CN105039548A - IL-4 detection primer, kit and semi-quantitative detection method - Google Patents
IL-4 detection primer, kit and semi-quantitative detection method Download PDFInfo
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Abstract
The invention provides an IL-4 detection primer. The IL-4 detection primer comprises a primer P1 with a sequence chart SEQ.ID.No.1 base sequence and a primer P2 with a sequence chart SEQ.ID.No.2 base sequence. Synthesis of mRNA based on the code IL-4 in the gene expression process of the primer is much earlier than that of a secreted IL-4 protein product, and the value of the protein expression index and the quantity of the mRNA of the corresponding code are in linear direct ratio; the mRNA of the corresponding code is amplified according to the RT-PCR method, and the quantity of the corresponding IL-4 protein product can be obtained according to the quantity of mRNA; the net content after secretion, adsorption, consumption and degradation of IL-4, but not the real-time fixed-point level of a regulation and control mechanism after genetic transcription and translation and after-translation in the IL-4 producing process, is detected; the real-time IL-4 expression level in cells or tissues can be truly and accurately reflected. On the basis of the primer, the invention further provides a method including the IL-4 detection primer detection kit and utilizing the primer for semi-quantitative detection.
Description
Technical field
The invention belongs to cytokines measurement technical field, be specifically related to a kind of IL-4 and detect primer, test kit and semi-quantitative detection method.
Background technology
Cytokine is the low-molecular-weight soluble protein that immunogen, mitogen or other stimulants induction various kinds of cell produces, and has and regulates the several functions such as inherent immunity and adaptive immunity, Growth of Cells and damaged tissue reparation.Cytokine can be divided into interleukin-, Interferon, rabbit, tumor necrosis factor superfamily, G CFS, chemokine, somatomedin etc.Wherein, important has IL-2, IL-3, IL-4, IL-5, IFN-γ, TNF-β and neuroleukin etc., in cell-cell interaction, immunomodulatory, hematopoiesis and inflammatory process, play important regulative.
Had many documents and achievement in research to show, IL-4 expression level and various diseases and immunity of organism closely related.By the detection to IL-4 expression level, the more early acquisition of a lot of essential information and the early diagnosis of some disease relevant can be beneficial to.And often use Radioimmunoassay of vascular endothelial growth in the detection of existing IL-4, the ELISA kit that such as rate of utilization is the highest; But the existing test kit that these detect or method, its detect for be IL-4 secretion, absorption, consume and degraded after net content, not genetic transcription in cytokine production process, translation and post-translational control mechanism submits necessary information, the result detected cytokine gene expression level real-time in cell or tissue can not be reflected, so can not fix a point to detect truth as IL-4 index in real time.
Summary of the invention
Object of the invention process is to overcome existing defect, provides a kind of and can detect primer, test kit and semi-quantitative detection method by the truer and accurate IL-4 detecting IL-4 expression level real-time in cell or tissue.
In order to realize foregoing invention object, the technical scheme of the embodiment of the present invention is as follows:
A kind of IL-4 detects primer, comprises the primer P1 with sequence table SEQ .ID.No.1 base sequence, the primer P2 with sequence table SEQ .ID.No.2 base sequence.
Based on above-mentioned detection primer, the present invention also proposes the IL-4 detection kit comprising above-mentioned primer.
Above-mentioned primer of the present invention based on the mRNA synthesis of the IL-4 that encodes in genetic expression process far prior to the IL-4 protein product of secretion, and protein expression amount number with the relation of the amount linear proportional of the corresponding mRNA encoded; Increased by RT-PCR mode the mRNA of corresponding coding, then can be obtained the amount of corresponding IL-4 protein product by the amount of mRNA; What it detected is not IL-4 secretion, absorption, consume and net content after degraded, but the real-time point level of genetic transcription in IL-4 production process, translation and post-translational control mechanism; It can be real-time in truer and accurate response cell or tissue IL-4 expression level.
