Method for detecting acute angina pectoris treating effect of nitroglycerin

[0062] Example 1

[0063] 1.1 Research object

[0064] We selected 80 Chinese Han nationality patients with a clear diagnosis of coronary heart disease. They all took nitroglycerin as medicine for angina pectoris. According to the effect of sublingual nitroglycerin administration, these patients can be divided into an effective group that is effective within ten minutes, and an ineffective group that is not effective within ten minutes. There are 59 patients in the effective group, including 47 males and 12 females. There were 21 patients in the ineffective group, including 13 males and 8 females. In these patients, from the perspective of gender relations, the effect of drug administration is not significantly related to the gender of the patient (p=0.107).

[0065] 1.2 Experimental methods and results

[0066] 1.2.1 DNA extraction

[0067] DNA was extracted from human blood by conventional phenol-chloroform method, and the concentration was adjusted to 20ng/ul, and then used for conventional PCR amplification.

[0068] 1.2.2 Design of PCR and sequencing primers

[0069] According to the ALDH2 gene sequence in GenBank, the following primers were designed and synthesized. The specific primers are shown in Table 1 below.

[0070] Primer name

[0071] 1.2.3 PCR amplification of ALDH2 gene

[0072] Using the extracted DNA as a template, Taq enzyme was used to perform PCR amplification on the GeneAmp 9700 PCR machine with the Touchdown program. The reaction conditions are: pre-denaturation at 94°C for 2 minutes, denaturation at 94°C for 30 seconds, annealing at 63°C for 40 seconds, and extension at 72°C for 40 seconds, a total of 10 cycles, with each cycle annealing temperature decreasing by 0.5°C; subsequent denaturation at 94°C for 30 seconds, Annealed at 58°C for 40 seconds, and extended at 72°C for 40 seconds, a total of 30 cycles; finally, it was extended at 72°C for 7 minutes. The PCR products were verified by agarose gel electrophoresis.

[0073] As a result, an amplified product of 358 bp was obtained.

[0074] 1.2.4 SNP discovery and detection

[0075] After the PCR product is purified by Resin resin, use ABI-PRISM TM 377 DNA sequencer (applied biosystems (ABI)) was used for fluorescent labeling end-termination of two-way sequencing, using Polyphred software (University of Washington http://droog.mbt.washington.edu/Polyphred.html) Interpretation and SNP confirmation.

[0076] As a result, the following SNP was found: 1951 G→A.

[0077]1.2.5 SNP genotyping and analysis

[0078] These 80 patients were genotyped for the 1951G/A site in the acetaldehyde dehydrogenase 2 gene using the above PCR and sequencing results.

[0079] The results showed that: in the effective group, the number of homozygous individuals of acetaldehyde dehydrogenase 2 genotype 1 (ALDH2*1) (that is, G/G at position 1951) was 40; while in the ineffective group, the number of acetaldehyde dehydrogenase The number of homozygous individuals with 2 genotype 1 (ALDH2*1) is 7. The number of homozygotes for acetaldehyde dehydrogenase 2 gene type 1 (ALDH2*1) was significantly different in the two different groups (x2=7.59, p=0.006). The results show that the effect of sublingual nitroglycerin administration in the treatment of acute angina pectoris is significantly greater for individuals who are homozygous for acetaldehyde dehydrogenase 2 gene type 1 (ALDH2*1) than for acetaldehyde dehydrogenase 2 gene type 2 (ALDH2*1). 2) Individuals who are homozygous (that is, A/A at position 1951), or heterozygous for acetaldehyde dehydrogenase 2 genotype 1 and type 2 (that is, G/A at position 1951) are more effective, and both have significant Sexual difference.

