CN104726608B - Application method of VEGFR2 gene Val297Ile site - Google Patents

Abstract

The invention discloses an application method of a Val297Ile locus of a VEGFR2 gene, and particularly relates to a kit for preparing a kit for detecting drug sensitivity of a Parkinson patient to levodopa treatment. The kit contains a probe and a primer for detecting the VEGFR2 gene Val297Ile site, and the probe and the primer can be specifically combined with a gene sequence before and after the VEGFR2 gene Val297Ile site and can amplify a gene sequence containing the VEGFR2 gene Val297Ile site. The kit can accurately, quickly and simply detect the gene locus (VEGFR2 gene Val297Ile polymorphism) related to the sensitivity of levodopa treatment Parkinson drugs.

Description

Application method of VEGFR2 gene Val297Ile site

technical field

The invention belongs to the field of molecular biology, and in particular relates to an application method of a VEGFR2 gene Val297Ile site related to drug sensitivity of Parkinson's disease treated with levodopa.

Background technique

Vascular endothelial growth factor receptor 2 (VEGFR2) belongs to the receptor tyrosine kinase (receptor tyrosine kinase, RTK) superfamily, mainly distributed in vascular endothelial cells and lymphatic endothelial cells, is the vascular endothelial growth factor-A (vascular endothelial growth factor, VEGF -A) Promote the division and proliferation of vascular endothelial cells, increase the main regulator of vascular permeability, and play a leading role in the signal transduction of VEGF-A.

Studies have reported that levodopa (levodopa, L-dopa) in the treatment of Parkinson's disease-induced dyskinesia complications may be related to the degeneration of the blood-brain barrier function, making it difficult for drugs to penetrate the substantia nigra and striatum through the blood-brain barrier, causing L- Due to changes in the effective concentration of dopa in the brain. A number of studies have confirmed that L-dopa-induced movement disorders are related to up-regulation of VEGF-A, induction of endothelialization of the blood-brain barrier, and changes in the permeability of the blood-brain barrier.

Studies have shown that VEGFR2 gene polymorphisms are associated with a variety of diseases, and different genotyping methods have been used to examine them. For example, some studies have confirmed that VEGFR2 (rs2071559) polymorphism is associated with increased risk of glioma by using mass spectrometry; some studies have used Taqman probe method to study the relationship between VEGFR2-604T/C polymorphism and the susceptibility and invasion of nasopharyngeal carcinoma. sexual relationship. Mass spectrometry is a high-throughput typing method for large samples and multiple sites, while Taqman probe method is the gold standard for medium-throughput typing, and has the advantages of high sensitivity and relatively low cost.

The Taqman probe method is to select a sequence between the upstream and downstream primers of PCR as a probe, and label the fluorescent group and the quenching group on the upper and lower sides of the probe. The probe can be combined with the template. In the extension reaction, when the primer is synthesized to When the probe is combined with the template, the 5' exonuclease activity of Taq enzyme can degrade the 5' end of the probe, so that the fluorescent group is separated from the quencher group, and the fluorescence is released. Compared with the traditional restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP), the Taqman probe method has the advantages of higher sensitivity, simpler operation, and higher access.

Contents of the invention

The present invention aims to provide the application method of the VEGFR2 gene Val297Ile site, specifically a test kit for detecting polymorphisms of genes related to levodopa treatment of Parkinson's drug sensitivity, which can be used for preparing detection and levodopa Dopa is an agent for the polymorphism of genes related to Parkinson's drug sensitivity.

In order to achieve the above object, the technical solution provided by the invention is:

The application method of the VEGFR2 gene Val297Ile site, wherein the VEGFR2 gene Val297Ile site is used to prepare a preparation for detecting the drug sensitivity of Parkinson's patients to levodopa treatment.

The above preparations include kits, especially kits using the Taqman probe method.

The kit contains probes and primers for detecting the Val297Ile site of the VEGFR2 gene, and the probes and primers can specifically bind to the gene sequences before and after the Val297Ile site of the VEGFR2 gene and amplify the DNA containing the Val297Ile site of the VEGFR2 gene. gene sequence.