The present invention is based on above-mentioned primer and test kit, propose a kind of above-mentioned primer structure competitive RT-PCR that adopts further and carry out IL-4 semi-quantitative detection method, comprise the steps:
Obtain IL-4 cellular control unit and IL-4 to be measured group of cell;
Extract the total serum IgE of described IL-4 cellular control unit, and the primer P1 of apparatus ordered list SEQ.ID.No.1 base sequence carries out RT-PCR with the competitive primer P3 with sequence table SEQ .ID.No.3 base sequence, obtains competitive template;
Extract the total serum IgE of described IL-4 to be measured group of cell, and carry out reverse transcription and obtain cDNA template to be measured;
After described cDNA template to be measured and competitive template mixing, the primer P1 of apparatus ordered list SEQ.ID.No.1 base sequence carries out PCR with the primer P2 with sequence table SEQ .ID.No.2 base sequence, obtains target sequence amplification product.
The process of aforesaid method, the IL-4cDNA target sheet segment length 474bp increased by the flat P1 of primer and primer P2, and with the long 454bp of house-keeping gene of primer P1 and primer P3 amplification, 22bp shorter in target fragment; Competitive template and target fragment sequence difference little, influence factor can be down to low-level when making competitive PCR, thus make detected result more can the truth of reflected sample; And competitive template used and target sequence electrophoresis position close, for judging that RT-PCR product electrophoretic band provides comparatively ideal object of reference, can avoid the erroneous judgement of non-specific band by measuring the density of electrophoresis band, can carry out quantitatively to the amplified production of target sequence, thus know the content of corresponding mRNA in institute's test sample basis by inference, more close to real expression level.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Example of the present invention proposes a kind of IL-4 and detects primer, comprises the primer P1 with sequence table SEQ .ID.No.1 base sequence, the primer P2 with sequence table SEQ .ID.No.2 base sequence.
The primer of above-mentioned detection of the present invention, different from the method for existing Radioimmunoassay of vascular endothelial growth and the Detection of content of ELISA kit.When conception of the present invention is based on cells protein, the mRNA of the IL-4 that encodes in genetic expression process synthesis far prior to the IL-4 protein product of secretion, and protein expression amount number with the relation of the amount linear proportional of the corresponding mRNA encoded; So the present invention adopts above-mentioned specific primer to be increased by RT-PCR mode the mRNA of corresponding coding, then the amount of corresponding IL-4 protein product can be obtained by the amount of mRNA.And half-quantitative detection primer of the present invention and method, compare existing detection method and test kit, what its advantage that can have was that it detects is not IL-4 secretion, absorption, consume and net content after degraded, but the real-time point level of genetic transcription in IL-4 production process, translation and post-translational control mechanism; It can reacting cells or the interior real-time IL-4 expression level of tissue.
Detect in primer containing having plenty of one group of upstream and downstream primer at above-mentioned IL-4, wherein primer P1 is upstream primer and primer P2 is downstream primer, singlely carry out RT-PCR amplification, result of its amplification is not when having internal reference or contrast, and the mRNA of single IL-4 is inconvenient to carry out quantitative accurately.So on the basis of the above, above-mentioned IL-4 of the present invention detects primer and comprises the competitive primer P3 with sequence table SEQ .ID.No.3 base sequence further.
The design of this competitive primer P3 is with the primer of the gene design of β-actin (Actin muscle), what wherein need explanation careful further be β-actin gene is belong to house-keeping gene, the albumen of being expressed by house-keeping gene coding in mammalian cell expression, their expression relative constancy in each tissue and cell, and its product is necessary to maintenance radical cellular activities, can as the comparison object of reference in sxemiquantitative.When carrying out Semiquatitative RT-PCR assay, select house-keeping gene by calculating the ratio of goal gene and internal reference, the relative concentration of genetic expression can be obtained, thus the whole system of auxiliary judgment RT-PCR and reaction conditions whether suitable, and the result situation of PCR.
Jointly primer pair is formed by the primer of this competitive primer P3 and above-mentioned primer P1, increase IL-4 gene fragment (i.e. target sequence) and β-actin gene fragment (internal reference sequence) simultaneously, in amplification procedure, target sequence and competitive sequence all increase with identical efficiency with identical primer pair, the initialize ratio of two sequences remains unchanged in whole reaction process, obtains emulative cDNA template.And then increase separately with IL-4 primer pair and β-actin primer pair respectively with this emulative cDNA template; By β-actin protein mRNA amplification output, (in amplification procedure, the primer of β-actin can designed, designed, or adopt primer P4 and P5 of the present invention following design) result, namely can be used as the contrast of the mrna expression level relatively determining IL-4, be convenient to the quantitative analysis in IL-4 of the present invention detection.
