CN109777879A - For detecting primer, kit and the detection method of NR1H3 gene 95bp missing alternative splicing body - Google Patents

Abstract

The present invention is provided to detect primer, kit and the detection method of NR1H3 gene 95bp missing alternative splicing body.The primer sequence for detecting NR1H3 gene 95bp missing alternative splicing body is as shown in SEQ ID NO:1-2.The present invention is separated the 95bp missing alternative splicing body of NR1H3 gene using specific primer from a variety of splicing forms of the gene, quantitative analysis is carried out to the expression of NR1H3 gene 95bp missing alternative splicing body, accurately, quickly, conveniently, there is very strong practicability.

Description

For detecting primer, kit and the inspection of NR1H3 gene 95bp missing alternative splicing body Survey method

Technical field

The present invention relates to field of biotechnology, specifically, being related to for detecting NR1H3 gene 95bp missing alternative splicing Primer, kit and the detection method of body.

Background technique

Liver X receptor alpha (LXR α/NR1H3) is the member of LXR core superrecepter family, as transcription regulatory factor, activity by To the adjusting of cholesterol degradation product cholesterol oxidized forms.The physiological function of NR1H3 mainly maintains cholesterol levels, carbon aquation Close the stable state of object metabolism, lipoprotein metabolism and Fatty synthesis.LXR has the function of adjusting tissue lipid biosynthesis, LXR α shadow The fat deposition and adipocyte lipid of sound of movement object are metabolized, and are lipogenetic positive regulating factors in muscle.Pig NR1H3 gene exists Wide expression in tissue, but its a variety of alternative splicing body identification and separation progress it is relatively slow, this is hindered to a certain extent The research of NR1H3 gene physiological function.Therefore identify a certain spliceosome tissue expression pattern using molecular biology method, into And illustrating with impetus to its biological function, it is significant.

Structurally, LXRs includes 5 structural domains, i.e. ligand-independent transcriptional activation domains, the DNA of aminoterminal The transcriptional activation domain of the ligand-dependent of binding structural domain, hinge area, ligand binding domain and c-terminus.DNA binding structural domain includes One highly conserved zinc fingers, the structural domain start the transcription of target gene in conjunction with target gene specific DNA, regulate and control target base Because of expression；Hinge area can keep the stability of receptor protein, make receptor while dimerization in conjunction with DNA, regulate and control target gene Expression.

Alternative splicing body is the one of the major reasons for influencing protein and functioning.Only find that a kind of NR1H3 is cut in pig Isomers is connect, and detection is cumbersome, time-consuming, accuracy rate is low, therefore needs to develop new detection technique means, deeply to probe into The biological function of spliceosome lays the foundation.

Summary of the invention

The object of the present invention is to provide for detect the NR1H3 gene 95bp missing primer of alternative splicing body, kit and Detection method.

Inventor has found that length is the 9th exon of 95bp during to pig NR1H3 gene cloning and functional study All missings provide a kind of primer for detecting NR1H3 gene 95bp missing alternative splicing body and the examination comprising the primer accordingly Agent box and detection method can effectively solve the problems, such as that existing detection method program is cumbersome, time-consuming, accuracy rate is low.

In order to achieve the object of the present invention, in a first aspect, NR1H3 gene 95bp missing is variable to cut the present invention is provided to detect The primer of junctor, including primer NX3-F and NX3-R, their nucleotide sequence is respectively as shown in SEQ ID NO:1-2.

Second aspect, the present invention provide the kit for containing the primer NX3-F and NX3-R.

Further, the kit further includes the primer 18S-F and 18S-R for expanding 18S reference gene, they Nucleotide sequence is respectively as shown in SEQ ID NO:3-4.

The third aspect, the present invention provides the primer NX3-F and NX3-R or the kit containing above-mentioned primer is detecting NR1H3 gene 95bp lacks the application in alternative splicing body.

