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DNA segment for efficient expression of egg white lysozyme by employing recombinant pichia pastoris and expression method thereof

A technology of egg white lysozyme and Pichia pastoris, which is applied in the field of biological genetic engineering, can solve the problems of limited preparation materials, difficult sources, difficult market demands, etc., to simplify the separation and purification process, improve the expression and activity, and increase the copy number. Effect

Inactive Publication Date: 2015-06-10
TIANJIN SHENGJI GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation materials of lysozyme are quite limited, and the source is extremely difficult. Due to the limited source of raw materials and the high cost of separation and purification, its application is limited, and industrial production cannot be carried out. It is difficult to meet the growing market demand. Therefore, other cheap production of egg white The way of lysozyme is very meaningful. In recent years, people are studying the use of molecular biotechnology to construct genetically engineered bacteria and produce lysozyme through fermentation. It will be one of the effective means to increase the production of lysozyme and reduce costs.

Method used

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  • DNA segment for efficient expression of egg white lysozyme by employing recombinant pichia pastoris and expression method thereof
  • DNA segment for efficient expression of egg white lysozyme by employing recombinant pichia pastoris and expression method thereof
  • DNA segment for efficient expression of egg white lysozyme by employing recombinant pichia pastoris and expression method thereof

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Experimental program
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Effect test

Embodiment 1

[0045] The method for fermenting egg white lysozyme by genetically engineered recombinant bacteria involved in the present invention is to use gene synthesis technology to directly obtain the cDNA sequence of egg white lysozyme, construct a recombinant expression vector containing the target gene lyz, and apply the Pichia pastoris expression system , under the induction of methanol, the egg white lysozyme lyz gene can be highly expressed outside the cell. The target gene used in the present invention is synthesized according to the egg white lysozyme protein sequence (ACCESSION NUMBER: GI345100466) published by NCBI, the vector used in the present invention is a shuttle plasmid pPIC9K, and the host cell of the recombinant vector is Pichia pastoris GS115. The constructed recombinant expression vector not only includes the DNA sequence encoding the enzyme, but also has the control elements required for expressing the gene.

[0046] The steps of the specific construction method a...

Embodiment 2

[0065] Enzyme activity determination of recombinant egg white lysozyme:

[0066] ① Substrate preparation

[0067] The substrate used in this experiment was Micrococcus lysodeikticus. Weighed 20 mg of dry enzyme powder, added 1 mL of phosphate buffer (pH6.2), ground it in a mortar for 3 min, and then added phosphate buffer (pH6.2). 2) Appropriate amount, so that the total volume is about 50mL, and the absorbance of the suspension measured at a wavelength of 450nm at 25±0.1°C is 0.70±0.005 (prepared before use).

[0068] ②Preparation of the test solution

[0069] Take an appropriate amount of this product (equivalent to about 10 mg of lysozyme), add an appropriate amount of phosphate buffer (pH 6.2) to dissolve, and dilute to a solution containing 50 μg of lysozyme per 1 mL.

[0070] ③Operation method

[0071] Precisely measure 3mL of the substrate suspension at 25±0.1°C, put it in a 1cm cuvette, measure the absorbance at a wavelength of 450nm, and take it as the zero-second re...

Embodiment 3

[0076] Determination of antibacterial activity of recombinant egg white lysozyme:

[0077] Add 2% agar to the LB medium, mix well, put it in a high-pressure sterilizer, sterilize at 0.12MPa for 30 minutes, cool to 50°C, pour it into a plate, add about 20mL of medium to each plate, place it horizontally, and wait for the medium to solidify. After the water film on the surface of the plate is completely evaporated, take and dilute to a bacterial count of about 5×10 7 100 μL of cfu / mL indicator bacteria suspension, spread on the plate, gently place the Oxford cup on the surface of the culture medium, gently squeeze it with tweezers to make the Oxford cup and the culture medium completely match, and suck 200 μL of the target lysozyme Add the solution into the Oxford cup, be careful not to overflow, then cover the plate, put it in a refrigerator at 4°C overnight, transfer it to an incubator, incubate at 35°C for 15 hours, and measure the inhibition zone.

[0078] Four main pathoge...

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Abstract

The invention belongs to the field of bio-genetic engineering and provides a DNA segment for efficient expression of egg white lysozyme by employing recombinant pichia pastoris and an expression method thereof. The method comprises the following steps: directly obtaining a gene of egg white lysozyme by gene synthesis means; carrying out operations such as digestion and connection, and then introducing the gene into an eukaryotic expression vector pPIC9K; transforming into pichia pastoris GS115, screening high-copy recons, and then realizing efficient expression of the recombinant egg white lysozyme in vitro through methanol induction. The method is not limited by a raw material source; the copy number of the lysozyme can be increased; the expression amount and the activity of the enzyme are improved; the later separation and purification technologies are simplified; large-scale industrialized production of the egg white lysozyme is realized; and the DNA segment has the characteristics of being stable, pure, and high in biological activity, and can be widely applied to many fields such as pharmacy and feeds.

Description

technical field [0001] The invention belongs to the field of biological genetic engineering, and relates to a high-efficiency expression of egg white lysozyme DNA fragments and an expression method using Pichia pastoris. Background technique [0002] Sub-therapeutic amounts of antibiotics and growth-promoting agents are added to feed in large quantities, resulting in the emergence of drug resistance in animals. Due to the emergence of such multi-resistant and stubborn bacteria, traditional antibiotics are facing great challenges. Facing the future, we must open up a new path. It has become an issue of increasing concern to seek active substances with antibacterial, anti-inflammatory and immune-enhancing properties from nature as substitutes. Wei Xi, a well-known expert in microbiology in my country, said: "The era after the glorious antibiotics will be the era of glorious ecological preparations." Lysozyme is the most promising product considered to replace antibiotics. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/36C12N15/81C12R1/84
Inventor 王连民刘珂飞荆玮
Owner TIANJIN SHENGJI GRP CO LTD
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