Further on the basis of the primer of above-mentioned detection, for the ease of the detection of β-actin protein mRNA amount in competitive RT-PCR, the primer for the amplification of β-actin protein mRNA is assisted in further proposition, comprises the primer P4 with sequence table SEQ .ID.No.4 base sequence, the primer P5 with sequence table SEQ .ID.No.5 base sequence.The primer pair that this group primer P4 and primer P5 form carries out with the competitive cDNA template amplification β-actin protein mRNA of above-mentioned structure the primer pair that detects, is convenient to analysis reference that IL-4 detects and comparison.
Certainly, the specificity of above-mentioned primer and the stability of amplification, repeatedly through BLEAST Determination and verification experimental verification repeatedly, stability and specificity relatively good, the object of detection and the requirement of semi-quantitative analysis can be reached.
Meanwhile, above-mentioned primer pair of the present invention is the primer based on RT-PCR and competitive RT-PCR design, so primer pair in use, technician carries out according to the implementation step of the conventional RT-PCR in field and competitive RT-PCR.Wherein, the information of primer is as shown in the table:
| Title | Base sequence (5 '-3 ') | Length | Sequence table is numbered |
| Primer P1 | AATTGCCTCACATTGTCACT | 20bp | SEQ.ID.No.1 |
| Primer P2 | CTCTCATGATCGTCTTTAGCC | 21bp | SEQ.ID.No.2 |
| Competitive primer P3 | CTCTCATGATCGTCTTTAGCCTACTCTGGTTGGCTTCCTTC | 41bp | SEQ.ID.No.3 |
| Primer P4 | CGCGAGAAGGTGACCCAGATC | 21bp | SEQ.ID.No.4 |
| Primer P5 | ATCACGATGCCAGTGGTACGG | 21bp | SEQ.ID.No.5 |
Detect the proposition of primer based on above-mentioned IL-4 of the present invention, the present invention also proposes a kind of IL-4 half-quantitative detection reagent kit product further; Reagent kit product of the present invention comprises and above-mentionedly has the primer P1 of sequence table SEQ .ID.No.1 base sequence and have the primer P2 of sequence table SEQ .ID.No.2 base sequence.In the process that this test kit is implemented, its mechanism and reason can see description corresponding to above-mentioned primer portion, and this part repeats no more.Certainly, in order to make test kit can carry out semiquantitative comparison, so also see the description of above-mentioned primer portion, above-mentioned competitive primer P3, primer P4 and primer P5 can be added in test kit in the process detected; Be convenient to the enforcement that technician carries out semiquantitative competitive RT-PCR in the detection further.
The above-mentioned half-quantitative detection test kit of further use carries out in the mrna expression level detection process of IL-4, its objective is for mRNA, very unstable and be very easy to the situation that is degraded in vitro based on RNA self, so the inhibitor of RNase can be added in test kit, such as DEPC (Diethypyrocarbonate, diethylpyrocarbonate); Carrying out adding in the mRNA extracted in the process detected, affect the accuracy of detection to avoid target RNA to be degraded.
State in the process of test kit enforcement further on the invention, because the object of its original material obtained and detection in the process of detection is generally peripheral blood mononuclear cell, if there is stage middle and later periods being some cancer patients relatively, the mRNA of the IL-4 so naturally containing higher level in mononuclearcell, can be relatively easy to detect, and if the very latent period of these disease pathology or morbidity early stage, the expression of mRNA in mononuclearcell of IL-4 is very micro-, may not reach the standard of detection.So can amplify, make it express and can reach detected degree; The starting materials to be measured of concrete acquisition is mononuclearcell, the mode of so amplifying can adopt PMA (phorbol exters) and/or calciumionophore (Calcium ionophore, purchased from the great biology in Shanghai) mononuclearcell is induced, make it on the basis of original metabolism, promote it and express multiple, and have test to show, PMA (phorbol exters) and/or calciumionophore (Calcium ionophore) carries out in the process of inducing, with the increase of above-mentioned two kinds of inducer concentrations and induction time, the transcript mRNA expression amount of IL-4 increases gradually; Certainly namely no longer to increase after reaching certain peak value and downwards, but utilize reach peak value before the amount that detects after the relation of linear growth and induction just can calculate to push back and obtain original expression amount.
