CN105886655A - Nucleotide sequence for detecting swine NLRP6 (nod-like receptor family pyrin domain-containing protein 6) and application of nucleotide sequence - Google Patents
Nucleotide sequence for detecting swine NLRP6 (nod-like receptor family pyrin domain-containing protein 6) and application of nucleotide sequence Download PDFInfo
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Abstract
The invention discloses a nucleotide sequence for detecting swine NLRP6 (nod-like receptor family pyrin domain-containing protein 6) and application of the nucleotide sequence. The nucleotide sequence for detecting the swine NLRP6 is shown as sequences SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 in a sequence table. The sequences SEQ ID NO.1 and the SEQ ID NO.2 respectively represent sense primers and antisense primers for detecting the swine NLRP6, and the sequences SEQ ID NO.3 represent fluorescent probes for detecting the swine NLRP6. The invention further discloses a method for detecting the swine NLRP6 by the aid of the primers and the probes. The nucleotide sequence, the application and the method have the advantages that as shown by results, the method is good in specificity and high in sensitivity, expression of the swine NLRP6 in swine tissue and organ samples can be quickly and accurately detected, the swine NLRP6 can be monitored in real time when NLRP6 inflammasome is researched, inflammation occurrence procedures and development procedures can be timely understood and mastered, and the nucleotide sequence, the application and the method have important theoretical significance and practical significance.
Description
Technical field
The invention belongs to farm animals Medical Technology research field, relate to based on TaqMan hydrolysis probes fluorescence RT-PCR
The detection nucleotide sequence of pig NLRP6 and method, it is adaptable to porcine tissue sample, including stomach, small intestinal, large intestine etc. digestive tract tissue with
And the accurate detection of NLRP6 in the histoorgan such as liver, spleen, lymph node, lungs, it is for porcine tissue organ in particular
The application of the aspects such as the expression analysis of middle NLRP6mRNA, inflammatory reaction Mechanism Study and gut barrier function research.
Background technology
NOD-sample receptor (NOD-like receptors, NLRs) is the big class cause of disease pattern recognition of in innate immunity
Receptor.Body relies on NLRs to identify the signal of pathogen infection, starts immunne response, plays the work of opposing cause pathogeny imcrobe infection
With.NLRP6 is a member of NLRs family, is widely present in digestive tract tissue, immune organ and tissue and immunocyte,
Participate in cause of disease identification and inflammatory body composition.Activate caspase (caspase-1) after the activation of inflammatory body, regulate and control interleukin 1
The generation of pro-inflammatory cytokine IL-1b, IL-18 and IL-33 in (interleukin-1, IL-1) family, participates in regulation body
Inflammatory reaction and gut muco-membranous barrier function.
The release of pro-inflammatory cytokine, it is possible to promote local inflammation cellular infiltration, alleviates or increases the weight of tissue inflammatory damage
State.Appropriateness inflammatory reaction contributes to eliminating interior external source paathogenic factor, makes organismic internal environment be in stable state；Chronic, the inflammation of imbalance
Disease reaction damaging cells and tissue the most constantly, promote the abnormal reconstruct of histoorgan, ultimately result in organ failure and even lose
Lose.Therefore, research different pathogens causes the mechanism of inflammation, is conducive to the process of generation and the inflammation controlled inflammation, makes inflammation
Disease reaction develops towards to the direction that body is useful, promotes that body returns to state and the level of health smoothly.
NLRP6 is to study more a kind of cause of disease identification receptor over the past two years, is concentrated mainly on grinding of animal pattern mice
On studying carefully, the research on people also only has a small amount of report, but the research on pig have not been reported.A lot of animal pathogens infect
After pig, all can cause inflammatory reaction, but its mechanism causing inflammatory reaction of different infection types is the most different.China is foster
Pig big country, the healthy aquaculture relation of pig and affect food-safety problem.Therefore, the generation of research research schweineseuche disease inflammatory reaction
Mechanism, the process of the reaction that controls inflammation, make pig get well early, just seem extremely important.On the one hand, it is possible to reduce medicine
The especially usage amount of antibiotics, it is ensured that the safety of charcuterie, on the other hand, it is also possible to reduce aquaculture cost, carry
The economic benefit of high pig industry.
