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Rare codon modified human znf580 gene, prokaryotic expressed protein and polyclonal antibody

A codon, rare technology, applied in the field of genetic engineering, can solve the problem that the human ZNF580 gene cannot be expressed in prokaryotic

Inactive Publication Date: 2016-08-17
LOGISTICS UNIV OF CAPF
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the deficiency that the human ZNF580 gene existing in the prior art cannot be expressed in prokaryotes, and to provide a human ZNF580 gene modified with rare codons that can be expressed in prokaryotes

Method used

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  • Rare codon modified human znf580 gene, prokaryotic expressed protein and polyclonal antibody
  • Rare codon modified human znf580 gene, prokaryotic expressed protein and polyclonal antibody
  • Rare codon modified human znf580 gene, prokaryotic expressed protein and polyclonal antibody

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Experimental program
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Effect test

Embodiment 1

[0020] Based on the human ZNF580cDNA, according to the codon preference of Escherichia coli, and while ensuring that the amino acid sequence of the expressed protein remains unchanged, the codon modification was carried out, and the human ZNF580 gene with rare codon modification was obtained, which is SEQ ID Shown in No.1.

Embodiment 2

[0022] 1. Materials and methods

[0023] 1.1 Materials

[0024] PCR kit, agarose gel DNA extraction kit, Escherichia coli BL21 (DE3), T4 DNA ligase and restriction endonuclease were purchased from Dalian Bao Biological Engineering Co., Ltd. The high-purity plasmid mini-extraction kit and enterokinase were purchased from TIANGEN BIOTECH, and the His·band protein purification kit was purchased from Novagen, Germany.

[0025] 1.2 Method

[0026] 1.2.1 Construction of expression plasmids

[0027] According to the characteristics of the expression plasmid pET30a used, the corresponding enterokinase sequence and restriction endonuclease site were designed, and the whole sequence was synthesized by Shanghai Sangon Biotechnology Service Co., Ltd. and connected to the plasmid pET30a, named pET30a-ZNF580. The prokaryotic expression vector pET30a-ZNF580 was successfully constructed.

[0028] 1.2.2 Expression of target protein

[0029] 1.2.2.1 Preparation of Competent Escherichia col...

Embodiment 3

[0059] 1. Preparation method of polyclonal antibody

[0060] 1.1 Antigen preparation

[0061] The purified ZNF580 protein obtained in Example 2 was diluted to 0.5 mg / ml with 0.01 M PBS with pH=7.4. Antigen emulsification adopts double syringe mutual pushing method.

[0062] 1.2 Immunization procedure

[0063] Take 4 New Zealand rabbits, 2 of them are used as the control group, and the control group is immunized with sterilized three-distilled water (the volume used is the same as that of the experimental group) according to the same immunization procedure as the experimental group below.

[0064] 1) Take 1ml of purified ZNF580 protein (0.5mg / ml), emulsify it with an equal volume of Freund's complete adjuvant, and inject it subcutaneously at multiple points on the back of New Zealand rabbits;

[0065] 2) One month after the second immunization, take 1ml of purified ZNF580 protein (0.5mg / ml), emulsify it with an equal volume of Freund's incomplete adjuvant, and inject it subc...

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Abstract

The invention discloses a rare codon-transformed human ZNF580 gene and a prokaryotic expression protein thereof and a polyclonal antibody. The rare codon-transformed human ZNF580 gene is shown by SEQ ID No.1. In the invention, the rare codon in the human ZNF580 gene is transformed to obtain the rare codon-transformed human ZNF580 gene; experiments prove that the transformed gene can generate a great quantity of ZNF580 proteins through prokaryotic expression, thus the problem that the ZNF580 gene can not be expressed in protokaryon in the prior art is solved; the ZNF580 protein is successfully expressed and purified; the polyclonal antibody is successfully prepared by taking the ZNF580 protein as an antigen.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a modified gene and its expression and application. Background technique [0002] Atherosclerosis (AS) is a common disease that seriously endangers human health and is an important pathological basis of ischemic cardiovascular and cerebrovascular diseases. The main features of AS plaques are the accumulation of intracellular and extracellular lipids, infiltration of monocytes / macrophages, formation of foam cells, hyperplasia of aortic smooth muscle, and accumulation of connective tissue components in the lesion. In recent years, with the changes in people's lifestyle and diet structure, the incidence of cardiovascular and cerebrovascular diseases caused by AS is on the rise in my country. AS is a polygenic disease induced by environmental factors and genetic factors, and there is still a lack of very effective treatment measures. Applying molecular biology techniques to a lar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/47C07K16/18
Inventor 张文成李海生牟心红
Owner LOGISTICS UNIV OF CAPF
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