So based on this situation, also PMA (phorbol exters) and/or the calciumionophore (Calcium ionophore) of above-mentioned two kinds of expression amounts induction lifting can be comprised in reagent kit product of the present invention, assist for when the transcript mRNA expression amount of IL-4 very low and cannot directly RT-PCR detects more modest result time, adopt after linear amplification is carried out to its expression amount and detect.
Based on above-mentioned primer of the present invention and test kit, the present invention also proposes the semi-quantitative detection method of a kind of IL-4, comprises the steps:
S10, obtains IL-4 cellular control unit and IL-4 to be measured group of cell;
S20, extracts the total serum IgE of IL-4 cellular control unit, and the primer P1 of apparatus ordered list SEQ.ID.No.1 base sequence carries out RT-PCR with the competitive primer P3 with sequence table SEQ .ID.No.3 base sequence, obtains competitive template;
S30, after extracting the total serum IgE of IL-4 to be measured group of cell, carries out reverse transcription and obtains cDNA template to be measured;
S40, after cDNA template to be measured and competitive template being mixed, the primer P1 of apparatus ordered list SEQ.ID.No.1 base sequence carries out PCR with the primer P2 with sequence table SEQ .ID.No.2 base sequence to interior standard amplification product;
S50, carries out RCR by the primer P4 of cDNA template apparatus ordered list SEQ.ID.No.4 base sequence to be measured and the primer P5 with sequence table SEQ .ID.No.5 base sequence, obtains contrasting amplified production;
S60, calculates the mRNA amount of IL-4 in cell to be measured according to the result of the contrast amplified production of the amplification of step S40 and step S50 amplification.
Among said process completes; wherein step S50 and S60 is only that a preparation is with reference to the process contrasted and calculate; whether higher, on the low side whether normal or these two steps are for when the RT-PCR product of cell to be measured does not contrast reference, can not obtain expressing problem.And in the step of step S50 and step S60, in order to contrast more can be convenient to and calculate, in step S50, in PCR system, adopt the amount of cDNA template to be measured, can be identical.The amount of such basic templates is identical, is convenient to comparing calculation.And in step S40 the amount of competitive template to remember in the process of adding the amount of adding number, the calculating after convenient.
Based on foregoing description, the amplification of contrast amplified production be the mRNA of β-actin albumen, be a kind of house-keeping gene, be all weigh to express in all cells, adopt it as the internal reference value of constant comparison, can obtain the situation of the relative expression of target fragment amplification product.
Certainly, the content of aforesaid method step, the IL-4 cellular control unit adopted in implementation process generally can adopt the mononuclearcell be separated in the blood sample of Healthy People, and IL-4 to be measured group of cell can be the blood sample mononuclearcell that doubtful sufferer or needs are determined, carry out with the expression of both contrasting the expression level that can obtain the IL-4 of to be measured group of cell.Certainly, wherein because Healthy People itself may express IL-4 hardly, if so with the mononuclearcell be separated in Healthy People blood sample in contrast, so obtain to use possibly after cell PMA (phorbol exters) and/or calciumionophore (Calcium ionophore) induce after could be used as cellular control unit.Or, also can adopt the mononuclearcell made a definite diagnosis and be separated in the blood sample of sufferer, then can induce and direct cell as a control group.
Therefore, first aforesaid method step of the present invention is may be used for the situation that in histocyte, IL-4 expresses, thus determines the situation of histiocytic related neoplasms or canceration further, helps early stage monitoring.But the method for the present invention is not merely for this situation, above-mentioned detection method of the present invention may be used in the fermentation culture of IL-4.Specifically in existing method, the preparation of IL-4 generally has two kinds of modes, and one carries out separation and Extraction from histocyte, and one is that cell fermentation is cultivated.Wherein, cell fermentation is cultivated because its turnout is large and the reason such as easy handling, is suitable for a large amount of manufactures carrying out IL-4; And wherein in the process of fermentation or before fermentation, the expression level of fermented cells IL-4 in seed cell and culturing process be confirmed, with defect underproduce after avoiding adopting the cell cultures of low expression level.So, just aforesaid method step of the present invention can be adopted to carry out the detection of the expression level of fermented cells IL-4 in seed cell and culturing process, detect the level of the expression of cell in the front seed cell of fermentation or fermenting process, thus be beneficial to productive rate and the benefit of fermentative production.