In our current research, we use TaqMan fluorescence RT-PCR method to establish the detection technique of detection pig NLRP6, right
The various conditions of reaction are optimized, including screening, the optimization of Mg2+ concentration, primer and the probe of primed probe
The optimization of concentration.Establish the special method that can be used in detecting pig NLRP6 mrna expression.
Summary of the invention
It is an object of the present invention to provide the nucleotide sequence of strong, the highly sensitive detection pig NLRP6 of a group-specific,
Including primer sequence and TaqMan hydrolysis probes sequence.
It is a further object to provide the side of detection pig NLRP6 mrna expression quick, accurate, easy to use
Method.
For achieving the above object, the invention discloses following technical scheme:
The nucleotide sequence of one group of detection pig NLRP6, it is characterised in that its nucleotide sequence such as sequence table SEQ ID NO.1 is extremely
Shown in SEQ ID NO.3, wherein SEQ ID NO.1 to SEQ ID NO.2 is respectively sense primer and the antisense of detection pig NLRP6
Primer, sequence SEQ ID NO.3 is the fluorescent probe of detection pig NLRP6.5 ' the end labellings of described probe sequence SEQ ID NO.3
Reporter fluorescence group FAM, 3 ' end labelling quenching fluorescence group TAMRA.
The present invention further discloses the method using the nucleotide sequence of one group of detection pig NLRP6 to carry out detecting, it is special
Levy and be, by following components as the indispensable material used by detection NLRP6:
1) lysate；
2) RT reactant liquor, including: 5 RT buffer, 25 mM MgCl2, 2.5 mM dNTPs(each), 200 mM random primers,
40 U/mL RNase inhibitor, reverse transcription 200 U/mL M-MLV；
3) qPCR reactant liquor, including: 10 PCR buffer, 25 mM MgCl2, 2.5 mM dNTPs(each), 2.5 U/mL
Taq archaeal dna polymerase, 20 mM sense primers, 20 mM antisense primers, 20 mM fluorescent probes；
4) DEPC water: obtain tri-distilled water with three distillations of tap water, add DEPC to final concentration of 0.1%, 37 ° of C in tri-distilled water
Stir process 12 h, 121 ° of C high pressure steam sterilization 15 min；
5) pig NLRP6 standard substance: for the RNA fragment of in vitro transcription, the sequence of pig NLRP6 standard substance is sequence table SEQ ID
Nucleotide sequence shown in NO.4；
Wherein, sense primer is the nucleotide sequence shown in sequence table SEQ ID NO.1, and antisense primer is sequence table SEQ ID
Nucleotide sequence shown in NO.2, fluorescent probe is the nucleotide sequence shown in sequence table SEQ ID NO.3, wherein 5 ' end labelling
Reporter fluorescence group FAM, 3 ' end labelling quenching fluorescence group TAMRA.In described RT reactant liquor, the concentration of each composition is 1
RT buffer, 2.5 mM MgCl2, 0.25 mM dNTPs, 20 mM random primers, the RNase inhibitor of 1 U/mL, 5 U/mL
Reverse transcription M-MLV.In described qPCR reactant liquor, the concentration of each composition is 1 PCR buffer, 3.0 mM MgCl2、
0.2 mM dNTPs, 0.05 U/mLTaq archaeal dna polymerase, 0.2 mM sense primer, 0.2 mM antisense primer, 0.1 mM fluorescence
Probe.
