CN105950715A - [Delta]42PD1 detection method, primers and kit - Google Patents
[Delta]42PD1 detection method, primers and kit Download PDFInfo
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Abstract
The invention provides a primer pair, wherein an amplified product and/or an amplification amount of the primer pair can distinguish [delta]42PD1 and a PD-1 sequence. The invention also provides an application of the primer pair in preparation of a product for detection of the [delta]42DP1. Experiments prove that the primer pair has the advantages of good specificity, high sensitivity, short detection time and wide application range; moreover, the [delta]42DP1 detection method established based on the primer pair can sensitively, accurately, conveniently and quickly detect the presence and/or content of the [delta]42DP1, and can highly distinguish the [delta]42DP1 and the PD-1 sequence.
Description
Technical field
The invention belongs to biological technical field, be specifically related to detect the primer of Δ 42PD1, test kit and detection method.
Background technology
Apoptotic cell death receptor 1 (programmed cell death 1, PD-1), has another name called CD279, belongs to
CD28 molecule families, being expressed in the T cell of activation, NKT cell, B cell and Macrophage Surface PD-1 has two parts, PD-
L1 (having another name called CD274 or B7-H1) and PD-L2 (having another name called CD273 or B7-DC), both of which is B7 family member.Result of study
Showing, PD-1/PD-L path (including PD-1/PD-L1 and PD-1/PD-L2) has immune suppression function.
A kind of brand-new PD-1 gene it is found that first during the gene pleiomorphism of state crowd PD-1 molecule under study for action
MRNA alternative splicing transcript.Compared with total length PD-1mRNA, this this in-frame deletion of new transcription exon 2 initiating terminal
42 bases (ACT CCC CAG ACA GGC CCT GGA ACC CCC CCA CCT TCT CCC CAG), this section of base is compiled
14 aminoacid (DSPDRPWNPPTFFP) of code PD-1 extracellular region.Therefore, the alternative splicing of this novel PD-1 gene is transcribed
Originally Δ 42PD1 it is named as.Research finds, the monoclonal antibody of conventional business-like PD-1 (EH12.2H7, MIH4,
EH12.1, J110 etc.) Δ 42PD1 can not be identified, there is notable change in the structure pointing out 14 amino acid whose disappearances to make Δ 42PD1
Change.Additionally, Δ 42PD1 can not combine with the part (PD-L1 and PD-L2) of PD-1.Result above shows, Δ 42PD1 with
PD-1 has different structures.Further study show that, Δ 42PD1 can induce human peripheral blood single nucleus cell and dendritic cell
The pro-inflammatory cytokines such as TNF secretion-α, IL-6 and IL-1 β.Additionally, Δ 42PD1 can be aobvious as molecule adjuvant in Mice Body
Writing and strengthen specific humoral immune response vaccine-induced for HIV Gag p24DNA and cellullar immunologic response, these results are all pointed out
Δ 42PD1 is likely to be of important immunoloregulation function.
But, Δ 42PD1 and the high consistency of PD-1 sequence, significantly increase exploitation this genetic transcription of specific detection
The technological means of level, limits the research to this new discovery immune modulatory molecules and application.
But, Δ 42PD1 and the high consistency of PD-1 sequence, significantly increase exploitation this genetic transcription of specific detection
The technological means of level, limits the research to this new discovery immune modulatory molecules and application.At present to gene transcription level
Conventional detection method includes regular-PCR and qPCR etc..Round pcr is end-point method analysis, needs the gel electrophoresis analysis after PCR to tie
Really, the longest, the dyestuff (such as EB etc.) of use has carcinogenecity, and its detection sensitivity is relatively low.QPCR is divided into again sonde method
With SYBR Green I method, both is without electrophoretic analysis and highly sensitive, can be quantitative.But sonde method needs to use more
Expensive specific probe, relatively costly, it is unfavorable for large-scale use；And though SYBR Green I method is without the most high for using
Expensive probe and be widely used, but the specificity of its primer and susceptiveness then become the key of design.
Summary of the invention
The technical problem to be solved in the present invention is to provide the side of a kind of sensitive, accurate, easy and quick detection Δ 42PD1
Method, present invention also offers the primer pair that specificity is good, highly sensitive, the detection time is short and applied widely for the method
With the test kit containing this primer pair and application thereof.
In order to solve above-mentioned technical problem, the present invention is achieved through the following technical solutions:
One aspect of the present invention discloses a kind of primer pair for detecting Δ 42PD1, the amplified production of described primer pair and/
Or amplification amount can distinguish Δ 42PD1 and PD-1 sequence.