Certainly, in the step S20 of above-mentioned detection, after also can adopting the total serum IgE extracting cell to be measured, inhibitor such as adding DEPC can be adopted to protect, prevent from being degraded after extracting and in the process of detection reaction, cause detected result inaccurate.
Certainly, based on the proposition of above-mentioned primer of the present invention, if on the basis of situation graphic representation being built with dissimilar or different steps cell expressing IL-4, also competitive RT-PCR can not be adopted to detect, and directly adopt normal RT-PCR to carry out, obtain cell to be measured, to extract after total serum IgE and directly carry out RT-PCR with the primer P1 with sequence table SEQ .ID.No.1 base sequence, the primer pair of primer P2 with sequence table SEQ .ID.No.2 base sequence.The process of this kind of conventional RT-PCR, more usually, technician can very simply can implement, and this part of this case repeats no more.
Adopt above-mentioned detection method of the present invention, compare with the detection of ELISA kit with the method for existing Radioimmunoassay of vascular endothelial growth, increased by RT-PCR mode the mRNA of corresponding coding, the amount of corresponding IL-4 protein product can be obtained again by the amount of mRNA, what it detected is not IL-4 secretion, absorption, consumption and the net content after degrading, but genetic transcription in IL-4 production process, the real-time point level of translation and post-translational control mechanism, avoid using IL-4 protein product as IL-4 secretion in direct-detection target, absorption, consume and degrade the result difference caused, and can not the expression level of real-time IL-4 in reacting cells or tissue.
The understanding of those skilled in the art can be easier to for the ins and outs and process approach that make above-mentioned enforcement of the present invention and implement reference, highlight the present invention simultaneously and utilize the feasibility of the detection of IL-4 expression level and the accuracy of result, be illustrated below by way of specific embodiment.
Embodiment 1
S11, preparation acquisition Healthy People routinely aseptic separating peripheral blood mononuclear cells bacterium liquid (roughly adjusts cell concn and is about 2 × 10
6/ ml);
S12, then by after the Calcium ionophore that adds 2.0 μm of ol/L in the cell bacterium liquid of above-mentioned preparation and 80ng/mlPMA, puts 37 DEG C, 5%CO with RPMI1640 complete culture solution
2after cultivating 7h respectively, centrifugal acquisition cell paste carries out following step;
S21, extracts the total serum IgE of the cell paste that test kit extraction step S12 obtains with RNA, add appropriate DEPC and carry out protection and prevent it from degrading, and calculate its absorbance with ultraviolet spectrophotometer, and calculate its concentration after extraction.
S22, after the concentration value calculated according to step S21, get total serum IgE 1.5 μ g and carry out reverse transcription, reverse transcription system is according to as follows: RNA1.5 μ g, oligo-dT (300pmol/ μ l) 0.5 μ l, dNTP (2.5mmol/L) 1 μ l, RNasin (40U/ μ l) 0.5 μ l, AMV reversed transcriptive enzyme (9U/ μ l) 0.5 μ l, 5 × AMVbuffer2 μ l, complement to 10 μ l with DEPC water;
According to above-mentioned system, 60min at carrying out 42 DEG C, 4min at 95 DEG C, 5min at 4 DEG C.
S23, the product after step S22 reverse transcription being completed, carries out pcr amplification; Amplification system is according to the reverse transcription cDNA template 3 μ l of step S30, each 1 μ l, the dNTP2 μ l of the primer P1 of 20pmol/ μ l, competitive primer P3, Taq enzyme 0.5 μ l (5U/ μ l), DD-H
2o12.5 μ l;
By above-mentioned system 60s, 66 DEG C of next 40s, 45s at 72 DEG C at 95 DEG C, after above step recirculation 28 times at 72 DEG C 5min.