The probes and primers are preferably specifically combined with the 500 bp gene sequence before and after the Val297Ile site of the VEGFR2 gene and can amplify the gene sequence including the Val297Ile site of the VEGFR2 gene.

The kit contains the following probes:

VIC-CCTTAACTATAGATGGTGTAACC-MGB;

FAM-CTTAACTATAGATGGTATAACC-MGB;

The kit contains the following primers:

5'-CAGTCTGGGAGTGAGATGAAGAAAT-3';

5'-GGTCATCAGCCCACTGGAT-3'.

VIC and FAM are reporter groups, VIC has an absorption wavelength of 515-535nm and an emission wavelength of 540-560nm; FAM has an absorption wavelength of 485-505nm and an emission wavelength of 515-530nm; MGB is a quenching group.

According to the similar fluorescence intensity of FAM and VIC in the detection of the above kit, it shows that the Val297Ile site of VEGFR2 gene is AG genotype; the fluorescence intensity of VIC is much higher than that of FAM, indicating that the Val297Ile site of VEGFR2 gene is GG genotype; the fluorescence intensity of FAM is much higher than that of VIC The fluorescence intensity indicates that the Val297Ile site of the VEGFR2 gene is the AA genotype.

The previous research of our research group showed that the VEGFR2 gene polymorphism Val297Ile (rs2305948) may affect the expression level of VEGFR2, which is related to the drug sensitivity of levodopa in the treatment of Parkinson's, but there is no related report before.

The extracellular domain of the VEGFR2 protein contains seven immunoglobulin-like loops. The first extracellular loop is the necessary site for binding to VEGF, and the second and third loops are the main sites for tight binding with VEGF. The Val297Ile site of the VEGFR2 gene encodes the third loop of the extracellular domain of VEGFR2. In Chinese Han population, the minimum allele frequency (MinorAlleleFrequency, MAF) is 29.71%. Since there is no relevant report on the Val297Ile gene polymorphism and its function, our research group recently found that it is related to the drug sensitivity of levodopa in the treatment of Parkinson's disease, so no one has tested the Val297Ile polymorphism of the VEGFR2 gene. There is no relevant test kit product available. Therefore, it is of far-reaching significance to explore methods and products for detecting polymorphisms of genes related to levodopa treatment of Parkinson's drug sensitivity.

The kit of the invention can accurately, quickly and simply detect the gene site (VEGFR2 gene Val297Ile polymorphism) related to the drug sensitivity of levodopa for treating Parkinson's.

Description of drawings

Figure 1 is the genotype of the VEGFR2 gene Val297Ile site detected by the Taqman probe method;

Fig. 2 is the genotype at Val297Ile site of VEGFR2 gene detected by mass spectrometry.

detailed description

The following examples are intended to further illustrate the present invention, rather than limit the present invention.

Example 1

1. Design Taqman probes and primers

1.1 Find the 500bp sequence before and after the Val297Ile site of the VEGFR2 gene

Search for VEGFR2 in the PUBMEDSNP database, enter Geneview, select the NC_000004.12 transcript and display the single nucleotide polymorphism (SNP) of the entire gene region, enter rs2305948, and obtain the front and rear 500bp of this polymorphic site in Fastasequence The sequences (SEQ ID NO.5 and SEQ ID NO.6) were used to find suitable probes and primers.

CAGGCTATGAATATTAGTTTTCTCTAGGTGATTACATCTTTCCACATTATGTCATTTCTCTGTTCTCCAAAGTTTTTGATCTACATTCCTTTTAAGGGAATTTCTCTTTAAGAGGTGGCATGAGATACACTGCTCCTTAAACAGTGGTCACATTTACTTGTGTTTCTGCAGTTTATATCCATCTCACTTTCACCACGTGAGGTTTTAAAAATCCTAATTCAGTTGGTTCCATTTATTTCTCCTGAAACAAAATATATTTGTTGTCTGCATGAGGTTAAAAGTTCTGGTGTCCCTGTTTTTAGCATTAAATAATGTTTACCAAAGCCCAGATTTAATTCTGTGTGTTACTAGAAGTTATTGGGTAATGTTATATGCTGTGCTTTGGAAGTTCAGTCAACTCTTTTTTTCAGCATCAGCATAAGAAACTTGTAAACCGAGACCTAAAAACCCAGTCTGGGAGTGAGATGAAGAAATTTTTGAGCACCTTAACTATAGATGGT(SEQIDNO.5)

