Anti-phosphorylation Oct4 protein antibody and its application

A phosphorylation and non-phosphorylation technology, applied in anti-animal/human immunoglobulin, material inspection products, biological testing, etc., can solve the problem that embryonic totipotent cells cannot maintain development and self-repair, lack of inner cell groups, etc. question

Inactive Publication Date: 2012-10-17
李凌松 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reason is the lack of inner cell mass formation, and the totipotent cells of the embryo cannot maintain development and self-repair

Method used

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  • Anti-phosphorylation Oct4 protein antibody and its application
  • Anti-phosphorylation Oct4 protein antibody and its application
  • Anti-phosphorylation Oct4 protein antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1 Preparation method of Oct4 phosphorylation site monoclonal antibody;

[0059] 1: Peptide synthesis

[0060] Synthetic phosphorylated polypeptide: Ala-Phe-Pro-Ser-Val-Pro-Val-p-Thr-Ala-Leu-Gly-Ser-Pro-Met-His50mg, non-phosphorylated polypeptide: Ala-Phe-Pro- Ser-Val-Pro-Val-Thr-Ala-Leu-Gly-Ser-Pro-Met-His 20mg.

[0061] 2: Serum Phase

[0062] After the rabbit was immunized for 5 times, the whole blood was collected, the serum was separated, and the titer of the serum was detected. Use part of the serum to detect the specificity of the reaction with the antigen by western blot method, and screen out the immunized rabbits with strong specificity.

[0063] 3: Cell Fusion

[0064] The selected spleen cells of rabbits and mouse myeloma cells SP2 / 0 were mixed together at a ratio of 5:1, added with 45% PEG solution, bathed in 37°C water for 90s, added culture medium to terminate the PEG effect, and cultured with selective medium. Only successfully fused cells...

Embodiment 2

[0071] Example 2 Preparation method of Oct4 phosphorylation site polyclonal antibody

[0072] 1: Peptide synthesis

[0073] Synthetic phosphorylated polypeptide Gly-Glu-Ala-Phe-Pro-Ser-Val-Pro-Val-p-Thr-Ala-Leu-Gly-Ser Pro-NH2 15mg, non-phosphorylated polypeptide Gly-Glu-Ala-Phe - Pro-Ser-Val-Pro-Val-Thr-Ala-Leu-Gly-Ser-Pro-NH 211 mg.

[0074] 2: Animal immunity

[0075] Select 2 healthy rabbits, collect serum 50-70ml / rat after 5ml / rat and 5 times of immunization.

[0076] 3: Identification of immune serum

[0077] Using enzyme-linked immunoassay, respectively, 10ug / ml antigen phosphorylated polypeptide Gly-Glu-Ala-Phe-Pro-Ser-Val-Pro-Val-p-Thr-Ala-Leu-Gly-Ser-Pro-NH 2 and non-phosphorylated polypeptide Gly-Glu-Ala-Phe-Pro-Ser-Val-Pro-Val-Thr-Ala-Leu-Gly-Ser-Pro-NH 2 Coat the reaction plate, add serum to the reaction plate for antigen-antibody reaction, and screen out animal sera that react positively with phosphorylated polypeptides but not negatively with phosphorylated...

Embodiment 3

[0078] Example 3 Analysis experiment of Oct4 phosphorylation site

[0079] First, the amino acid residues of Oct4 protein were analyzed using NetPhos software 2.0 (Blom et al., 1999). The result of the analysis was that T343 at the C-terminus was a potential phosphorylation site.

[0080] Design primers, use the polymerase chain reaction (PCR) method to truncate the T343 site, and construct the N6 protein expression plasmid. The schematic diagram of the plasmid construction is as attached figure 1 As shown, the constructed N6 protein expression plasmid was transferred into 293 cells for expression, and the cell lysate was collected, immunoprecipitated with Oct4 antibody, transferred to the hybridization membrane, and hybridized with the antibody against phosphorylated threonine. At the same time, a control group was designed. The control group was the full-length Oct4 group. figure 2 As shown, at the same time, the thickness and brightness of the bands were scanned by the Od...

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Abstract

The invention relates to Oct4 protein phosphorylation sites and an anti-phosphorylation Oct4 protein antibody, concretely relates to Oct4 protein 343rd site phosphorylation and an antibody aimed at Oct4 protein 343rd site phosphorylation. The antibody is a monoclonal antibody or a polyclonal antibody. The invention also relates to an application of the antibody used for detecting phosphorylation of Oct4 protein 343rd site threonine. When the Oct4 protein 343rd site threonine is subjected to phosphorylation, the positive regulation and control capability of Oct4 protein on downstream genes Nanong and Sox2 is decreased, thereby the capability for keeping self-replicating type stem cells and pluripotent differentiation is lost. The application of the invention comprises the application of the antibody in detection of stem cells proliferation, cell differentiation, stem cell pluripotency and stem cells self-replicating capability.

Description

technical field [0001] The present invention relates to an antibody against phosphorylated Oct4 protein, in particular to an antibody against phosphorylation at position 343 of Oct4 protein. Background technique [0002] Stem cells are divided into totipotent, pluripotent and multipotent stem cells according to their differentiation ability. Oct4 is a marker of totipotent or pluripotent stem cells. It is positively expressed in embryonic stem cells, embryonic germ cells, and embryonic / germ cell tumors, but its expression is reduced or disappeared in differentiated tissues, suggesting that it is in embryonic stem cells, germ cells, and embryos. / expression in reproductive tumors is closely related to the pluripotent differentiation properties of these cells. Positive expression of Oct4 was also found in adult tissues and somatic tumors. So far, the expression of Oct4 in adult tissues is mainly restricted to cells with stem cell properties, such as individual cells in the b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18G01N33/68
Inventor 李凌松文锦华
Owner 李凌松
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