[0080] Example 2

[0081] Activity determination of wild-type and mutant acetaldehyde dehydrogenase 2

[0082] From individuals who are heterozygous for the acetaldehyde dehydrogenase 2 genotype, reverse transcription was used to obtain two different genotypes of reverse transcription DNA (cDNA) of wild type and 1951 G→A mutant, and sequenced The obtained sequence was verified. Subsequently, the nested PCR (nest-PCR) method was used to amplify the target sequence and introduce restriction sites. Then, the amplified sequence was recombined into a plasmid (recombinantplasmid), and the plasmid was transfected into insect cells sf9 (purchased from Invitrogen). The sequence recombined into the plasmid was also verified by sequencing, and they are SEQ ID NO: 5 (wild type, 1510 position is G) and SEQ ID NO: 6 (mutant type, 1510 position is A). Using the conventional immunoblotting method, the expression of the acetaldehyde dehydrogenase 2 gene of the plasmid infected into the cell was verified.

[0083] The recombinantly expressed acetaldehyde dehydrogenase 2 gene protein was purified by affinity chromatography. It can be subjected to 12% acrylamide gel electrophoresis and stained with Coomassie blue to see a protein of about 54kDa.

[0084] Two different genotypes of acetaldehyde dehydrogenase 2 gene proteins were recombinantly expressed, and directly from acetaldehyde dehydrogenase 2 gene type 1 homozygous individuals and aldehyde dehydrogenase 2 gene type 1 and type 2 heterozygous individuals The purified acetaldehyde dehydrogenase 2 gene protein was obtained, and the ability of the four samples to convert nitroglycerin was tested by radiochemical methods.

[0085] The results are shown in the table below:

[0086] Enzyme genotype

[0087] It was found that the protein of acetaldehyde dehydrogenase 2 gene type 1 (ie wild type) had a lower Km and a higher Vmax than the protein of acetaldehyde dehydrogenase 2 gene type 2 in terms of converting nitroglycerin. . The protein homozygous for the acetaldehyde dehydrogenase 2 gene type 1 protein also has a lower Km and a higher Vmax than the type 1 and type 2 heterozygous protein.

[0088] It can be seen that the acetaldehyde dehydrogenase 2 gene type 1 protein has a higher physiological activity of converting nitroglycerin. The acetaldehyde dehydrogenase 2 gene type 2 protein, due to the 1951G→A mutation, changes the amino acid sequence of the protein, which affects the function and activity of the protein. The acetaldehyde dehydrogenase 2 gene type 2 protein cannot effectively convert nitroglycerin, which affects the effect of nitroglycerin on the downstream cGMP pathway, and cannot effectively relax the vascular smooth muscle, thereby reducing the effect of nitroglycerin on acute angina pectoris. .

[0089] Example 3

[0090] A kit for predicting the effectiveness of nitroglycerin in the treatment of acute angina pectoris

[0091] As described in Example 1, the G→A mutation at position 1951 in SEQ ID NO:1 is closely related to the efficacy of nitroglycerin in the treatment of acute angina pectoris. Therefore, based on this mutation, ALDH2 gene-specific primers can be designed to use the DNA of the individual to be tested as a template for amplification for prediction.

[0092] Name

[0093] Take 3ml of blood from the male patient to be tested, and use conventional methods (or use a specific kit) to extract DNA from the blood. Dilute the PCR primers in the kit for predicting the efficacy of nitroglycerin in the treatment of acute angina pectoris to 1μmol/μl, and use the extracted DNA as a template to perform a PCR reaction with the provided primers. After the PCR product is purified, use ABI-PRISM TM 377 DNA sequencer performs two-way sequencing with fluorescent label end termination method, and uses Polyphred software for sequence interpretation and SNP confirmation.

[0094] Alternatively, chromatographic analysis of the amplified product and the normal control using a denaturing high performance liquid chromatograph (DHPLC) can also detect the 1951 position G→A.

[0095] The results showed that the effectiveness of nitroglycerin in the treatment of acute angina in 1951 G→A test subjects (using ten minutes as the standard) is lower than that of ordinary people with acute angina.