Fourth aspect, the present invention provide a kind of method of detection NR1H3 gene 95bp missing alternative splicing body, including following Step:

1) sample to be tested total serum IgE is extracted, reverse transcription is at cDNA；

2) using the cDNA of step 1) as template, 18S reference gene is expanded using primer 18S-F and 18S-R qPCR, simultaneously Alternative splicing body is lacked using primer NX3-F and NX3-R qPCR amplification NR1H3 gene 95bp；

3) amplification curve and melting curve analysis are carried out to the qPCR real-time monitoring result of step 2), using 2^-△△CTRelatively Quantitative approach calculates expression quantity of the NR1H3 gene 95bp missing alternative splicing body in different tissues, with SPSS version 20.0 carry out significance test of difference and are mapped by GraphPad Prism 5.

The reaction system of qPCR are as follows: 2 × SYBR Premix Ex Taq II, 9~11 μ L removes RNA enzyme water 7~8 μ L, 10 μ M upstream primer 0.3~1 μ L, 10 μM of 0.3~1 μ L, cDNA template of downstream primer, 0.5~2.0 μ L.

Preferably, reaction system are as follows: 2 × SYBR Premix Ex Taq II, 10 μ L removes 7.4 μ L of RNA enzyme water, on 10 μM Swim primer 0.3 μ L, 10 μM of 0.3 μ L, cDNA template of downstream primer, 2.0 μ L.

The response procedures of qPCR are as follows: 92~96 DEG C of initial denaturation 30s~6min；92~96 DEG C of denaturation 5~12s, 55~65 DEG C Anneal 25~35s, 35~45 circulations.

Preferably, response procedures are as follows: 95 DEG C of initial denaturation 30s；95 DEG C of denaturation 5s, 59 DEG C of annealing 30s, 45 recycle.

Wherein, 2 × SYBR Premix Ex Taq II includes Ex Taq HS, dNTPs, chlorine containing archaeal dna polymerase Change magnesium, SYBR Green I and PCR reaction buffer.

The amplified production size of primer 18S-F and 18S-R are 119bp, the amplified production size of primer NX3-F and NX3-R For 87bp.

Method above-mentioned uses relative quantitation method in step 3), and using lung tissue as outer ginseng, (the outer ginseng of setting is to disappear Except different batches test error) to calculate the spliceosome opposite in different tissues NR1H3 gene differential expression in different tissues is compared using one-way analysis of variance, as a result with Average value ± standard error indicates, carries out Multiple range test using Duncan ' s method, P < 0.05 indicates that significant difference, P < 0.01 indicate poor Heteropolar significant, all statistical analysis are completed with 20.0 software of SPSS version.

It is for NR1H3 gene for detecting the primer of pig NR1H3 gene 95bp missing alternative splicing body in the present invention The sequence (5 '-of part the 8th exon and exon10 junction after the whole 9th Exon deletion 95bp of mRNA level in-site TCTGCAGGACCGACTGATGTTCC-3 ', SEQ ID NO:5), design identifies the specific primer NX3- of the alternative splicing body R；The upstream primer NX3-F of the alternative splicing body is designed on exon10.Primer pair NX3 in this way can only be augmented with 95bp and lack The spliceosome of mistake, to avoid the interference of other known or unknown NR1H3 gene alternative splicing bodies.

By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that

The 95bp of NR1H3 gene is lacked a variety of montages of the alternative splicing body from the gene using specific primer by the present invention It is separated in form, the expression progress quantitative analysis to NR1H3 gene 95bp missing alternative splicing body, accurate, quick, It is convenient, there is very strong practicability.

Detailed description of the invention

Fig. 1 is the techniqueflow schematic diagram of the embodiment of the present invention 1.

Fig. 2 is the amplification curve of NR1H3 gene and 18S reference gene in the embodiment of the present invention 1.

Fig. 3 is the melting curve of NR1H3 gene and 18S reference gene in the embodiment of the present invention 1.