More detailed description of the present invention is as follows:
Select the particular sequence of pig NLRP6 gene as target region, on the basis of Multiple sequence alignments, carry out primer and probe
Design.Primer length is about 20 bases, without secondary structure and repeatability in primer, and sequence complementary with nothing in primer between primer
Row, the melting temperature (Tm value) of primer is at 60 ° of about C, and the Tm value difference between primer is less than 2 DEG C.The length of probe is at 25 alkali
About base, the Tm value of probe, at about 70 DEG C, uses TaqMan to modify, and mark fluorescent group.
The nucleotide sequence of one group of detection pig NLRP6, as follows:
1) 5-TCAACCGCCTCTTCAGCC-3 (SEQ ID NO.1)
2) 5-CGCCCAGTCGTACAGGATTT-3 (SEQ ID NO.2)
3) 5-[FAM]-ACCTCTGACCGTGGTGCTGCAGG-[TAMRA]-3 (SEQ ID NO.3)
Wherein sequence 1) and 2) be respectively sense primer and antisense primer, the sequence 3 detecting pig NLRP6) for detection pig NLRP6's
The fluorescent probe that TaqMan modifies, 5 end labelling reporter fluorescence group FAM(6-carboxy-fluoresceins of probe), 3 end labellings
Quenching fluorescence group TAMRA.
We use TaqMan fluorescence RT-PCR detection technique to establish the detection method of detection pig NLRP6, to reaction
Various conditions are optimized, including screening, the Mg of primed probe2+The optimization of concentration, primer and probe use dense
When the optimization of degree, PCR amplification, primer annealing temperature and the optimization of extension of time, obtain following detection method.
Nucleotides sequence list:
A kind of method detecting pig NLRP6 that the present invention provides, is made up of herein below:
1) RNA extracts test kit: purchased from TIANGEN Biotech (Beijing) Co., Ltd..
2) RT reactant liquor, table 1 is RT reactant liquor formula:
Table 1 RT reactant liquor formula
Reverse transcription M-MLV(200 U/mL), 5 RT buffer, random primer (200 mM), RNase inhibitor (40 U/mL)
Purchased from Dalian treasured biotinylated biomolecule Engineering Co., Ltd；DNTPs(2.5 mM each), MgCl2(25 mM) is purchased from sky root biochemistry section
Skill (Beijing) company limited.
3) qPCR reactant liquor, table 2 is qPCR reactant liquor formula:
Table 2 qPCR reactant liquor formula
Taq archaeal dna polymerase (2.5 U/mL), 10 PCR buffer, dNTPs(2.5 mM each), MgCl2(25 mM) is from sky
Root biochemical technology (Beijing) company limited, primer and the synthesis of probe equal student on commission work biological engineering (Shanghai) limited company.
4) DEPC water, obtains tri-distilled water with three distillations of tap water, adds DEPC to final concentration of 0.1% in tri-distilled water,
37 ° of C stir process 12 h, 121 ° of C high pressure steam sterilization 15 min.
5) pig NLRP6 standard substance: for the RNA fragment of in vitro transcription.NLRP6 RT-PCR amplification obtained is a length of
315bp coding sequence, in pGEM-T carrier, converts TOP10 competent cell, extracts plasmid with the little extraction reagent kit of plasmid
DNA, obtains positive recombiant plasmid, named pGEM-NLRP6 after PCR and enzyme action identify.With the plasmid of purification as template, enzyme
After tangent linearization, try with the Ribo MAXTM Large Scale RNA Production System-T7 of Promega company
Agent box carries out in vitro transcription；Survey after Trizol extracts after in vitro transcription product DNase is removed DNA profiling therein
Fixed, i.e. obtain preparing the RNA standard substance mother solution needed for pig NLRP6 standard substance.
6) gene order of pig NLRP6 standard substance is as shown in SEQ ID NO.4.
7) RT reaction condition: 30 ° of C 10 min, 42 ° of C 1 h, 99 ° of C 5 min, 16 ° of C 1 min.
8) qPCR reaction condition: 95 ° of C 4 min；95 ° of C 10 sec, 62 ° of C 15 sec, 72 ° of C 15 sec, 40 are followed
Ring, collects fluorescence when each cycle annealing extends.