In a preference, two primer sequences of the primer pair of detection Δ 42PD1 all match completely with Δ 42PD1, and
The splice site of the Δ 42PD1 formed during wherein primer sequence is when matching with PD-1 sequence by PD-1 sequence cuts off.
In a preference, at the primer sequence that splice site is cut off, it is divided into the upstream region of primer after partition and draws
The downstream area of thing, the base of described upstream region and the base ratio of downstream area are 1:4～4:1.
In a preference, a sequence of the primer centering of detection Δ 42PD1 comprises the nucleoside shown in SEQ ID NO:1
Acid sequence.
In a preference, another primer sequence comprises the nucleotide sequence shown in SEQ ID NO:2.
In a preference, the described method used by differentiation is fluorescence quantifying PCR method.
In a preference, the described method used by differentiation is quantitative fluorescent PCR SYBR Green I dye method.With
Time differentiating method be not limited to fluorescence quantifying PCR method, differentiating method also includes the methods such as chip hybridization, order-checking, gel electrophoresis.
Second aspect present invention discloses primer described above to the application in the product of preparation detection Δ 42PD1.
In a preference, the product of described detection Δ 42PD1 includes PCR amplifing reagent.
In a preference, described PCR amplifing reagent is quantitative fluorescent PCR reagent.
Third aspect present invention discloses a kind of test kit, including primer pair recited above.
In a preference, test kit also includes PCR amplifing reagent.
In a preference, described PCR amplifing reagent is quantitative fluorescent PCR reagent.
In a preference, described quantitative fluorescent PCR reagent include from the article No. of TaKaRa company be RR820A'sPremix Ex TaqTMThe reagent that II test kit is comprised.
In a preference, test kit also includes positive control and negative control.
In a preference, test kit also includes that RNA extracts reagent.
In a preference, described RNA extract reagent include from the article No. of TIANGEN company be Cat.#DP419's
The reagent that RNAsimple Totle RNA Kit total RNA extraction reagent box is comprised.
In a preference, test kit also includes reverse transcription reagents.
In a preference, described reverse transcription reagents include from the article No. of TaKaRa company be Code No.6110A's
Reverse Transcriptase kitThe reagent that 1st Strand cDNA Synthesis Kit is comprised.
Fourth aspect present invention discloses the test kit recited above application in detection Δ 42PD1.
Fifth aspect present invention discloses and a kind of detects the method for Δ 42PD1 in testing sample, described above including using
The step of primer pair.
In a preference, the method for Δ 42PD1 in detection testing sample, described testing sample for include DNA and/or
The blood of RNA, cell or tissue, or be plasmid, or be DNA, cDNA, mRNA or cDNA fragment, or be containing DNA, cDNA,
The reagent of mRNA or cDNA fragment；
In a preference, when testing sample is DNA, cDNA or cDNA fragment, or it is containing DNA, cDNA or cDNA sheet
The reagent of section, or when be plasmid, by its directly as template and described primer to carrying out real-time fluorescence PCR reaction；Then judge
The existence of Δ 42PD1 and/or content in testing sample.
In a preference, when testing sample is the blood containing DNA and/or RNA, cell or tissue, or it is mRNA, or
During for reagent containing mRNA, it is translated into DNA, cDNA or cDNA fragment and carries out real-time fluorescence PCR reaction detection again；Then
Judge existence and/or the content of Δ 42PD1 in testing sample.
In a preference, when described testing sample is blood, also include the step of the Total RNAs extraction to blood and right
Total serum IgE carries out the step of reverse transcription.
In a preference, the method for Δ 42PD1 in detection testing sample, utilize used by Fluorescence PCR detection is anti-
Answer system as follows:
In a preference, the program of Fluorescence PCR recited above is as follows: 98 DEG C of denaturations 1 minute；98 DEG C of degeneration
10s, 60 DEG C of annealing 30s, 40 circulations.
The beneficial effect comprise that
(1) the present invention is directed to Δ 42PD1 design amplimer specificity good, highly sensitive, the detection time is short and applicable
Scope is wide.
(2) detection method of the Δ 42PD1 that the present invention sets up, can be sensitive, accurate, easy and the most qualitative and/or fixed
The existence of the detection Δ 42PD1 of amount and/or content, can highly distinguish Δ 42PD1 and PD-1 sequence.