S24, after step S23 has increased, whether the band of the amplification obtained has had the cDNA of target sequence, and the amount of this cDNA obtained is how many, needs to carry out verifying and calculating, and carries out in this step S24, specifically can be according to:
The amplified production obtained by step S23 is through 3% agarose electrophoresis, object band is cut under uv analyzer, competitive template is reclaimed by test kit description operation, measure content, the competitive template simultaneously reclaimed for primer pair with primer P1 and primer P2 respectively carries out PCR, in order to determine the suitable amounts through competitive template, frozen as competitive template-80 DEG C, for subsequent use after packing;
S30, obtain the peripheral blood mononuclear cell (being roughly also about 2 × 106/ml according to the cell concn identical with step S11) of a patient to be measured, then directly total serum IgE (A260nm/A280nm>1.7) is extracted with RNA test kit, and carry out reverse transcription with reference to the system that step S22 is identical, obtain cDNA template to be measured;
S40, the competitive template mixing prepared by the cDNA template 3 μ l and same amount step S24 to be measured that step S30 obtains, carries out pcr amplification with primer P1 and primer P2 after mixing; Wherein the system of PCR and detailed process are carried out see the implementation detail of step S23.
S50, in order to step S40 contrasts, the cDNA template 3 μ l primer P4 and primer P5 to be measured that step S30 obtains is carried out pcr amplification, in this step, the PCR system of PCR amplification system also refer step S23 is carried out;
The PCR of this step S50 is the situation of the mRNA amount in order to detect β-actin in cDNA template to be measured, is used as the contrast of IL-4 in step S40.
S60, by the product of above-mentioned steps S40 and being at war with property of step S50 RT-PCR, through 3% agarose electrophoresis, SX-100imagesystem makes a video recording, makes density quantitatively with the calibration of the competitive template of concentration known, know the relative content of corresponding mRNA by inference.
Through calculating and comparison, in above-mentioned steps S11, Healthy People is after the Calcium ionophore and 80ng/mlPMA induction 7h of 2.0 μm of ol/L, in its cell, the mRNA of IL-4 is that 8.10 ± 0.61ng is as a comparison with reference to (healthy cell after selecting induction is because the mRNA of IL-4 is 0.18 ± 0.13ng in the cell directly surveyed without induction as reference, it shows that the IL-4 of Healthy People mononuclearcell does not express substantially, and this detected result name to show variance excessive, therefore not too qualitative reference can be used as), and the mRNA of IL-4 is 6.73 ± 0.58ng in cell to be measured.
In the cell to be measured that the ELISA immunoassay kit detection above-mentioned steps S30 detecting IL-4 equally again obtains, the mRNA of IL-4 is 4.18 ± 0.43ng, the result that this result obtains than the method for above-mentioned detection is obviously low about 38%, so above-mentioned detection of the present invention more may can embody real expression level.
The process of wherein carrying out detecting in the above embodiment of the present invention 1 is the situation for detecting histiocytic related neoplasms or canceration, and further aforesaid method process of the present invention for detecting the IL-4 expression level of the cell of the fermentation culture of a large amount of manufactures of IL-4 and seed cell time, also contrast and to be measured be can build by above-mentioned equal method, thus the cell of the fermentation culture that will detect and the IL-4 expression level of seed cell obtained.Concrete implementation process is replaced see said process and is implemented, and this part repeats no more.
Adopt the IL-4cDNA target sheet segment length 474bp that the process of aforesaid method is increased by the flat P1 of primer and primer P2, and with the long 454bp of house-keeping gene of primer P1 and primer P3 amplification, 22bp shorter in target fragment; Competitive template and target fragment sequence difference little, influence factor can be down to low-level when making competitive PCR, thus make detected result more can the truth of reflected sample; And competitive template used and target sequence electrophoresis position close, for judging that RT-PCR product electrophoretic band provides comparatively ideal object of reference, can avoid the erroneous judgement of non-specific band by measuring the density of electrophoresis band, can carry out quantitatively to the amplified production of target sequence, thus know the content of corresponding mRNA in institute's test sample basis by inference, more close to real expression level.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. IL-4 detects a primer, it is characterized in that, comprises the primer P1 with sequence table SEQ .ID.No.1 base sequence, the primer P2 with sequence table SEQ .ID.No.2 base sequence.
