRIM35, TRIM47, TRIM65 and TRIM68 gene；
2. the peripheral blood of collection activity tuberculosis patient, tuberculosis latent infection person and Healthy People, cracks peripheral red blood cells,
RNA is extracted, is saved backup；
3. Optimal Experimental condition, primer is designed for target gene, detected using qRT-PCR method in three groups of crowds
The expression of TRIM35, TRIM47, TRIM65 and TRIM68 gene；
4. use 2-ΔΔCtMethod is analyzed data, by decision tree analysis TRIM35, TRIM47, TRIM65 and
TRIM68 genes are respectively used to differentiate the diagnosis effect of active tuberculosis.
Another aspect of the present invention there is provided it is a kind of be used for differentiate, diagnose and/or examination active tuberculosis examination
Agent box, mentioned reagent box includes the product of TRIM genes and/or protein level in detection sample.
Preferably, mentioned reagent box includes being selected from gene magnification primer, probe, genetic chip, antibody, protein core
One or more of products in piece or antibody.
It is highly preferred that mentioned reagent box includes being used to expand one kind in TRIM35, TRIM47, TRIM65 or TRIM68
Or the PCR primer of several genes.
It is further preferred that mentioned reagent box includes a pair or several to PCR primer in SEQ ID No.1-8.
Above-mentioned PCR primer is preferably qRT-PCR primers.
Normally, any necessary part, such as kit specification are also included in mentioned reagent box.
Another aspect of the present invention there is provided a kind of mentioned reagent box and prepare discriminating, diagnosis and/or examination activity
Application in property tuberculosis.
Beneficial effects of the present invention：
Because IFN-r release tests (IGRAs) have quick, the features such as susceptibility is high, at present clinically using more
Extensively, but the maximum defect of the method is cannot distinguish between latent infection person and active tuberculosis patient, and the maximum of the present invention
Advantage is to make up this defect.
In addition, sample of the present invention is readily available, with quick, efficiently, high sensitive, high accuracy operates relative ease,
Easy to spread the characteristics of.
Brief description of the drawings
Fig. 1 is TRIM genes relative expression levels in active tuberculosis patient, latent infection person and normal healthy controls person
Scatter diagram, wherein Figure 1A be TRIM 35, Figure 1B be TRIM 47, Fig. 1 C be TRIM65, Fig. 1 D be TRIM 68, wherein, TB is
Active tuberculosis patient, LTBI is latent infection person, and HC is normal healthy controls person；
Fig. 2 is the decision tree analysis schematic diagram of TRIM genes, and wherein Fig. 2A is TRIM 35, and Fig. 2 B are TRIM 47, Fig. 2 C
For TRIM 65, Fig. 2 D are TRIM 68, and wherein N is sample number, and TB is active tuberculosis patient, and LTBI is latent infection person.
Sequence explanation
SEQ ID No.1-2 are respectively the upstream and downstream PCR primer sequence of TRIM 35 in the present invention；
SEQ ID No.3-4 are respectively the upstream and downstream PCR primer sequence of TRIM 47 in the present invention；
SEQ ID No.5-6 are respectively the upstream and downstream PCR primer sequence of TRIM 65 in the present invention；
SEQ ID No.7-8 are respectively the upstream and downstream PCR primer sequence of TRIM 68 in the present invention.
Embodiment
The invention discloses the purposes of detection TRIM genes and/or the product of protein level, those skilled in the art can be with
Present disclosure is used for reference, technological parameter realization is suitably modified.It is important to note that all similar replacements and change are to ability
It is that, it will be apparent that they are considered as being included in the present invention, and related personnel can substantially not take off for field technique personnel
From being modified or suitably being changed with combining to content described herein on the basis of present invention, spirit and scope, to realize
With apply the technology of the present invention.
In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology
The implication that personnel are generally understood that.Also, the laboratory operation step such as cell biology used herein, molecular biology is equal
For widely used conventional steps in corresponding field.
In order that those skilled in the art more fully understands technical scheme, with reference to specific embodiment pair
The present invention is described in further detail.
In this embodiment, term " active tuberculosis patient " refers to clinical symptoms lungy, through applying
Piece or culture are positive, and C-XF is abnormal, do not start or just start cycle chemistry drug therapy make a definite diagnosis tuberculosis patient.