Accompanying drawing explanation
The gene order contrast of the variant part of Fig. 1 Δ 42PD1 Yu PD-1, and the sequence of Δ 42PD1 forward primer
(SEQ ID NO.1) schematic diagram in position corresponding for Δ 42PD1 with PD-1.
Fig. 2 SEQ ID NO.1, the SEQ ID NO.2 primer specificity analyses result schematic diagram to detection Δ 42PD1.
Fig. 3 SEQ ID NO.1, the SEQ ID NO.2 primer sensitivity analysis result schematic diagram to detection Δ 42PD1.
Fig. 4 SEQ ID NO.1, SEQ ID NO.2 primer are to the Ct value of detection by quantitative Δ 42PD1 and Δ 42PD1 copy number
Linear regression analysis.
Fig. 5 every ml human peripheral Δ 42PD1mRNA copy number testing result schematic diagram.
Detailed description of the invention
Unless specifically indicated, the general sense during term used herein has art of the present invention.
Below with reference to specific embodiments and the drawings, the present invention will be described, it should be noted that these embodiments are only
It is illustrative, and is not considered as limiting the invention.Unreceipted concrete technology or condition in embodiment, according to ability
The technology described by document or condition in territory or carry out according to product description.Agents useful for same or the unreceipted factory of instrument
Shang Zhe, be can by city available from conventional products.
Embodiment 1
Present embodiments provide a kind of primer detecting people's Δ 42PD1 transcriptional level to and application.
The design of primer pair: forward primer (SEQ ID NO.1) comprises 20 oligonucleotide, the most front 16 nucleotide positions
On first exon of Δ 42PD1, latter 4 are positioned on second exon, and this design can get rid of genome PD-1DNA
Interference to testing result；On this external PD-1 total length transcript, front 16 oligonucleotide of forward primer and rear 4 oligonucleoside
There are 42 bases (as shown in Figure 1) between nucleotide sequence corresponding to acid, to get rid of the PD-1mRNA interference to testing result,
I.e. forward primer sequence is crossed over PD-1 and is formed the splice site of Δ 42PD1, is that this can be with specific detection Δ to primer
The key point of 42PD1mRNA.
For obtaining sensitivity and the best primer of specificity, our initial design at least 5 crosses over the upper of splice sites
Trip primer, experimental result finds, although the detection sensitivity of these at least 5 forward primer is suitable, but forward primer (SEQ ID
NO.1) specificity is significantly better than other 4 primers.
Primer is as follows to particular sequence:
Forward primer F:5 '-CCAGGATGGTTCTTAGCCCT-3 ' (SEQ ID NO.1),
Downstream primer R:5 '-GCTTGTCCGTCTGGTTGCTG-3 ' (SEQ ID NO.2).
Above-mentioned primer synthesizes by Shenzhen Huada Genetic Technology Co., Ltd.
Above-mentioned PCR primer can be applicable to the product of preparation detection Δ 42PD1, can carry out Δ 42PD1 and PD-1 sequence
Highly distinguish；The product of described detection Δ 42PD1 can also include PCR amplifing reagent；Described PCR amplifing reagent is preferably fluorescence
Quantitative PCR reagent.
Embodiment 2
Present embodiments providing a kind of test kit and application thereof, described test kit includes:
(1) forward primer F and downstream primer R (from embodiment 1)；
(2) article No. from TaKaRa company is RR820A'sPremix Ex TaqTMII test kit is comprised
Reagent.
It is demonstrated experimentally that it is above-mentionedPremix Ex TaqTMII test kit, forward primer F and the connection of downstream primer R
Close and use, have save time, high, high sensitivity economic, repeatable and specific feature, effect is more superior.
Mentioned reagent box can be applicable to detect on Δ 42PD1, it is possible to height distinguishes Δ 42PD1 and PD-1 sequence.
Embodiment 3
Present embodiments provide and a kind of detect the method for Δ 42PD1 in testing sample, such as Δ in detection testing sample
The existence of 42PD1 and/or content, the method use RPA primer and the test kit of embodiment 2 of embodiment 1.
