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Method for cloning unique micro-molecule polypeptide ES61 encoding gene of embryonic stem cell

A technology of small molecule polypeptides and embryonic stem cells, which is applied in the field of genetic engineering, can solve the problem of difficult to complete quantitative detection of ES61 polypeptide genes, and achieve the effect of convenient quantitative detection.

Inactive Publication Date: 2014-06-18
广州达恩基因科技有限公司
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Problems solved by technology

[0003] At present, no one has reported the cloning method for the gene encoding the polypeptide ES61, so it is difficult to complete the quantitative detection of the ES61 polypeptide gene in order to further study the function of the polypeptide

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  • Method for cloning unique micro-molecule polypeptide ES61 encoding gene of embryonic stem cell
  • Method for cloning unique micro-molecule polypeptide ES61 encoding gene of embryonic stem cell
  • Method for cloning unique micro-molecule polypeptide ES61 encoding gene of embryonic stem cell

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Embodiment Construction

[0022] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention.

[0023] 1 Total RNA extracted from human villi tissue and transcribed into cDNA

[0024] 1.1 Extraction of RNA

[0025] (1) Pre-treatment of the sample: Take about 30mg of tissue and grind it with liquid nitrogen, put it into a 1.5ml EP tube, add 0.5ml BioPure, and mix for 5-10 minutes to fully lyse the tissue. Cell samples do not need to be milled, and can be directly carried out in the next step.

[0026] (2) Phase separation: add 100 μl of chloroform (1 / 5 of the total volume of BioPure), fully invert and mix well, let stand at room temperature for 5 minutes, centrifuge at 12000 rpm at 4°C for 15 minutes, separate the solution, and carefully pipette the upper part containing RNA. Transfer to a new centrifu...

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Abstract

The invention provides a method for cloning unique micro-molecule polypeptide ES61 encoding gene of an embryonic stem cell. The method comprises the following steps: (1) extracting total RNA of human embryonic chorion tissue, and reversely transcribing the total RNA to be cDNA; (2) designing a PCR amplification primer to amplify a target gene; (3) recycling and purifying a PCR product; (4) performing enzyme digestion and connecting transformation on the gene. By adopting the method, the ES61 encoding gene can be effectively cloned, the quantitative determination of ES61 polypeptide gene is facilitated, and foundation is laid for researching the function of the ES61 polypeptide gene.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for cloning a gene encoding embryonic stem cell-specific small molecule polypeptide ES61. Background technique [0002] Human embryonic stem cells (Embryonic Stem cells, ES cells) are a type of cells that exist in the early stages of embryonic development. They are totipotent and can differentiate into various tissues and organs of organisms; RP4-792G4.2 (NCBI accession number HG492132.1) gene It is a type of ncRNA (non-coding RNA, ncRNA) that is specifically expressed in ES cells and other pluripotent stem cells. Studies have found that the RP4-792G4.2 gene can encode a protein consisting of 61 amino acids The polypeptide is named as ES61 polypeptide. The nucleotide sequence encoding ES61 polypeptide consists of 183 bases and encodes 61 amino acids. This amino acid has a typical secretion signal peptide sequence after bioinformatics analysis. [0003] At ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/79
Inventor 张茂雷陈杰
Owner 广州达恩基因科技有限公司
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