Term " latent infection person " refers to without clinical chief complaint, tuberculin skin test (TST) experiment and T-SPOT
Experiment is the positive, C-XF negative patient.
Term " normal healthy controls person " refers to that TST experiments and T-SPOT experiments are feminine gender, chest X without clinical chief complaint
Line piece negative patient.
Above crowd excludes diabetes, HIV and other autoimmune diseases.
Above crowd comes from attached BJ Chest Science Hospital of the Capital University of Medical Sciences.
In this embodiment, sample is divided into three groups by inventor：Active tuberculosis group (TB, 39), latent sense
Dye group (LTBI, 29) and healthy control group (HC, 33).
The preparation of the Peripheral Blood Nucleated Cells of embodiment 1
The peripheral blood 2mL of collection each subject is in the heparin tube of liquaemin anti-freezing on an empty stomach.Using erythrocyte cracked liquid
(solarbio, article No. R1010) prepares karyocyte in 6h, comprises the following steps that：
1 according to fresh whole blood：Erythrocyte cracked liquid volume ratio is 1:3, add 6mL erythrocyte cracked liquids, gently be vortexed or
It is reverse to mix.
2 place 15min on ice, and be gently vortexed mixes twice therebetween, and after erythrocyte splitting, solution should be limpid transparent
's.
3 collect cell：4 DEG C, 450 × g centrifuges 10min to precipitate leucocyte, discards supernatant.
4 add the erythrocyte cracked liquid of 2 times of volumes (4mL) into leukocyte cell pellet, and leucocyte is resuspended.
5 collect cell：4 DEG C, 450 × g centrifugation 10min precipitation leucocytes suck carefully and thoroughly supernatant.
6 add 1mLTRIZOL reagents (Invitrogen, article No.：15596) cell lysis, is then transferred in centrifuge tube,
- 80 DEG C are stored in, for subsequent extracted RNA.
The expression change of the application qRT-PCR method detection TRIM genes of embodiment 2
1.RNA extracting
The cell pyrolysis liquid that will be obtained in embodiment 1, melts at room temperature, and often pipe adds 0.2mL chloroforms, with lower play on hand
Strong vibration 15s, produces pink turbid solution without lamination, is stored at room temperature 3min.12000r/min, 4 DEG C of centrifugation 15min, takes
Go out centrifuge tube, sample is divided into three layers：Colourless supernatant aqueous phase, middle white layer and pink lower floor's organic phase.It is careful to inhale
Take colorless supernatant aqueous phase to move to new centrifuge tube, 1/2TRIZOL volumes (500mL) isopropanol be added to obtained supernatant water,
Gently mix, be stored at room temperature 10min.12000r/min, 4 DEG C of centrifugation 10min, discards supernatant, adds 1mL75% ethanol (nothing
RNase water is prepared), gently mix.7500r/min, 4 DEG C of centrifugation 5min, carefully exhaust supernatant, and drying at room temperature precipitates 5min, plus
Enter 30 μ L without RNase water dissolve RNA precipitate, in -80 DEG C preserve, it is stand-by.
2. reverse transcription
Using the PrimeScriptTMRT reagent Kit (article No.s of TAKARA companies：RR037Q), obtained in taking 1)
The μ g of RNA 2, carry out reverse transcription, and total system is 20 μ L.Reaction system is prepared and carried out on ice, in order to ensure reaction solution preparation
Accuracy, reduces the error caused during packing, should prepare reaction solution according to the volume more slightly larger than actual amount, be eventually adding RNA
Sample.System is as follows：
The reaction system prepared, carries out following react in PCR instrument：37 DEG C, 15min；85 DEG C, 5s；After completion of the reaction,
40 μ L ddH are added into each cDNA samples2O, after mixing, is stored in 4 DEG C.
3. real-time fluorescence quantitative PCR
3.1 primer sequence
3.2 real-time fluorescence quantitative PCR testing goal genes
CDNA to be obtained in 2) is used as templatePremix Ex TaqTMII kits (TAKARA, goods
Number：RR820Q the expression of TRIM35, TRIM47, TRIM65 and TRIM68 gene) is detected, while being internal reference from GAPDH
Gene, while setting the negative control hole for being not added with template.Detected using ABI7500 real-time fluorescence quantitative PCR instrument.Reactant
System is as follows：
Reaction condition is：95 DEG C of pre-degeneration 40s, 1 circulation；95 DEG C of denaturation 5s, 60 DEG C of annealing 34s, 40 circulations.