Y (Y refers to the VEGFR2 gene Val297Ile site)

TAACCCGGAGTGACCAAGGATTGTACACCTGTGCAGCATCCAGTGGGCTGATGACCAAGAAGAACAGCACATTTGTCAGGGTCCATGGTAAGCTATGGTCTTGGAAATTATTCTGTGCCTTGACAAGTGAGATAATTTAAATAAATTTAGGTCACTTAGTGATTCCTATTTTGTTCATTCAGAAGATAGTTTCTAGTTTTTCTTGTTAGGGAGGCCACATGACCTAGAGGTCAAGAGCATAGCTTTGTAGTCAGGAACTTGGGTTCAAACCTCAACTTTAAAGATGAGATGTGCTGATATACAGTAAGAGTTCATTTAGTATTACTTATTATAGTTATTGCTGCTATTAGGATTGTTACTATGATAAATAGTATTAGCTAAGGTAGTTTTTAAATTTTCATTTTATTGCAAGGCTGAGAGGCCTACTTGAATAAGCATGAGCTTTGCAAACTGGGGAAACATTTAGCAATATACAGTTGACCTGTGAGCAACTCAGGGAT(SEQIDNO.6)

1.2 Probe and primer design using PrimerExpress3.0

(1) Probe design principles:

1. Make sure the G-C content is between 20%-80%.

2. The first base at the 5' end of the probe cannot be G.

3. The probe Tm value calculated by PrimerExpress software should be between 68-70°C.

4. The probe should be as short as possible, but not shorter than 13 bases.

5. Avoid too many repetitions of the same base, especially G, no more than 4 or more.

6. Try to use probes that contain more C than G. If C is less than G, the probe on the complementary strand is used.

7. If it is a SNP, the polymorphic site should be located in the center of the probe as much as possible.

(2) Primer design principles:

1. Choose primers after the probe is determined.

2. The primers should be as close to the probe as possible, but not overlapping.

3. Keep the G-C content between 20%-80%.

4. Avoid repeating the same base too much. Especially G, no more than 4 and above.

5. The Tm value calculated by PrimerExpress software should be between 58-60°C.

6. Among the 5 bases at the 3' end, the sum of G and C bases should not exceed 2.

(3) PrimerExpress3.0 operation method

Enter PrimerExpress3.0 software, File/New/select TaqmanMGBAllelic. Paste the 500bp sequence before and after the SNP site, mark the SNP position and select the variation type. Then select Primer/Probe Search, and the software starts to search for suitable primers and probes. If there are no matching primers and probes, they can be designed manually. Determine the Taqman probe and primers at the Val297Ile site of the VEGFR2 gene as follows:

Probes (Applied Biosystems):

Probe1 (G): VIC-CCTTAACTATAGATGGTGTAACC-MGB (SEQ ID NO.1);

Probe2 (A): FAM-CTTAACTATAGATGGTATAACC-MGB (SEQ ID NO.2);

The absorption wavelength of VIC is 515-535nm, and the emission wavelength is 540-560nm;

FAM absorption wavelength 485-505nm, emission wavelength 515-530nm;

MGB is a quenching group.

Primers (Applied Biosystems):

Primer-F: 5'-CAGTCTGGGAGTGAGATGAAGAAAT-3' (SEQ ID NO.3);

Primer-R: 5'-GGTCATCAGCCCACTGGAT-3' (SEQ ID NO. 4).

2. SNP detection

2.1 PCR amplification system and program

2x Master Mix 12.5μl

20X Assay Working Stock 1.25 μl

(Including Probe1, 2 and Primer-F, Primer-R)

Nuclease-freewater 10.25μl

gDNA 1μl (1–20ng)

Total volume25μl

Program (Applied Biosystems7500)

AmpliTaq UP, Enzyme Activation: 95°C, 10 minutes, HOLD

[Denaturation: 95℃, 15seconds;

Annealing/Extension: 60℃, 1minute] 40cycles

Amplified product (108bp):

CAGTCTGGGAGTGAGATGAAGAAATTTTTGAGCACCTTAACTATAGATGGT

Y (Y refers to the VEGFR2 gene Val297Ile site)

TAACCCGGAGTGACCAAGGATTGTACACCTGTGCAGCATCCAGTGGGCTGATGACC (SEQ ID NO. 7).