The present invention further discloses the method for the nucleotide sequence detection pig NLRP6 using detection pig NLRP6 in pig group
Knit the application in terms of NLRP6 mrna expression in organ.Experimental result shows: the fluorescence qPCR detection method that the present invention is set up
Viral RNA can be carried out relative quantification, represent Ct value according to standard curve Y=-4.23X+36.13(X, Y represents the phase of NLRP6
To content), it is known that Ct value is the least, and the expression of NLRP6 is the highest.In morbidity pig different tissues, the expression of NLRP6 is above
The expression of NLRP6 in normal pig different tissues, and with the expression of NLRP6 in morbidity pig lymph node for the highest.
The positive effect that the nucleotide sequence of detection pig NLRP6 disclosed by the invention and application are compared with prior art had
Fruit is:
The present invention establishes the fluorescence RT-PCR method for quick of detection pig NLRP6, and this method specificity is good, highly sensitive, energy
NLRP6 in enough porcine tissue organ samples of detection fast and accurately, can be carried out NLRP6 when studying NLRP6 inflammatory body in real time
Monitoring, understands and grasp generating process and the development process of inflammation in time, and uses anti-inflammatory drug to intervene the effect of inflammatory reaction
Really, there is important theory significance and practice significance.
Accompanying drawing explanation
The test of NLRP6 mRNA relative quantification and linear regression result in Fig. 1 pig different tissues；
The specific test result of Fig. 2 NLRP6 fluorescence PCR detecting method.
Detailed description of the invention
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.It addition, embodiment is interpreted as illustrative, and the unrestricted present invention
Scope, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
On the premise of invention spirit and scope, the various changes carrying out the material component in these embodiments and consumption or change are also
Belong to protection scope of the present invention.The present invention is raw materials used and reagent is commercially available.
Embodiment 1
NLRP6 mRNA relative quantification test in pig different tissues
(1) porcine tissue sample treatment: cut off tissue sample to be checked about 1.0 g with aseptic shears and tweezers and fully grind in mortar
Mill, adds 5 mL PBS mixings, is then proceeded to by tissue suspension in aseptic Eppendorf pipe, number standby.
(2) extraction of RNA: extract test kit with RNA and extract the RNA of different tissues, enter according to test kit operating instruction
OK, the RNA of extraction saves backup on ice.
(3) RNA reverse transcription is cDNA: from refrigerator take out M-MLV(200 U/mL), 5 RT buffer, random primer
(200 mM), RNase inhibitor (40 U/mL), dNTPs(2.5 mM each), MgCl2(25 mM), after at room temperature melting,
2 000 rpm are centrifuged 10 sec.If required PCR pipe number is n(n=sample number+1 pipe negative control), each test reaction system needs
Want 10 mL RT reactant liquors, wherein contain 4 mL 5 RT buffer, 2mLMgCl2(25 mM), 2 mL dNTPs(2.5 mM
Each), 1 mL random primer (200 mM), 0.5mLRNase inhibitor (40 U/mL), 0.5mL reverse transcription M-MLV(200
U/mL).Calculate the usage amount of each reagent, add in a proper volume test tube, be sufficiently mixed uniformly, each in each PCR pipe
Subpackage 10 mL.In the regular-PCR pipe of each setting, it is separately added into each 10 mL of RNA solution of preparation, covers tightly lid, in 1000
Rpm is centrifuged 10 sec.PCR pipe is sequenced and puts into regular-PCR instrument and carry out reverse transcription.Response parameter is arranged: 30 ° of C 10 min,
42 ° of C 1 h, 99 ° of C 5 min, 16 ° of C 1 min.After reverse transcription terminates, take out cDNA and save backup on ice.