3.3 data processings and statistical analysis
According to qRT-PCR result, result is analyzed with ABI7500software v2.0.6, with GAPDH genes
For reference gene, pass through 2-△△CtThe method person that calculates latent infection and normal healthy controls person relative to active tuberculosis patient
Expression quantity.Using the analysis method of one way ANOVA combination Bonferroni coefficient correlations, threshold value is set to P<0.05,
With significant difference.As a result as shown in figs. 1A-d, wherein abscissa represents different crowd, and ordinate represents relative expression quantity,
Ordinate value shows that more greatly its expression is higher.
As a result show, TRIM35, TRIM47, TRIM65 and TRIM68 gene express water in active tuberculosis patient
Averagely it is substantially less than the expression of latent infection person and normal healthy controls person.(in Figure 1A-D, TB is active tuberculosis patient,
LTBI is latent infection person, and HC is normal healthy controls, and horizontal line is average value ± standard error, and wherein ns represents p>0.05, * represents p=
0.01-0.05, * * represent p<0.01, * * * represent p<0.001, * * * * represent p<0.0001.)
The TRIM genes of embodiment 3 are used for diagnostic activities decision method lungy
According to the experimental result of embodiment 2, qRT-PCR diagnostic activities tuberculosis patient and tuberculosis latent infection can be passed through
Person.The present invention specifically provides four kinds of independent diagnositc decision methods, respectively using TRIM35, TRIM47, TRIM65 and
TRIM68 relative fold expression, is analyzed by ROC curve, it is determined that the optimal threshold of each gene.It is specific as follows：
Using TRIM35 relative fold expression when, if TRIM35 relative fold expression be less than 1.939, testing result
For the positive, that is, it is determined as active tuberculosis；If during more than or equal to 1.939, testing result is feminine gender, that is, it is determined as that tuberculosis is hidden
Infection.The judgement is used in 68 samples (including 39 active tuberculosis patients and 29 latent infection persons) of the present embodiment
The sensitiveness of method diagnosis is 92.31%, and specificity is 96.55%, and differentiation correctness is 94.12% (Fig. 2A).
Using TRIM47 relative fold expression when, if TRIM47 relative fold expression be less than 2.059, testing result
For the positive, that is, it is determined as active tuberculosis；If during more than or equal to 2.059, testing result is feminine gender, that is, it is determined as that tuberculosis is hidden
Infection.The judgement is used in 68 samples (including 39 active tuberculosis patients and 29 latent infection persons) of the present embodiment
The sensitiveness of method diagnosis is 94.87%, and specificity is 96.55%, and differentiation correctness is 95.59% (Fig. 2 B).
Using TRIM65 relative fold expression when, if TRIM65 relative fold expression be less than 2.567, testing result
For the positive, that is, it is determined as active tuberculosis；If during more than or equal to 2.567, testing result is feminine gender, that is, it is determined as that tuberculosis is hidden
Infection.The judgement is used in 68 samples (including 39 active tuberculosis patients and 29 latent infection persons) of the present embodiment
The Sensitivity and Specificity of method diagnosis is 100%, and differentiation correctness is 100% (Fig. 2 C).
Using TRIM68 relative fold expression when, if TRIM68 relative fold expression be less than 2.516, testing result
For the positive, that is, it is determined as active tuberculosis；If during more than or equal to 2.516, testing result is feminine gender, that is, it is determined as that tuberculosis is hidden
Infection.The judgement is used in 68 samples (including 39 active tuberculosis patients and 29 latent infection persons) of the present embodiment
The sensitiveness of method diagnosis is 94.87%, and specificity is 100%, and differentiation correctness is 97.06% (Fig. 2 D).