2.2 Verification of the accuracy of the experimental method

51 samples of unknown VEGFR2 gene Val297Ile locus genotype were detected by this detection method, and the experimental results are shown in Figure 1, among which 3 representative samples were taken: the fluorescence intensity of FAM and VIC of sample No. 45 was similar, indicating that it was AG Genotype; No. 46 sample VIC fluorescence intensity (533-580nm) was significantly greater than FAM fluorescence intensity (465-510nm), indicating that it was GG genotype; No. 47 sample FAM fluorescence intensity (465-510nm) was significantly greater than VIC fluorescence intensity (533 -580nm), indicating that it is AA genotype; NoTemplateControl is a negative control. The genotypes of the above 51 samples were detected by mass spectrometry (Fig. 2), among which, sample No. 45 was of AG genotype; sample No. 46 was of GG genotype; and sample No. 47 was of AA genotype. The genotypes of the corresponding samples detected by the Taqman probe method we established were completely consistent with the known genotypes, indicating that our Taqman SNP detection method was successfully established.

2.3 SNP detection of experimental samples

According to the Taqman probe method established above to detect the polymorphism of VEGFR2 gene Val297Ile, we performed typing experiments on 51 samples. The experimental results are shown in Table 1. Call in Table 1 represents the experimental results of the analysis (Homozygous1/1 is the GG genotype, Homozygous2/2 is the AA genotype, and Heterozygous1/2 is the AG genotype), and 36 of the 51 samples detected were One case was GG genotype, 14 cases were AG genotype, and 1 case was AA genotype. The distribution of the three genotypes was concentrated within the group in Figure 1, and the distribution among groups was significantly different. Further explaining that we have successfully established a Taqman probe method for the detection of VEGFR2 gene Val297Ile polymorphism.

The kit of the invention can quickly and quickly detect the polymorphism of VEGFR2 gene Val297Ile, and has high sensitivity.

In our previous study, we collected 69 samples of patients with Parkinson's disease, including 32 cases in the experimental group (with L-dopa treatment within 5 years), 32 cases in the control group (with L-dopa treatment for more than 8 years without dyskinesia) 37 example. Genotyping experiments were performed on the VEGFR2 gene Val297Ile (rs2305948), and it was found that the maximum L-dopa dosage (mg/day) [565.00±163.55, P=0.038] of the AA type at rs2305948 was significantly higher than that of the AG type [396.88±200.39] and GG type [300.00±80.18]; and the MAIMS scale value of AA type [17.00±5.24, P=0.026] was significantly higher than that of AG type [8.94±6.53] and GG type [7.86±4.45]. The results show that the dose of L-dopa needed for Parkinson's patients with AA type is higher than that of patients with AG and GG genotypes, and the MAIMS scale value of AA type is significantly increased, suggesting that AA type has an important role in the treatment of L-dopa. There may be a correlation with motor dysfunction in the process. Therefore, the VEGFR2 gene Val297Ile has certain clinical guiding significance for the treatment of L-dopa in Parkinson's patients.

Table 1 The results of the Taqman probe method to detect the VEGFR2 gene Val297Ile polymorphism

Claims (1)

1. the preparation that detects VEGFR2 gene Val297Ile loci polymorphism judges disturbances in patients with Parkinson disease pair in preparationApplication on the preparation of the drug susceptibility in the time adopting levodopa treatment, is characterized in that, preparation comprises examinationAgent box, contains following probe in described kit:

VIC-CCTTAACTATAGATGGTGTAACC-MGB；

FAM-CTTAACTATAGATGGTATAACC-MGB；

In described kit, contain following primer:

5’-CAGTCTGGGAGTGAGATGAAGAAAT-3’；

5’-GGTCATCAGCCCACTGGAT-3’；

VIC and FAM are reporter group, VIC absorbing wavelength 515-535nm, emission wavelength 540-560nm;FAM absorbing wavelength 485-505nm, emission wavelength 515-530nm; MGB is quenching group.