Fig. 4 is that pig NR1H3 gene 95bp missing alternative splicing body is relatively fixed in different tissues in the embodiment of the present invention 1 Measure expression.In histogram, the value of pillar is average ± standard error；There is no significant (the P < of identical lowercase letter indication difference 0.05), indicate that difference is extremely significant (P < 0.01) without identical capitalization.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW, Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.

The method that embodiment 1 detects NR1H3 gene 95bp missing alternative splicing body

The techniqueflow schematic diagram of the present embodiment is as shown in Figure 1, mainly comprise the steps that

1, the acquisition of tissue sample

1 monthly age length pig 3, from Datong City, Shanxi Province kind pig farm.After butchering, respectively acquire biceps muscle of thigh (GJ), Greater psoas muscle (YJ), longissimus dorsi muscle (BJ), dorsal sc fat (BZ), abdominal subcutaneous fat (PZ), heart (X), liver (G), spleen Dirty (P), lungs (F), kidney (S), stomach (W), small intestine (XC) etc. are put into liquid nitrogen immediately, and it is standby to be subsequently placed at -80 DEG C of refrigerators preservations With.

2, tissue RNA is extracted and cDNA is synthesized

The total serum IgE of each tissue is extracted using TaKaRa RNAiso Plus kit, extracting method is referring to kit explanation Book.The integrality of total serum IgE is detected by agarose gel electrophoresis and is determined；Total rna concentration is micro ultraviolet by NanoDrop1000 Spectrophotometric determination.

Using PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) Kit is by RNA reverse transcription at cDNA.

3, design of primers

Pig NR1H3 gene mRNA forecasting sequence (the GenBank Accession No.XM_ announced according to NCBI 013994345.2) design can identify the primer pair NX3 of NR1H3 gene 95bp missing alternative splicing body；It is announced according to NCBI 18S gene order design primer is to 18S.

Primer pair NX3 is (SEQ ID NO:1-2):

Upstream primer, NX3-F:5 '-GCAGGACCGACTGATGTT-3 '；

Downstream primer, NX3-R:5 '-GCAAACACTTGCTCTGAGTG-3 '；

Primer pair 18S is (SEQ ID NO:3-4):

Upstream primer, 18S-F:5 '-ATGCCAGAGTCTCGTTCGTTAT-3 '；

Downstream primer, 18S-R:5 '-CGGACAGGATTGACAGATTGAT-3 '.

It is complete for NR1H3 gene mRNA levels for detecting the primer of pig NR1H3 gene 95bp missing alternative splicing body The sequence (5 '-of part the 8th exon and exon10 junction after the 9th Exon deletion 95bp of portion TCTGCAGGACCGACTGATGTTCC-3 '), design identifies the specific primer NX3-F of the alternative splicing body；It is shown outside the 10th The downstream primer NX3-R of the alternative splicing body is designed on son.Primer pair NX3 in this way can only be augmented with the spliceosome of 95bp missing, To avoid the interference of other known or unknown NR1H3 gene alternative splicing bodies.

4, fluorescent quantitative PCR

Quantitative fluorescent PCR reaction system is using mixing sample-adding method, the i.e. various components according to needed for each reaction system Quantity and 1 secondary response needed for PCR react number, calculate the total amount of various reactive components, be added to 1 and go RNA enzyme In 1.5mL centrifuge tube, rear brief centrifugation is mixed well, then is dispensed into dedicated 8 union of quantitative fluorescent PCR, is then added respectively Enter template cDNA, then carry out fluorescent quantitative PCR after brief centrifugation, whole operation process is protected from light as far as possible；Quantitative fluorescent PCR is anti- System is answered to be shown in Table 1.Response procedures are as follows: 95 DEG C of initial denaturation 30s；95 DEG C of denaturation 5s, 59 DEG C of annealing 30s, 45 recycle.