TRIM35, TRIM47, TRIM65 and TRIM68 are in active tuberculosis patient and latent infection person in the present embodiment
With significant differential expression.QRT-PCR detections are carried out by stencil design specific primer of the TRIM genes, available for living
In the quick detection of dynamic property tuberculosis patient peripheral blood, with higher sensitiveness and accuracy, relative ease, Ke Yiti are operated
The early diagnostic rate of high active tuberculosis.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>Attached BJ Chest Science Hospital of the Capital University of Medical Sciences
<120>Detect the purposes of the product of TRIM genes and/or protein level
<130> None
<160> 8
<170> PatentIn version 3.5
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Claims (10)
1. detect the purposes of the product of TRIM genes and/or protein level, it is characterised in that the products application reflects in preparation
Not, the product of diagnosis and/or examination active tuberculosis.
2. purposes according to claim 1, it is characterised in that the gene be selected from TRIM35, TRIM47, TRIM65 or
One or more in TRIM68.
3. purposes according to claim 1, it is characterised in that the albumen be selected from TRIM35, TRIM47, TRIM65 or
The albumen expressed by one or more of genes in TRIM68.
4. the purposes according to claim 1-3 any one, it is characterised in that in the detection sample TRIM genes and/
Or the product of protein level is the product of quantitatively detection TRIM genes and/or protein level.
5. purposes according to claim 4, it is characterised in that TRIM genes and/or protein level in the detection sample
Product be one or more in gene magnification primer, probe, genetic chip or antibody.
6. purposes according to claim 5, it is characterised in that the product be for expand TRIM35, TRIM47,
The PCR primer of one or more of genes in TRIM65 or TRIM68.
7. purposes according to claim 6, it is characterised in that the PCR primer is one in SEQ ID No.1-8
Pair or it is several to PCR primer.
8. it is a kind of be used for differentiate, diagnose and/or examination active tuberculosis kit, it is characterised in that the kit bag
Include the product of TRIM genes and/or protein level in detection sample as claimed in claim 4.
9. kit according to claim 8, it is characterised in that TRIM genes and/or albumen water in the detection sample
Flat product is a pair or several to PCR primer in SEQ ID No.1-8.
10. a kind of kit as claimed in claim 8 or 9 is preparing discriminating, diagnosis and/or examination active tuberculosis product
In application.
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|---|---|---|---|
| CN201710718522.9A CN107267659B (en) | 2017-08-21 | 2017-08-21 | Application of product for detecting TRIM gene and/or protein level |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710718522.9A CN107267659B (en) | 2017-08-21 | 2017-08-21 | Application of product for detecting TRIM gene and/or protein level |
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| CN107267659A true CN107267659A (en) | 2017-10-20 |
| CN107267659B CN107267659B (en) | 2020-10-16 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112143792A (en) * | 2020-09-30 | 2020-12-29 | 中国医学科学院病原生物学研究所 | Application of RNF181 as tuberculosis diagnosis molecular marker |
| CN112725434A (en) * | 2021-01-20 | 2021-04-30 | 首都医科大学附属北京胸科医院 | Rifampicin-resistant tuberculosis molecular marker, detection reagent and application thereof |
| CN115873942A (en) * | 2022-12-29 | 2023-03-31 | 徐州医科大学 | Novel target of TRIM35 as anti-adenovirus infection therapeutic drug and application thereof |
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| CN102150043A (en) * | 2008-06-25 | 2011-08-10 | 贝勒研究院 | Blood transcriptional signature of mycobacterium tuberculosis infection |
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| CN102150043A (en) * | 2008-06-25 | 2011-08-10 | 贝勒研究院 | Blood transcriptional signature of mycobacterium tuberculosis infection |
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| Y. CHEN等: "Gene expression profiling of the TRIM protein family reveals potential biomarkers for indicating tuberculosis status", 《MICROBIAL PATHOGENESIS》 * |
| Y. HAN等: "TRIM47 overexpression is a poor prognostic factor and contributes to carcinogenesis in non-small cell lung carcinoma", 《ONCOTARGET》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112143792A (en) * | 2020-09-30 | 2020-12-29 | 中国医学科学院病原生物学研究所 | Application of RNF181 as tuberculosis diagnosis molecular marker |
| CN112725434A (en) * | 2021-01-20 | 2021-04-30 | 首都医科大学附属北京胸科医院 | Rifampicin-resistant tuberculosis molecular marker, detection reagent and application thereof |
| CN115873942A (en) * | 2022-12-29 | 2023-03-31 | 徐州医科大学 | Novel target of TRIM35 as anti-adenovirus infection therapeutic drug and application thereof |
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