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Cloning of egg white lysozyme gene (RJM) and yeast expression method thereof

A technology for the expression of egg white lysozyme and yeast, which is applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problem that egg white lysozyme has not been successfully expressed in yeast systems, and achieve cost savings and avoid The effect of killing and simplifying the operation steps

Inactive Publication Date: 2015-09-30
JIANGSU AOZHONG BIOLOGICAL TECH CO LTD
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the successful expression of egg white lysozyme (RJM) in yeast system

Method used

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  • Cloning of egg white lysozyme gene (RJM) and yeast expression method thereof
  • Cloning of egg white lysozyme gene (RJM) and yeast expression method thereof
  • Cloning of egg white lysozyme gene (RJM) and yeast expression method thereof

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Embodiment Construction

[0052] In order to make the technical solutions of the present invention clearer and clearer to those skilled in the art, the present invention will be described in further detail below in conjunction with specific examples, but the embodiments of the present invention are not limited thereto.

[0053] A kind of cloning of egg white lysozyme gene (RJM) and yeast expression method thereof, comprises the steps:

[0054] Step 1: Get egg white lysozyme gene (RJM)

[0055] Use the Primer primer design software to design a pair of primers to synthesize the egg white lysozyme gene (RJM) sequence. The sequence of the synthesized lysozyme-F is shown in Sequence Table 1, wherein the CTCGA in the underlined part in Sequence Table 1 is XhoI digestion site, the sequence of the synthetic lysozyme-R is shown in Sequence Table 2, wherein TCTAGA in the underlined part in Sequence Table 2 is the XbaⅠ restriction site; the egg white lysozyme gene was obtained through RT-PCR amplification and PCR...

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Abstract

The invention belongs to the field of biological gene engineering, and discloses cloning of an egg white lysozyme gene (RJM) and a yeast expression method thereof. The method comprises the following steps: acquiring egg white lysozyme gene (RJM), determining nucleic acid sequence and amino acid sequence of the lysozyme gene, constructing a gene expression vector, identifying the gene expression vector, transforming a yeast expression plasmid into yeast cells, inducing the expression of the egg white lysozyme, and verifying by an immune imprinting experiment, thereby implementing the cloning of the egg white lysozyme gene (RJM) and the yeast expression thereof. The method can implement high-density yeast fermentation and abundant expression of the egg white lysozyme gene. Since the microzyme belongs to eukaryon microbes, the expressed lysozyme protein does not have a killing action on the microzyme, thereby effectively preventing the lysozyme protein from killing the engineering bacterium in the prokaryotic microorganism expression process. The cloning belongs to secretion expression, can easily acquire the target protein, and can simplify the operation steps. Compared with the chemically synthesized target protein, the method has the advantage of cost saving.

Description

technical field [0001] The invention relates to a clone of a gene and a yeast expression method thereof, in particular to a clone of an egg white lysozyme gene (RJM) and a yeast expression method thereof, belonging to the field of biological genetic engineering. Background technique [0002] Lysozyme (Lysozyme) is an alkaline enzyme that can hydrolyze mucopolysaccharides in pathogenic bacteria. Its chemical name is N-acetylmuramic glycan hydrolase, which is a hydrolase that specifically acts on microbial cell walls, also known as cell wall lytic enzyme . Lysozyme is widely present in egg whites of birds and poultry, and it is also widely present in mammalian tears, saliva, plasma, urine, milk, white blood cells and other body fluids (such as lymph) and tissue (such as liver, kidney) cells , which is the most abundant in egg white, and most commercial lysozymes are extracted and purified from egg white. Given that the use of antibiotics will bring negative effects such as d...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/81C12N9/36G01N33/573
Inventor 勾连军徐雪松宋海泉赵光伟张立武
Owner JIANGSU AOZHONG BIOLOGICAL TECH CO LTD