1 quantitative fluorescent PCR reaction system of table

5, quantitative fluorescent PCR data are analyzed

The amplification for carrying out NR1H3 gene 95bp missing alternative splicing body and reference gene 18S to fluorescent quantitative PCR result is bent Line (Fig. 2) and melting curve (Fig. 3) analysis.Each gene magnification curve is in serpentine and reaches plateau in Fig. 2, is illustrated to have Effect amplification；Each gene melting curve peak value is single in Fig. 3, illustrates amplified production special, primer free dimer and other are non-specific Property amplification, the analysis of subsequent result can be carried out.The CT value respectively organized according to pig, using lung tissue as outer ginseng (the outer ginseng of setting It is to eliminate different batches test error), it is calculated using relative quantitation method

6, distribution expression pattern charts

According to the calculated result of relative quantification, significance test of difference is carried out using 20.0 software of SPSS version, and The tissue expression map of the spliceosome is made by GraphPad Prism 5, as a result as shown in Figure 4.Individual repeat number is in figure 3, qPCR loading techniques repeat to be 3.

The 95bp of NR1H3 gene can be lacked alternative splicing body from the gene using specific primer provided by the invention It is separated in a variety of splicing forms, quantitative analysis is carried out to the expression of NR1H3 gene 95bp missing alternative splicing body, it is quasi- Really, quickly, conveniently, there is very strong practicability.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Sequence table

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Claims (8)

1. the primer for detecting NR1H3 gene 95bp missing alternative splicing body, which is characterized in that including primer NX3-F and NX3-R, their nucleotide sequence is respectively as shown in SEQ ID NO:1-2.

2. the kit containing primer described in claim 1.

3. kit according to claim 2, which is characterized in that the kit further includes for expanding 18S internal reference base The primer 18S-F and 18S-R of cause, their nucleotide sequence is respectively as shown in SEQ ID NO:3-4.

4. kit described in primer or Claims 2 or 3 described in claim 1 can be changed in detection NR1H3 gene 95bp missing and cut Application in junctor.

5. a kind of method of detection NR1H3 gene 95bp missing alternative splicing body, which comprises the following steps:

1) sample to be tested total serum IgE is extracted, reverse transcription is at cDNA；

2) using the cDNA of step 1) as template, 18S reference gene is expanded using primer 18S-F and 18S-R qPCR, is utilized simultaneously Primer NX3-F and NX3-R qPCR expand NR1H3 gene 95bp and lack alternative splicing body；

3) amplification curve and melting curve analysis are carried out to the qPCR real-time monitoring result of step 2), using 2^-△△CTRelative quantification Method calculates expression quantity of the NR1H3 gene 95bp missing alternative splicing body in different tissues, with SPSS version 20.0 into Row significance test of difference is simultaneously mapped by GraphPad Prism 5.

6. according to the method described in claim 5, it is characterized in that, in step 2) qPCR reaction system are as follows: 2 × SYBR 9~11 μ L of Premix Ex Taq II goes 7~8 μ L of RNA enzyme water, 10 μM of 0.3~1 μ L of upstream primer, 10 μM of downstream primers 0.3 0.5~2.0 μ L of~1 μ L, cDNA template；

Preferably, reaction system are as follows: 2 × SYBR Premix Ex Taq II, 10 μ L removes 7.4 μ L of RNA enzyme water, and 10 μM of upstreams are drawn Object 0.3 μ L, 10 μM of 0.3 μ L, cDNA template of downstream primer, 2.0 μ L.

7. according to the method described in claim 5, it is characterized in that, in step 2) qPCR response procedures are as follows: 92~96 DEG C are pre- It is denaturalized 30s~6min；92~96 DEG C of 5~12s of denaturation, 55~65 DEG C of 25~35s of annealing, 35~45 recycle；

Preferably, response procedures are as follows: 95 DEG C of initial denaturation 30s；95 DEG C of denaturation 5s, 59 DEG C of annealing 30s, 45 recycle.

8. according to the method described in claim 6, it is characterized in that, the Ex Taq of 2 × SYBR Premix described in step 2) II Including Ex Taq HS, dNTPs, magnesium chloride, SYBR Green I and PCR reaction buffer containing archaeal dna polymerase.