(4) qPCR reaction: take out Taq archaeal dna polymerase (2.5 U/mL), 10 PCR buffer, sense primer from refrigerator
(20 mM), antisense primer (20 mM), fluorescent probe (20 mM), dNTPs(2.5 mM each), MgCl2(25 mM), in room
After the lower thawing of temperature, 2 000 rpm are centrifuged 10 sec.If it is positive that required PCR pipe number is n(n=sample number+1 pipe negative control+10 pipe
Standard substance), each test reaction system needs 15mL qPCR reactant liquor, wherein contains 2 mL 10 PCR buffer, 2.4 mL
MgCl2(25 mM), 1.6 mL dNTPs(2.5 mM each), 0.4 mLTaq archaeal dna polymerase (2.5 U/mL), 0.2 mL is just
Justice primer (20 mM), 0.2 mL antisense primer (20 mM), 0.1 mL fluorescent probe (20 mM), 8.1 mL DEPC water.Calculate
The usage amount of good each reagent, adds in a proper volume test tube, is sufficiently mixed uniformly, each subpackage 15mL in each qPCR pipe.
In the qPCR pipe of each setting, it is separately added into each 5mL of cDNA of preparation, covers tightly lid, be centrifuged 10 sec in 1000 rpm.Will
QPCR bank of tubes puts into well fluorescent PCR detector, and order put by record sample.Response parameter is arranged: 95 ° of C 4 min；95°C
10 sec, 62 ° of C 15 sec, 72 ° of C 15 sec, 40 circulations, collect fluorescence when each cycle annealing extends.
(5) judgement of result: interpretation of result condition sets, reads testing result.Threshold value setting principle is with threshold line just
Exceed the peak of normal negative controls amplification curve.Different instruments can be adjusted according to instrument noise situation.Quality Control mark
Standard, negative control is without Ct value and without amplification curve.The Ct value of positive control answers 35.0, and specific amplification curve occurs.As
Negative control and positive control conditions are unsatisfactory for conditions above, and it is invalid that this time experiment is considered as.
(6) NLRP6 mRNA relative quantification statistical analysis in pig different tissues: to 3 health pig and 3 pathogenic large intestines
The detection data of the tissue internal organs qPCR that infected pigs is different carry out statistical analysis, the results are shown in Table 3.
Table 3 NLRP6 qPCR detects the result statistics of same head pig different tissues internal organs
The fluorescence qPCR detection method that we set up can carry out relative quantification to viral RNA, according to standard curve Y=-4.23X+
36.13(X represents Ct value, and Y represents the relative amount of NLRP6), it is known that Ct value is the least, and the expression of NLRP6 is the highest.From table
3 it can be seen that in morbidity pig different tissues the expression of NLRP6 be above the expression of NLRP6 in normal pig different tissues, and with
In morbidity pig lymph node, the expression of NLRP6 is the highest.
Embodiment 2
The specific test of detection method
By method described in embodiment 1, several pathogen common in Intestinum Sus domestica tissue and environment (are included escherichia coli, Salmonella
Bacterium, staphylococcus and porcine reproductive and respiratory syndrome virus) detect, result shows set up method and the equal nothing of pathogen
Cross reaction, specificity is good (Fig. 2).
SEQUENCE LISTING
<110>Tianjin City Livestock Raising and Veterinary Inst.
<120>a kind of nucleotide sequence detecting pig NLRP6 and application
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>artificial sequence
<400> 1
tcaaccgcct cttcagcc 18
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
cgcccagtcg tacaggattt 20
<210> 3
<211> 23
<212> DNA
<213>artificial sequence
<400> 3
acctctgacc gtggtgctgc agg 23
<210> 4
<211> 315
<212> DNA
<213>artificial sequence
<400> 4
acaccttcaa ccgcctcttc agccgcgacg cggagggcca gagacctctg accgtggtgc 60
tgcagggccc ggcgggcatc ggcaagacca tggccgctaa gaaaatcctg tacgactggg 120
cggcgggcaa gctgtaccac ggccaggtgg acttcgcctt cttcatgccg tgccgcgagc 180
ttctagagcg atcgggcacg tgcagcctgg ccgacctgat cttggaacag tgccccgacc 240
gcagcgcgcc ggtgccgcag atactagcgc aggccgagcg gctgcttttc attctggacg 300
gtgtggaggaactgc 315
Claims (6)
1. the nucleotide sequence of one group of detection pig NLRP6, it is characterised in that change nucleotide sequence such as sequence table SEQ ID NO.1
To shown in SEQ ID NO.3, wherein SEQ ID NO.1 to SEQ ID NO.2 is respectively the sense primer of detection pig NLRP6 with anti-
Justice primer, sequence SEQ ID NO.3 is the fluorescent probe of detection pig NLRP6.
