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Swine GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) protein antibody and preparation method and application thereof

A protein and antibody technology, applied in the field of biological genetic engineering, can solve the problem of lack of porcine GAPDH antibody and other problems

Inactive Publication Date: 2013-12-18
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

GAPDH antibodies have been well studied in both humans and mice. For example, Tan Xiangwen et al. (2008) cloned a partial fragment of the human GAPDH gene and constructed a recombinant plasmid pUC18-GAPDH, and the mouse GAPDH monoclonal antibody Commercial production has started, but pig GAPDH antibody is extremely lacking

Method used

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  • Swine GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) protein antibody and preparation method and application thereof
  • Swine GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) protein antibody and preparation method and application thereof
  • Swine GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) protein antibody and preparation method and application thereof

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Experimental program
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Embodiment 1

[0034] S1. Cloning of porcine GAPDH gene: In the present invention, firstly, total RNA of porcine skeletal muscle satellite cells was extracted, reverse-transcribed to obtain cDNA, and porcine GAPDH gene was cloned using this as a template.

[0035] S11. For the extraction of total RNA from porcine skeletal muscle satellite cells, refer to the instruction manual of RNAiso Plus Total RNA Extraction Reagent from TAKARA Company.

[0036] S12. Reverse transcription of total RNA: The reverse transcription of RNA was performed according to the instructions of the TIANScript cDNA first-strand synthesis kit from TIANGEN, and the specific steps were as follows:

[0037] Add the following reaction mixture to a nuclease-free centrifuge tube on ice:

[0038]

[0039] Reaction conditions: heat at 70°C for 5 minutes, then quickly cool on ice for 2 minutes, briefly centrifuge to collect the reaction solution, and then add the following components:

[0040]

[0041] Reaction condition...

Embodiment 2

[0115] The specificity of the anti-pig GAPDH antibody prepared in Example 1 of the present invention was compared with the commercially available anti-human GAPDH antibody. The specific method was: compare the anti-pig GAPDH antibody prepared by the present invention with the commercially available anti-human GAPDH antibody by Western blot As can be seen from Figure (14), the anti-pig GAPDH antibody prepared in Example 1 of the present invention can well recognize the GAPDH protein of pigs and mice; Human GAPDH antibody can only limitedly recognize mouse GAPDH protein, and basically cannot recognize pig GAPDH protein.

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Abstract

The invention discloses a swine GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) protein antibody and a preparation method and application thereof. According to the swine GAPDH protein antibody and the preparation method thereof, an immunogen is prepared through expressing and purifying swine GAPDH protein, and a swine GAPDH protein resisting rabbit antibody is prepared through immunizing purebred New Zealand rabbits, so that the gap that swine specific antibodies are lacking is made up, and materials are provided for the scientific research on the genetics and breeding of swine; compared with the human GAPDH resisting antibodies purchased on the market, which can distinguish mouse GAPDH proteins only in a limited manner and can hardly distinguish swine GAPDH proteins, the swine GAPDH protein antibody prepared by the method has the advantage that GAPDH proteins of the swine and mice can be distinguished excellently.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and in particular relates to a porcine GAPDH protein antibody and a preparation method and application thereof. Background technique [0002] At present, the preparation of antibodies against pigs at home and abroad is very rare, and with the deepening of pig molecular genetics and breeding, the demand for pig-specific antibodies is becoming more and more intense. In studies using pigs as experimental materials, it is often encountered that no suitable antibody can be found when detecting the expression of a certain protein, which makes many studies unable to be carried out comprehensively. Therefore, it is very urgent to prepare pig protein antibodies. [0003] GAPDH gene is a very important internal reference gene, which is widely used as a standard in mRNA and protein detection. GAPDH antibodies have been well studied in both humans and mice. For example, Tan Xiangwen ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18G01N33/68
Inventor 王翀徐建田志浩区锦馨曾芳童雄李加琪吴珍芳张哲
Owner SOUTH CHINA AGRI UNIV
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