2. IL-4 as claimed in claim 1 detects primer, it is characterized in that, also comprises the competitive primer P3 with sequence table SEQ .ID.No.3 base sequence.
3. an IL-4 detection kit, is characterized in that, the IL-4 comprised described in claim 1 or 2 detects primer.
4. IL-4 detection kit as claimed in claim 3, it is characterized in that, described test kit also comprises the primer P4 with sequence table SEQ .ID.No.4 base sequence, the primer P5 with sequence table SEQ .ID.No.5 base sequence.
5. the IL-4 detection kit as described in claim 3 or 4, is characterized in that, described test kit also comprises RNase inhibitor, and this RNase inhibitor is diethylpyrocarbonate.
6. the IL-4 detection kit as described in claim 3 or 4, is characterized in that, described test kit also comprises PMA and/or Calcium ionophore.
7. an IL-4 semi-quantitative detection method, is characterized in that, comprises the steps:
Obtain IL-4 cellular control unit and IL-4 to be measured group of cell;
Extract the total serum IgE of described IL-4 cellular control unit, and the primer P1 of apparatus ordered list SEQ.ID.No.1 base sequence carries out RT-PCR with the competitive primer P3 with sequence table SEQ .ID.No.3 base sequence, obtains competitive template;
Extract the total serum IgE of described IL-4 to be measured group of cell, and carry out reverse transcription and obtain cDNA template to be measured;
After described cDNA template to be measured and competitive template mixing, the primer P1 of apparatus ordered list SEQ.ID.No.1 base sequence carries out PCR with the primer P2 with sequence table SEQ .ID.No.2 base sequence, obtains target sequence amplification product.
8. IL-4 semi-quantitative detection method as claimed in claim 8, it is characterized in that, after described cDNA template to be measured and competitive template mixing, the primer P1 of apparatus ordered list SEQ.ID.No.1 base sequence and the primer P2 with sequence table SEQ .ID.No.2 base sequence also comprises after carrying out PCR step:
The primer P4 of described cDNA template apparatus ordered list SEQ.ID.No.4 base sequence to be measured and the primer P5 with sequence table SEQ .ID.No.5 base sequence are carried out RCR, obtains contrasting amplified production.
9. IL-4 semi-quantitative detection method as claimed in claim 8, it is characterized in that, after the primer P4 of described cDNA template apparatus ordered list SEQ.ID.No.4 base sequence to be measured and the primer P5 with sequence table SEQ .ID.No.5 base sequence are carried out RCR step, also comprise:
The mRNA amount of IL-4 in described IL-4 to be measured group of cell is calculated according to target sequence amplification product and contrast amplified production.
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|---|---|---|---|---|
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102186590A (en) * | 2008-10-17 | 2011-09-14 | 布鲁塞尔自由大学 | Device, kit and method for pulsing biological samples with an agent and stabilising the sample so pulsed |
| CN103320447A (en) * | 2013-05-20 | 2013-09-25 | 深圳市亚太兴实业有限公司 | Recombinant human interleukin 4 gene, and fermentation method of engineering bacteria comprising recombinant human interleukin 4 gene |
-
2015
- 2015-07-28 CN CN201510448282.6A patent/CN105039548A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102186590A (en) * | 2008-10-17 | 2011-09-14 | 布鲁塞尔自由大学 | Device, kit and method for pulsing biological samples with an agent and stabilising the sample so pulsed |
| CN103320447A (en) * | 2013-05-20 | 2013-09-25 | 深圳市亚太兴实业有限公司 | Recombinant human interleukin 4 gene, and fermentation method of engineering bacteria comprising recombinant human interleukin 4 gene |
Non-Patent Citations (1)
| Title |
|---|
| 朱晴晖等: "定量测定IL-4和IFN-γ mRNA的RT-竞争PCR法及其应用", 《中华微生物学和免疫学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113293202A (en) * | 2021-07-02 | 2021-08-24 | 广东莱恩医药研究院有限公司 | Real-time fluorescent quantitative PCR kit for quantitatively detecting mRNA content in organism, detection method and application |
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