2. the nucleotide sequence of the detection pig NLRP6 described in claim 1, it is characterised in that: described probe sequence SEQ ID
5 ' the end labelling reporter fluorescence group FAM of NO.3,3 ' end labelling quenching fluorescence group TAMRA.
3. the method for the nucleotide sequence detection pig NLRP6 that a kind uses described in claim 1, it is characterised in that by following group
It is allocated as detecting the indispensable material used by NLRP6:
1) lysate；
2) RT reactant liquor, including: 5 RT buffer, 25 mM MgCl2, 2.5 mM dNTPs(each), 200 mM random primers,
40 U/mL RNase inhibitor, 200 U/mL reverse transcription M-MLV；
3) qPCR reactant liquor, including: 10 PCR buffer, 25 mM MgCl2, 2.5 mM dNTPs(each), 2.5 U/mL
Taq archaeal dna polymerase, 20 mM sense primers, 20 mM antisense primers, 20 mM fluorescent probes；4) DEPC water: use tap water three times
Distillation obtains tri-distilled water, adds DEPC and steam to final concentration of 0.1%, 37 ° of C stir process 12 h, 121 ° of C high pressure in tri-distilled water
Vapour sterilizing 15 min；
5) pig NLRP6 standard substance: for the RNA fragment of in vitro transcription, the sequence of pig NLRP6 standard substance is sequence table SEQ ID
Nucleotide sequence shown in NO.4；
Wherein, sense primer is the nucleotide sequence shown in sequence table SEQ ID NO.1, and antisense primer is sequence table SEQ ID
Nucleotide sequence shown in NO.2, fluorescent probe is the nucleotide sequence shown in sequence table SEQ ID NO.3, wherein 5 ' end labelling
Reporter fluorescence group FAM, 3 ' end labelling quenching fluorescence group TAMRA.
4. the detection method described in claim 3, it is characterised in that in described RT reactant liquor, the concentration of each composition is 1 RT
Buffer, 2.5 mM MgCl2, 0.25 mM dNTPs, 20 mM random primers, the RNase inhibitor of 1 U/mL, 5 U/mL
Reverse transcription M-MLV.
5. the detection method described in claim 3, it is characterised in that in described qPCR reactant liquor, the concentration of each composition is 1
PCR buffer, 3.0 mM MgCl2, 0.2 mM dNTPs, 0.05 U/mL Taq archaeal dna polymerase, 0.2 mM sense primer,
0.2 mM antisense primer, 0.1 mM fluorescent probe.
6. described in claim 3, use the method for nucleotide sequence detection pig NLRP6 of detection pig NLRP6 in porcine tissue organ
Application in terms of NLRP6 mrna expression.
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| CN110736838A (en) * | 2018-07-18 | 2020-01-31 | 北京正旦国际科技有限责任公司 | Application of NLRP6 as disease state marker of type 2 diabetes |
| CN114958918A (en) * | 2022-06-24 | 2022-08-30 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Double-luciferase reporter gene vector based on pig NLRP6 gene promoter region and construction method and application thereof |
| CN114958918B (en) * | 2022-06-24 | 2023-08-25 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Double-luciferase reporter gene vector of porcine NLRP6 gene promoter region |
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