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Preparation method of carp amino acid transporter transport subunit rBAT rabbit anti-antiserum

An amino acid and 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 rBAT gene, and achieve the solution of inability to express, high titer, specificity, etc. strong effect

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

AI Technical Summary

Problems solved by technology

The full-length rBAT gene of carp intestine is 2040 bp, and its rBAT protein contains a transmembrane domain, so it is difficult to express the full-length rBAT gene recombinantly

Method used

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  • Preparation method of carp amino acid transporter transport subunit rBAT rabbit anti-antiserum
  • Preparation method of carp amino acid transporter transport subunit rBAT rabbit anti-antiserum
  • Preparation method of carp amino acid transporter transport subunit rBAT rabbit anti-antiserum

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Embodiment Construction

[0016] Embodiments are described in detail with reference to the accompanying drawings.

[0017] 1. Cloning and Expression of Carp Intestinal rBAT

[0018] (1) PCR primer design

[0019] Two pairs of primers were designed using Primer Primern 5.0, and a pair of specific primers rBAT–F and rBAT–R were designed based on the full-length sequence of the carp intestinal rBAT gene obtained in our laboratory:

[0020] rBAT–F: 5'-GGGACTGAAGGAAGCAAG-3'

[0021] rBAT–R: 5'-GTCGCTTTCTATCTTCAACACTT-3'

[0022] The other pair are specific primers rBAT-F2 and rBAT-R2 containing Kpn ? and Not ? double restriction sites:

[0023] rBAT-F2: 5'-GG GGTACC ATGAGTTCGACCAAAAAA-3'

[0024] Kpn?

[0025] rBAT-R2: 5'-ATAAGAAT GCGGCCGC TCAGCGGTAGCAGGCTTTCC-3'

[0026] Not?

[0027] The underlined parts are the enzyme cutting sites of Kpn? and Not? respectively.

[0028] (2) Cloning and sequence analysis of the full-length...

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Abstract

The invention discloses a preparation method of a carp amino acid transporter transport subunit rBAT rabbit antiserum. A key point of a technical scheme provided by the invention lays in that the preparation method of carp amino acid transporter transport subunit rBAT rabbit antiserum comprises the following steps: (1) cloning a carp intestinal amino acid transporter transport subunit rBAT gene; (2) constructing gene engineering strains of rBAT; (3) conducting inducible expression and identifying the gene engineering strains of amino acid transporter transport subunit rBAT; (4) preparing an amino acid transporter transport subunit rBAT antigen; (5) immunizing rabbits; (6) collecting a blood sample and determining titer of the carp intestinal amino acid transporter transport subunit rBAT; (7) collecting blood of carotid artery and preparing antiserum amino acid transporter transport subunit rBAT rabbit antiserum; 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 preparation method for rBAT rabbit antiserum of carp amino acid transport carrier transport subunit. 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] Amino acid transport carrier rBAT / b 0 +AT is a heterodimer containing rBAT and b 0 The two subunits of +AT are the main carrier protein for transporting basic amino acids in the kidney and the apical membrane of the small intestine. rBAT is the transport subunit, b 0 +AT is the catalytic subunit. The rBAT protein is a type II membrane glycoprotein...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K16/06
Inventor 聂国兴王俊丽明红王贝刘起丽段艳艳
Owner HENAN NORMAL UNIV
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