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Human aminopeptidase 1 mutant protein and application thereof

A technology of aminopeptidase-like and mutant protein, which is applied in the field of human aminopeptidase-like 1 mutant protein, and can solve problems such as limiting the application of protein hydrolyzate

Inactive Publication Date: 2018-08-21
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

However, its hydrolyzate usually produces bitter peptides with a bitter taste, which limits the application of protein hydrolyzate

Method used

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  • Human aminopeptidase 1 mutant protein and application thereof
  • Human aminopeptidase 1 mutant protein and application thereof
  • Human aminopeptidase 1 mutant protein and application thereof

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Experimental program
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Embodiment 1

[0023] Embodiment 1, the preparation of gene chip

[0024] First, the human aminopeptidase-like 1 mutant protein encoding gene can be determined according to the detection method described in the Chinese invention patent with the application number "201710429915.8" and the patent name "A method and device for detecting a mutant protein". Its nucleotide sequence is shown in SEQ ID NO: 2; correspondingly, human aminopeptidase-like 1 mutein is determined according to the coding gene, and its amino acid sequence is shown in SEQ ID NO: 1.

[0025] Secondly, according to the human aminopeptidase-like 1 mutant protein and its coding gene, the preparation of the gene chip is realized in the following manner.

[0026] 1. Design of nucleotide probes

[0027] (1) Design of the nucleotide probe: the nucleotide probe is compared with the nucleotide sequence of the human aminopeptidase 1 normal protein according to the nucleotide sequence of the human aminopeptidase 1 mutant protein The r...

Embodiment 2

[0048] Embodiment 2, the application of gene chip

[0049] 1. Sample processing

[0050] (1) Collect 1-3mL of blood from the test subject.

[0051] (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.

[0052] (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.

[0053] (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.

[0054] (5) W...

Embodiment 3

[0109] Example 3. Preparation of a monoclonal antibody that specifically recognizes human aminopeptidase-like 1 mutein

[0110] 1. Design an upstream primer as shown in SEQ ID NO:4 according to the base sequence of human aminopeptidase 1 mutein (as shown in SEQ ID NO:2), and a downstream primer as shown in SEQ ID NO:5 :

[0111] Upstream primer (F): gatgaagcga gactgcg

[0112] Downstream primer (R): cacaagcctg cgtctcttgg

[0113] 2. The DNA of the test object is used as a template for PCR amplification

[0114] 10×Buffer

[0115] PCR amplification was performed using the DNA of the test object as a template to obtain the complete fragment of the human aminopeptidase-like 1 mutein 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 antibody can specifically bind to the amino acid sequence shown in S...

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Abstract

The invention relates to human aminopeptidase 1 mutant protein and an application thereof. By using several leukemia patients as research cases and conducting genetic testing and analysis of the cases, the human aminopeptidase 1 mutant protein is identified, and gene chips, monoclonal antibodies and ELISA (enzyme linked immunosorbent assay) kits are prepared according to the human aminopeptidase 1mutant protein. The human aminopeptidase 1 mutant protein has the advantages of providing guidance for genetic diagnosis of the leukemia and realizing diagnosis and treatment of the leukemia.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a human aminopeptidase-like 1 mutant protein and its application. Background technique [0002] Protein is an important nutrient substance. In recent years, the research on the function and development of protein has been paid more and more attention. Protein hydrolysis is an important way of modern food processing. However, its hydrolyzate usually produces bitter peptides with a bitter taste, which limits the application of protein hydrolyzate. Aminopeptidase is a kind of enzyme that hydrolyzes amino acids one by one from the nitrogen terminal of the polypeptide chain, and plays an important role in the debittering of protein hydrolyzate and the deep hydrolysis of protein. [0003] Recent studies have found that there is also a certain relationship between the occurrence of leukemia and the mutation of the gene encoding aminopeptidase-like 1 (isoform CRA_d). There is a cert...

Claims

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Application Information

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IPC IPC(8): C12N9/48C12N15/57C12Q1/6886C07K16/40G01N33/577G01N33/574G01N33/573
CPCC12N9/485C07K16/40C12Q1/6886C12Q2600/156G01N33/573G01N33/57426G01N33/57484G01N33/577G01N2333/948
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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