C9ORF135 gene, its encoding protein and application of antibody thereof

A gene and protein technology, applied in the application field of gene C9ORF135

Inactive Publication Date: 2012-12-19
李凌松 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] And in the report of prior art, also do not have the report of the function of this gene and its coded protein, in order to further study the function of this gene and its coded protein, inventor has carried out in-depth research

Method used

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  • C9ORF135 gene, its encoding protein and application of antibody thereof
  • C9ORF135 gene, its encoding protein and application of antibody thereof
  • C9ORF135 gene, its encoding protein and application of antibody thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Preparation of polyclonal antibody of gene C9ORF135

[0048] Use the conserved amino acid sequence encoded by the gene to prepare polyclonal antibody (rabbit source).

[0049] 1. Antibody Preparation

[0050] 1.1 Synthesize a short peptide, amino acid sequence: VERKGSLTLRSHHKKY, as an antigen.

[0051] 1.2 Before the antigen injection, it is necessary to collect some normal serum from rabbits, and use it as a negative control when the detection antibody is prepared. After the rabbit is stable in the new environment, it will take about four days, and blood can be taken from the ear artery, with a blood volume of about 5ml.

[0052] 1.3 Purify the antigen used for immunization, and the antigen can be injected after being fully emulsified by FCA or FIA. After mixing the antigen solution and the adjuvant in equal proportions, place it on a mixer and oscillate vigorously to emulsify the antigen fully. After oscillating, centrifuge at 1000rpm for one minute. If t...

Embodiment 2

[0066] Example 2 Preparation of monoclonal antibody against gene C9ORF135

[0067] 1. Animal Selection and Immunization

[0068] 1.1 Selection of animals

[0069] Purebred BALB / C mice are more docile, have a small range of activities outside the nest, are weak, eat less food and discharge less sewage, and can be raised and lived in laboratories with a generally clean environment. At present, laboratories developing hybridoma technology mostly use purebred BALA / C mice.

[0070] 1.2 Immunization scheme

[0071] Initial immunization: 1-50 μg of antigen plus Freund's complete adjuvant in mice with subcutaneous multi-point injection or intrasplenic injection (generally 0.8-1ml, 0.2ml / point);

[0072] The second immunization: the dose is the same as above, plus Freund's incomplete adjuvant subcutaneous or intraperitoneal injection (the intraperitoneal dose should not exceed 0.5ml).

[0073] 2. Cell Fusion

[0074] 2.1 Preparation before cell fusion

[0075] 2.1.1 Selection of ...

Embodiment 3

[0104] Example 3 Subcellular Localization

[0105] The cloning antibody (rabbit source) of the gene C9ORF135 prepared in Example 1 was used to detect the expression of endogenous proteins in human embryonic stem cells (ES), and the results of human embryonic stem cells (ES) were detected by cell immunofluorescence and Western Blot tests, and it was confirmed that C9ORF135 was new. The gene encodes a membrane protein of human ES cells. in figure 1 It shows the localization of C9ORF135-encoded protein in human ES cells. The upper picture shows self-made antibody staining, and the lower picture shows negative control. From left to right, DAPI-stained nuclei, C9ORF135-encoded protein fluorescent staining, and the synthesis of the former two.

[0106] The new gene C9ORF135 was linked to the Flag tag, overexpressed in HEK293T cells, and then the overexpressed protein was detected with Flag antibody, figure 2 Confocal microscopy results. Among them, from left to right are DAPI-st...

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Abstract

The invention relates to an application of a gene C9ORF135, specifically to the gene C9ORF135, its encoding protein, an application of an antibody against the protein and a kit thereof. The invention brings forward an application of the gene C9ORF135, its encoding protein and the antibody against the protein in diagnosis, detection or treatment of sperm abnormality, an application in a latent target site as a male contraceptive, an application in diagnosis, detection or treatment of sperm vitality reduction and sperm abnormality caused by aging, and an application in detection of differentiation of embryonic stem cells. The invention also brings forward a kit for detecting sperm abnormality, a kit for screening male contraceptives, a kit for detecting differentiation of embryonic stem cells and their usage method.

Description

technical field [0001] The present invention relates to the application of the gene C9ORF135, specifically, the application of the gene C9ORF135, the protein encoded by it, the antibody of the protein and the kit thereof. Background technique [0002] The new gene C9ORF135 is located on human chromosome 13. The coding protein region (CDS) is 690 nucleosides long and encodes 229 amino acids. After analyzing the sequence of amino acid residues with the SWISSPORT tool, it was found that there is a single transmembrane domain. It is divided into extracellular region, transmembrane region and intracellular region, and has a potential N-glycosylation site (Table 1), among which Liu T, Qian W.-J., Gritsenko M.A., et al. Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry. J. Proteome Res. 4: 2070-2080 (2005) suggested that the protein is a glycosylated protein. The predicted domains of this protein are shown in the table ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/53G01N33/577G01N21/31
Inventor 李凌松周士新文锦华
Owner 李凌松
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