Method of in-vitro expression of gekko japonicus Hoxc10 (homebox gene c10) protein and preparation of polyclonal antibody

A polyclonal antibody, hoxc10 technology, applied in botanical equipment and methods, biochemical equipment and methods, from serum immunoglobulins, etc., to achieve high serum titers and good specificity.

Inactive Publication Date: 2011-10-05
NANTONG UNIVERSITY
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Problems solved by technology

There are mostly antibodies against Hoxc10 in mice, humans and rats on the market. These antibodies have certain cross-reactivity

Method used

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  • Method of in-vitro expression of gekko japonicus Hoxc10 (homebox gene c10) protein and preparation of polyclonal antibody
  • Method of in-vitro expression of gekko japonicus Hoxc10 (homebox gene c10) protein and preparation of polyclonal antibody
  • Method of in-vitro expression of gekko japonicus Hoxc10 (homebox gene c10) protein and preparation of polyclonal antibody

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Embodiment Construction

[0042] Step 1), obtaining the EST sequence of the gecko gecko Hoxc10

[0043] According to the homologous comparison of Hoxc10 gene sequences in mouse, human, chicken and other species, a pair of degenerate primers were designed with the following sequences:

[0044] Upstream primer F: 5'-TGTCTCTCAACACCTACCCATC-3';

[0045] Downstream primer R: 5'- GTTCTCCCCK(G / T)GTTCATTTTCT -3' ,

[0046] Gene cloning obtained the EST sequence of Hoxc10 of the gecko gecko.

[0047] Step 2) Acquisition of the full-length sequence of gecko gecko Hoxc10

[0048] The 5'cDNA and 3'cDNA sequences of Gecko gecko gecko Hoxc10 were obtained by RACE method, and the full-length sequence of Hoxc10 was obtained after splicing with the EST sequence, which contained a total of 2156 bp, and its ORF region was located at 112-1242 bp, encoding a total of 376 amino acids. It was confirmed to be the Hoxc10 gene of the gecko gecko by biological correlation analysis. GenBank accession number: GQ267528.1

[00...

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Abstract

The invention discloses a method of the in-vitro expression of a gekko japonicus Hoxc10 (homebox gene c10) protein and the preparation of a polyclonal antibody, comprising the following steps of: obtaining an EST (Expressed Sequence Tag) sequence of gekko japonicus Hoxc10; obtaining an overall length sequence of the gekko japonicus Hoxc10 through an RACE (rapid amplification of cDNA ends) method; predicting an antigenic epitope of the gekko japonicus Hoxc10 protein on line by adopting DNASTAR software and SWISS-PLOT; constructing a prokaryotic expression vector of the gekko japonicus Hoxc10 by selecting superior epitope peptides; inducing the expression of fusion proteins in vitro; purifying the fusion proteins; preparing the polyclonal antibody, and the like. In the invention, an in-vitro gekko japonicus Hoxc10 expression system constructed on the basis of pGEX-4T1 can efficiently express a target protein in BL21 and further purify the target protein; and a large quantity of GST-Hoxc10 fusion proteins can be conveniently obtained; in addition, a rabbit gekko japonicus Hoxc10 antiserum prepared by immunizing a New Zealand rabbit through the GST-Hoxc10 fusion proteins has high titer and good specificity.

Description

technical field [0001] The invention relates to an in vitro expression of gecko gecko Hoxc10 protein and a preparation method of a polyclonal antibody. Background technique [0002] Homeobox gene (Hox), encoding a family of highly conserved transcription factors, these transcription factors play a key role in the morphogenesis of embryonic development, and can maintain normal somite formation, rhombomere formation and development , and the normal differentiation and development of the hindbrain and various organs, tissues and limbs. Hoxc10 is an important member of the Hox gene family. Studies have shown that Hoxc10 not only affects the embryonic development of vertebrates, especially the development of the nervous system, but also affects the regeneration process of amputated limbs and tails of vertebrates. There are mostly antibodies against Hoxc10 in mice, humans and rats on the market. These antibodies have certain cross-reactivity with sheep, rabbits, monkeys, etc. Th...

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Application Information

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IPC IPC(8): C12N15/12C12N15/70C07K14/435C07K16/18C07K16/06
Inventor 顾星星周晓芳陈海莲陆仁飞
Owner NANTONG UNIVERSITY
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