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Human serine/threonine kinase PINK1 mutant protein and application thereof

A technology of threonine protein and mutant protein, applied in the field of human serine/threonine protein kinase PINK1 mutant protein

Inactive Publication Date: 2018-11-23
天津市湖滨盘古基因科学发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The function of PINK1 is not yet fully understood

Method used

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  • Human serine/threonine kinase PINK1 mutant protein and application thereof
  • Human serine/threonine kinase PINK1 mutant protein and application thereof
  • Human serine/threonine kinase PINK1 mutant protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, the preparation of gene chip

[0030] First, according to the detection method described in the Chinese invention patent with the application number "201710429915.8" and the patent name "a detection method and device for a mutant protein", the identity of the human serine / threonine protein kinase PINK1 mutant protein can be determined. Encoding gene, its nucleotide sequence is shown in SEQ ID NO:2; Correspondingly, according to this encoding gene, determine the human serine / threonine protein kinase PINK1 mutant protein, its amino acid sequence is shown in SEQ ID NO:1 .

[0031] Secondly, according to the human serine / threonine protein kinase PINK1 mutant protein and its coding gene, the preparation of the gene chip is realized in the following manner.

[0032] 1. Design of nucleotide probes

[0033] (1) Design of nucleotide probe: The nucleotide probe is based on the nucleotide sequence of the human serine / threonine protein kinase PINK1 mutant protein, a...

Embodiment 2

[0062] Embodiment 2, the application of gene chip

[0063] 1. Sample processing

[0064] (1) Collect 1-3mL of blood from the test subject.

[0065] (2) Take the DEPC-treated 1.5mL EP tube and use it as the test sample processing tube. Add 300 μL of the blood of the test subject to the tube, then add 700 μL of Trizol, mix well, and place at room temperature for 10 minutes.

[0066] (3) Add 140 μL of chloroform, close the cap tightly, shake vigorously, place at room temperature for 3-5 minutes, place in a centrifuge at 12000 r / min, 4 °C for 15 minutes, carefully absorb the supernatant in the centrifuge tube, and remove the supernatant Transfer to a clean centrifuge tube.

[0067] (4) Add an equal volume of isopropanol to the centrifuge tube containing the supernatant in step (3), gently invert the centrifuge tube to fully mix the liquid, place at room temperature for 10 minutes, centrifuge at 12000r / min, 4°C for 15 minutes, and carefully absorb All supernatants.

[0068] (5)...

Embodiment 3

[0123] Example 3. Preparation of monoclonal antibody specifically recognizing human serine / threonine protein kinase PINK1 mutein

[0124] 1. According to the base sequence (as shown in SEQ ID NO:2) of human serine / threonine protein kinase PINK1 mutein, design upstream primers as shown in SEQ ID NO:6, and downstream primers as shown in SEQ ID NO: 7 shows:

[0125] Upstream primer (F): gagatccagg caattttta

[0126] Downstream primer (R): gtcggatttc aggtctctg

[0127] 2. The DNA of the test object is used as a template for PCR amplification

[0128] 10×Buffer

[0129] PCR amplification was performed using the DNA of the detection object as a template to obtain the complete fragment of the human serine / threonine protein kinase PINK1 mutant protein gene, which was connected to pMD19-T Vector (Takara Company) for sequencing. Then the antibody is prepared by a specialized biological company, which is a humanized or chimeric antibody. Wherein, the prepared monoclonal ant...

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Abstract

The invention relates to a human serine / threonine kinase PINK1 mutant protein and application thereof. A plurality of patients suffering from leukemia and parkinson's disease are used as studying cases, gene detection and analysis on the cases are performed, the human serine / threonine kinase PINK1 mutant protein is determined, a gene chip, a monoclonal antibody and an ELISA kit are prepared according to the human serine / threonine kinase PINK1 mutant protein, a guidance function is provided for gene diagnosis of patients suffering from leukemia and parkinson's disease, and a scientific basis isprovided for diagnosis and treatment of leukemia and parkinson's disease.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a human serine / threonine protein kinase PINK1 mutant protein and its application. Background technique [0002] PINK1 (PTEN induced putative kinase 1) is a protein kinase expressed in cells throughout the body, especially in energy-intensive organs such as the heart, muscle and brain. In cells, it is mainly located in the inner membrane of mitochondria. The function of PINK1 is not fully understood. It is hypothesized to function to protect mitochondria within cells during periods of cellular stress, such as when cells have abnormally high energy demands. [0003] Recently, studies have found that the mutation of the PINK1 gene is associated with the occurrence of leukemia, and the mutation of the PINK1 gene can lead to autosomal recessive early-onset Parkinson's disease. Therefore, the human PINK1 serine / threonine protein kinase PINK1 The detection can provide certain guida...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C07K16/40C12Q1/6886C12Q1/6883C40B40/08G01N33/577G01N33/574G01N33/573
CPCC12N9/1205C07K16/40C12Q1/6883C12Q1/6886C40B40/08G01N33/573G01N33/57426G01N33/57484G01N33/577G01N2800/2835
Inventor 张耀洲吴玉乾冯建华李冬梅张树军陈玉皎王文雅
Owner 天津市湖滨盘古基因科学发展有限公司
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