Above-mentioned testing sample can be to include the blood of DNA and/or RNA, cell or tissue, or is plasmid, or is
DNA, cDNA, mRNA or cDNA fragment, or be the reagent containing DNA, cDNA, mRNA or cDNA fragment.When testing sample is
DNA, cDNA or cDNA fragment, or be the reagent containing DNA, cDNA or cDNA fragment, or when being plasmid, by it directly as mould
Plate and described primer are to carrying out real-time fluorescence PCR reaction；Then existence and/or the content of Δ 42PD1 in testing sample are judged；
When testing sample is the blood containing DNA and/or RNA, cell or tissue, or it is mRNA, or is containing mRNA's
During reagent, it is translated into DNA, cDNA or cDNA fragment and carries out real-time fluorescence PCR reaction detection again；Then testing sample is judged
The existence of middle Δ 42PD1 and/or content.Such as when testing sample is blood, in addition it is also necessary to the Total RNAs extraction of blood and right
Total serum IgE carries out reverse transcription, and to obtain cDNA sample, testing sample is originally as DNA, cDNA or cDNA fragment in a word, or for containing
The reagent of DNA, cDNA or cDNA fragment, or do not deal with during for plasmid, remaining is both needed to be processed into DNA, cDNA or cDNA fragment
Sample.
Said method comprises the steps:
1. real-time fluorescence PCR reaction
According toPremix Ex TaqTMThe explanation preparation of II (Code No.RR820A, TaKaRa) test kit is anti-
Answer liquid: take 12.5 μ L SYBR Premix Ex Taq II, 1 μ L forward primer F (10 μMs), 1 μ L downstream primer R (10 μMs), 0.5
(copy number is 10 for μ L ROX Reference Dye II, 1 μ L DNA or cDNA or cDNA fragment1To 109), 9 μ L ultra-pure waters add
Enter to 0.2mL PCR reaction tube, with ultra-pure water as negative control sample, the brief centrifugation several seconds after vibration mixing.By reaction tube
It is placed in ViiATMReal-time fluorescent PCR amplification and detection, institute is carried out on 7 real-time fluorescence quantitative PCR instrument (Applied Biosystems)
The program stating real-time fluorescent PCR amplification is as follows: 98 DEG C 1 minute；98 DEG C 10 seconds, 60 DEG C 30 seconds, 40 circulations.As shown in table 1.
Table 1
2. judge existence and/or the content of Δ 42PD1 in testing sample
Collecting fluorescence signal after 60 DEG C of stages of each circulation terminate and carry out the analysis of melting curve, result is positive
(Ct≤33) represent containing Δ 42PD1 in testing sample, and result then represents in testing sample not containing Δ for negative (Ct ＞ 33)
42PD1。
When needing the content judging Δ 42PD1 in testing sample, then also need to set known copy number in above-mentioned steps 1
Positive control.
Embodiment 4
The present embodiment to having carried out specificity verification, specifically includes following steps to the primer of embodiment 1:
1. prepared by sample
By Δ 42PD1 and PD-1 full-length cDNA (wherein PD-1 derives from the ID NM_005018 of Genbank) insertion vector
In pVAX1 carrier (article No. V260-20, American I nvitrogen company), build eukaryon expression plasmid pVAX-Δ 42PD1 and
pVAX-PD1.Use plasmid is measured and extract test kit (article No. D0018, the green skies, Shanghai Bioisystech Co., Ltd), and according to
Plasmid pVAX-Δ 42PD1 and pVAX-PD1 is prepared in test kit explanation.Plasmid is measured respectively with trace ultraviolet spectrophotometer
The concentration of pVAX-Δ 42PD1 and pVAX-PD1, calculates copy number by its Molecular weights, and then preparing copy number is 1 × 109/
The sample of μ L.
2.PCR reacts
According toPremix Ex TaqTMThe explanation preparation of II (Code No.RR820A, TaKaRa) test kit is anti-
Answer liquid: take 12.5 μ L SYBR Premix Ex Taq II, 1 μ L forward primer F (10 μMs), 1 μ L downstream primer R (10 μMs), 0.5
μ L ROX Reference Dye II, 1 μ L sample, 9 μ L ultra-pure waters add to 0.2mL PCR reaction tube, with ultra-pure water as the moon
Property control sample, vibration mixing after the brief centrifugation several seconds.Reaction tube is placed in ViiATM7 real-time fluorescence quantitative PCR instrument (Applied
Biosystems) carrying out real-time fluorescent PCR amplification and detection on, the program of described real-time fluorescent PCR amplification is as follows: 98 DEG C 1 point
Clock；98 DEG C 10 seconds, 60 DEG C 30 seconds, 40 circulations.As shown in table 1.
Table 1
3. result:
Testing result is as in figure 2 it is shown, Fig. 2 shows 109The Ct value of the Δ 42PD1 sample of copy number is 10.065,109Copy
The Ct value of the PD1 sample of number is 31.572, and negative control can't detect Ct value.This experimental result illustrates with this primer pair amplifies Δ
The efficiency of 42PD1 is about the 2 of the efficiency of amplification PD-121(231.572-10.065) times, there is the strongest specificity.
