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Expressions of recombined swine lactoferrin and its peptide by pichia

A technology for porcine lactoferrin and lactoferrin, which is applied in the field of expression system of Pichia pastoris for expressing recombinant porcine lactoferrin and its peptides, can solve the problems of high price of LF, complicated methods and low purity.

Inactive Publication Date: 2005-02-23
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Extract natural LF from milk, the method is complicated and the purity is not high, which makes LF expensive

Method used

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  • Expressions of recombined swine lactoferrin and its peptide by pichia

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Experimental program
Comparison scheme
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Embodiment Construction

[0013] The following takes porcine lactoferrin as an example to introduce the specific operation method:

[0014] (1) Extract 50-100 mg of mammary gland tissue from Dudu long-lactating pigs, place it in a mortar that has been baked at 200°C and cooled to room temperature, add 1 / 3 of the liquid nitrogen in the mortar, grind it into powder and transfer it to Put into a 1.5ml Eppendorf tube, add 1ml Trizol solution, mix well, let stand on ice for 5 minutes, then add 0.2ml chloroform, cover the tube cap tightly, shake vigorously and centrifuge at 12000rpm for 30 seconds at 4°C, take the upper aqueous phase in Add 0.5ml of isopropanol to a new eppendorf tube, mix well, let stand at -20°C for 30 minutes, centrifuge at 12,000rpm for 10 minutes, discard the supernatant, wash the precipitate with 1ml of 75% ethanol and dry it at room temperature. After drying, dissolve the precipitate in 20-100 μL of enzyme-free water to obtain a total RNA extract, use appropriate primers to perform re...

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Abstract

A bichia methanol saccharomyces expressing recombinant swine lactoferrin and its peptide are disclosed. It includes: amplifying LF gene, constructing and identifying recombinant saccharomyces expressing carrier, sieving and detecting PMAD11 or PMAD16 and Ade+Muts phenotypic conversion, inducing expressing Ade+Muts strain, purifying and identifying objective protein, transferring into fermenter to grow after identifying. It achieves simple process and high purity for feed additives.

Description

technical field [0001] The invention relates to a method for expressing recombinant porcine lactoferrin and its peptide using a Pichia methanolic yeast expression system. Background technique [0002] Lactoferrin (LF for short), isolated from cow milk in 1960, is an iron-binding glycoprotein with a molecular weight of about 80KDa. LF has the functions of transporting iron ions and sterilizing and antiviral. Its expression in mammary glands The amount is the highest, and the content in human milk is the highest, and the content in cow's milk is relatively small. Its unique mechanism of action has attracted people's attention, and it is separated and purified from milk and added to infant milk powder. Lactoferrin peptide (Lactoferricin, referred to as Lfcin) is the product of lactoferrin degraded by protease in the digestive tract of animals, and some of its biological activities are stronger than lactoferrin. Natural LF has a wide range of biological functions, especially i...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/12C12N15/81
Inventor 汪以真许梓荣黄海青胡迎利刘光富
Owner ZHEJIANG UNIV
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