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Method for preparing carp small peptide transporter protein PepT1 rabbit antiserum

A carrier protein, antiserum technology, applied in the direction of serum immunoglobulin, anti-animal/human immunoglobulin, etc., can solve the problems of recombinant expression of the full length of PepT1 gene, etc. high price effect

Inactive Publication Date: 2013-07-24
HENAN NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The full length of the carp intestinal PepT1 gene is 2172bp, and its PepT1 protein has 12 transmembrane domains, so it is difficult to express the full length of the PepT1 gene recombinantly

Method used

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  • Method for preparing carp small peptide transporter protein PepT1 rabbit antiserum
  • Method for preparing carp small peptide transporter protein PepT1 rabbit antiserum
  • Method for preparing carp small peptide transporter protein PepT1 rabbit antiserum

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Embodiment Construction

[0014] Embodiments are described in detail with reference to the accompanying drawings.

[0015] 1. Cloning and Expression of Carp Intestinal PepT1

[0016] (1) PCR primer design

[0017] Using Primer Primern 5.0 to design two pairs of primers, based on the carp intestinal tract obtained in our laboratory PepT1 The full-length sequence of the gene, design a pair of specific primers PepT1 –F and PepT1 –R:

[0018] PepT1 –F: 5'-GCAGCCTCAGTCAAAGCA-3'

[0019] PepT1 –R: 5'-ACAGCCACCAGGAGACAT-3'

[0020] The other pair is specific primers containing BamH I and EcoR I double restriction sites PepT1 -F2 and PepT1 -R2:

[0021] PepT1 -F2: 5'-CGGGATCCACAGTCCCCAAC-3' (BamH I)

[0022] PepT1 -R2: 5'-CGGAATTCTCACTGGAAGTAAGCTG-3' (EcoR I)

[0023] The underlined parts are Eco R I and Hind Restriction site for III.

[0024] (2) full length PepT1 Gene cloning and sequence analysis

[0025] Total RNA was extracted from carp foregut tissue by Trizol method, and ...

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Abstract

The invention discloses a method for preparing a carp small peptide transporter protein PepT1 rabbit antiserum. A key point of a technical scheme provided by the invention lays in that the preparation method of the carp small peptide transporter protein PepT1 rabbit antiserum comprises the following steps: (1) cloning gene of a carp intestinal small peptide transporter protein PepT1; (2) constructing gene engineering strains of the small peptide transporter protein PepT1; (3) conducting induced expression and identifying the gene engineering bacteria of the small peptide transporter protein PepT1; (4) preparing a small peptide transporter protein PepT1 antigen; (5) immunizing rabbits; (6) collecting a blood sample and determining titer of the carp intestinal small peptide transporter PepT1; (7) collecting carotid artery blood and preparing rabbit antiserum of the small peptide transporter protein PepT1; and (8) detecting by an immunohistochemical method. The invention adopts partial gene fragment expressing a transmembrane protein and having an antigenic determinant as an antigen protein to substitute a full-length gene, and solves the problem that the transmembrane protein can not be expressed.

Description

technical field [0001] The invention relates to the technical field of biological products, in particular to a method for preparing rabbit antiserum of carp small peptide transporter protein PepT1. Background technique [0002] carp( Cyprinus carpio ), also known as carp kidnapper and carp, belongs to fish class, Cyprinidae, Cyprinidae. There are many types, including red carp, group carp, grass carp, lotus carp, koi carp, fire carp, hibiscus carp, purse carp and so on. Carp has strong adaptability to the water body environment and is a traditional fine species of freshwater aquaculture in my country. [0003] The small peptide transporter protein PepT1 belongs to the proton-dependent small peptide transporter, and it mainly transports most dipeptides and tripeptides, as well as some peptide drugs. It is a H + Gradient is the driving force, a protein that transports small peptides in the intestinal lumen and other tissues from extracellular to intracellular, and plays a ...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K16/06
Inventor 聂国兴张建新刘起丽闫潇孙君君
Owner HENAN NORMAL UNIV
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