Embodiment 5
The present embodiment to having carried out susceptiveness checking, specifically includes following steps to the primer of embodiment 1:
1. prepared by sample
The dense of plasmid pVAX-Δ 42PD1 (embodiment 4 is shown in the preparation of plasmid) is measured respectively with trace ultraviolet spectrophotometer
Degree, calculates copy number by its Molecular weights, is then sequentially prepared 10 times of gradient dilutions to the sample of position copy number, and altogether 7
Individual gradient, copy number is: 1 × 106To 1 × 100/μL。
2.PCR reacts
According toPremix Ex TaqTMThe explanation preparation of II (Code No.RR820A, TaKaRa) test kit is anti-
Answer liquid: take 12.5 μ L SYBR Premix Ex Taq II, 1 μ L forward primer F (10 μMs), 1 μ L downstream primer R (10 μMs), 0.5
μ L ROX Reference DyeII, 1 μ L sample, 9 μ L ultra-pure waters add to 0.2mL PCR reaction tube, with ultra-pure water as the moon
Property control sample, vibration mixing after the brief centrifugation several seconds.Reaction tube is placed in ViiATM7 real-time fluorescence quantitative PCR instrument (Applied
Biosystems) carrying out real-time fluorescent PCR amplification and detection on, the program of described real-time fluorescent PCR amplification is as follows: 98 DEG C 1 point
Clock；98 DEG C 10 seconds, 60 DEG C 30 seconds, 40 circulations.As shown in table 2.
Table 2
3. result: testing result is as it is shown on figure 3, Fig. 3 shows that the detection detecting Δ 42PD1 with the qPCR of described primer is rolled off the production line
It is 1 (100) copy, and negative control can't detect Ct value.And have the strongest linear between Ct value and Δ 42PD1 copy number
Relation (P < 0.0001, r=0.999, Fig. 4), shows that the susceptiveness with the qPCR detection Δ 42PD1 of the primer pair of the present invention is very
By force.
Embodiment 6
Present embodiments provide the primer provided in embodiment 1 to detection by quantitative human peripheral Δ 42PD1mRNA reverse transcription
The copy number of rear cDNA.
1. prepared by sample
Take 24 Healthy People vein anticoagulation (EDTA anticoagulant) 250 μ L, with RNAsimple Totle RNA Kit total serum IgE
Extract test kit (Cat.#DP419, TIANGEN), reference reagent box description, extract blood cell total serum IgE, be dissolved in 50 μ L without
In the ultra-pure water of RNase.Take 12.5 μ L total serum IgE, use Reverse Transcriptase kit1st Strand cDNA
Synthesis Kit (Code No.6110A, TaRaKa), reference reagent box description, carry out reverse transcription and synthesize 20 μ L cDNA.
The standard substance of positive control are prepared sample as described in Example 4 and are prepared.
2.PCR reacts
According toPremix Ex TaqTMThe explanation preparation of II (Code No.RR820A, TaKaRa) test kit is anti-
Answer liquid: take 12.5 μ L SYBR Premix Ex Taq II, 1 μ L forward primer F (10 μMs), 1 μ L downstream primer R (10 μMs), 0.5
μ L ROX Reference Dye II, 1 μ L cDNA template (being prepared by Healthy People anticoagulation), 9 μ L ultra-pure waters add to 0.2mL
In PCR reaction tube；With ultra-pure water for negative control template, do positive control experiment with standard substance are parallel.Vibration is instantaneous after mixing
The centrifugal several seconds.Reaction tube is placed in ViiATMCarry out the most glimmering on 7 real-time fluorescence quantitative PCR instrument (Applied Biosystems)
Light PCR amplification and detection, the program of described real-time fluorescent PCR amplification is as shown in table 2.
3. result
Testing result is as it is shown in figure 5, Fig. 5 shows that in 24 example healthy adult human peripherals, Δ 42PD1cDNA copy number is average
Value is 7.92 × 107, minimum and maximum value is respectively 2.85 × 106With 1.91 × 108。
Claims (10)
1. the primer pair being used for detecting Δ 42PD1, it is characterised in that the amplified production of described primer pair and/or amplification amount
Δ 42PD1 and PD-1 sequence can be distinguished；
Optional, two primer sequences of described primer pair all match completely with Δ 42PD1, and wherein primer sequence with
Cut off by the splice site of the Δ 42PD1 formed in PD-1 sequence during the pairing of PD-1 sequence；
Optional, at the primer sequence that splice site is cut off, after partition, it is divided into the upstream region of primer and the catchment of primer
Territory, the base of described upstream region and the base ratio of downstream area are 1:4～4:1.
Primer pair the most according to claim 1 a, it is characterised in that sequence of primer centering comprises SEQ ID NO:1
Shown nucleotide sequence；
Optional, another sequence comprises the nucleotide sequence shown in SEQ ID NO:2；
Optional, the described method used by differentiation is fluorescence quantifying PCR method；
Optional, the described method used by differentiation is quantitative fluorescent PCR SYBR Green I dye method.
Primer the most according to claim 1 and 2 is to the application in the product of preparation detection Δ 42PD1；
Optional, the product of described detection Δ 42PD1 also includes PCR amplifing reagent；
Optional, described PCR amplifing reagent is quantitative fluorescent PCR reagent.
4. a test kit, it is characterised in that include the primer pair described in claim 1 or 2.
Test kit the most according to claim 4, it is characterised in that: also include PCR amplifing reagent；
Optional, described PCR amplifing reagent is quantitative fluorescent PCR reagent；
Optional, described quantitative fluorescent PCR reagent include from the article No. of TaKaRa company be RR820A'sPremix
Ex TaqTMThe reagent that II test kit is comprised；
Optional, also include positive control and negative control；
Optional, also include that RNA extracts reagent；
Optional, described RNA extracts reagent and includes the RNAsimple that article No. is Cat.#DP419 from TIANGEN company
The reagent that Totle RNA Kit total RNA extraction reagent box is comprised；
Optional, also include reverse transcription reagents；
Optional, described reverse transcription reagents includes the reverse transcription reagents that article No. is Code No.6110A from TaKaRa company
BoxThe reagent that 1st Strand cDNA Synthesis Kit is comprised.
6. the application in detection Δ 42PD1 of the test kit described in claim 5.
7. one kind is detected the method for Δ 42PD1 in testing sample, it is characterised in that include using drawing described in claim 1 or 2
The step of thing pair.
Method the most according to claim 7, it is characterised in that: described testing sample is the blood including DNA and/or RNA
Liquid, cell or tissue, or be plasmid, or be DNA, cDNA, mRNA or cDNA fragment, or be containing DNA, cDNA, mRNA or
The reagent of cDNA fragment；
Optional, when testing sample is DNA, cDNA or cDNA fragment, or it is the reagent containing DNA, cDNA or cDNA fragment, or
During for plasmid, by its directly as template and described primer to carrying out real-time fluorescence PCR reaction；Then Δ in testing sample is judged
The existence of 42PD1 and/or content；
Optional, when testing sample is the blood containing DNA and/or RNA, cell or tissue, or it is mRNA, or is containing mRNA
Reagent time, be translated into DNA, cDNA or cDNA fragment and carry out real-time fluorescence PCR reaction detection again；Then judge to treat test sample
The existence of Δ 42PD1 and/or content in product；
Optional, when described testing sample is blood, also includes the step of the Total RNAs extraction to blood and total serum IgE is carried out
The step of reverse transcription.
Method the most according to claim 8, it is characterised in that:
The reaction system of described real-time fluorescence PCR reaction is as follows:
Method the most according to claim 9, it is characterised in that: the program of described real-time fluorescence PCR reaction is as follows: 98 DEG C
Denaturation 1 minute；98 DEG C of degeneration 10s, 60 DEG C of annealing 30s, 40 circulations.
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| CN107338305A (en) * | 2017-07-23 | 2017-11-10 | 嘉兴允英医学检验有限公司 | A kind of kit for the detection of the expressions of PD 1 |
| WO2023083377A1 (en) * | 2021-11-15 | 2023-05-19 | Versitech Limited | Humanized monoclonal antibody for restoring dysfunctional human t and b cells against cancer and viral infection |
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| CN104250302A (en) * | 2013-06-26 | 2014-12-31 | 上海君实生物医药科技有限公司 | Anti-PD-1 antibody and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107338305A (en) * | 2017-07-23 | 2017-11-10 | 嘉兴允英医学检验有限公司 | A kind of kit for the detection of the expressions of PD 1 |
| WO2023083377A1 (en) * | 2021-11-15 | 2023-05-19 | Versitech Limited | Humanized monoclonal antibody for restoring dysfunctional human t and b cells against cancer and viral infection |
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