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Method for producing recombinant sea cucumber antalzyme and recombinant sea cucumber antalzyme produced thereby

A production method and technology of lysozyme, applied in the field of molecular biology pharmacy, can solve the problems of high price, limited sources, complicated production process, etc.

Inactive Publication Date: 2009-05-06
DALIAN POLYTECHNIC UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, lysozyme is mainly extracted from egg white. Due to limited sources and complicated production process, the output of lysozyme is very limited and the price remains high, which makes it difficult to meet the growing market demand.

Method used

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  • Method for producing recombinant sea cucumber antalzyme and recombinant sea cucumber antalzyme produced thereby
  • Method for producing recombinant sea cucumber antalzyme and recombinant sea cucumber antalzyme produced thereby
  • Method for producing recombinant sea cucumber antalzyme and recombinant sea cucumber antalzyme produced thereby

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Embodiment Construction

[0050] 1. Preparation of recombinant sea cucumber lysozyme

[0051] Step 1: Cloning and sequence analysis of sea cucumber lysozyme

[0052] (1) Sea cucumber intestinal tissue was taken from local fresh sea cucumber, and total RNA was extracted from it with TRIZOL REAGENT (Invitrogen) reagent; the conserved cDNA region of sea cucumber lysozyme was amplified with TaKaRa One Step RNA PCR Kit (AMV). According to the known sequences of lysozyme genes of different biological species, degenerate primers were designed, and the sequences were as follows:

[0053] HS1-1 5'-ATGGA(CT)GT(GC)GG(CAT)TC(AT)CT(AG)TCGTGTG-3'

[0054] HS1-2 5′-GCCGTTGTG(TAG)ATACGGGCAAA(AG)TC-3′

[0055] Firstly, the reverse transcription reaction was carried out at 50°C for 30 minutes; after PCR amplification, the reaction conditions were: 94°C, 2 minutes; 94°C, 30s; 40°C, 30s; 72°C, 1.5min cycle 5 times; then 94°C ℃, 30s; 55℃, 30s; 72℃, 1.5min cycle 28 times. Recover the PCR product, connect it to the vecto...

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Abstract

The invention relates to a production method for recombinant holothurian lysozyme and the recombinant holothurian lysozyme produced by the method. The method comprises the following steps: collecting a holothurian lysozyme gene (SEQ ID NO.1) to a eukaryotic expression vector pPIC9K to construct recombinant plasmid, transforming the formed recombinant plasmid into Pichia pastoris SMD 1168, inducing the recombinant plasmid by methanol and expressing the recombinant plasmid to obtain the recombinant lysozyme after purification for cation resin chromatographic column. The method adopts genetically engineered vaccine strains to produce lysozyme, is suitable for large-scale culture and extraction separation, and has the advantages of simple processing technology, simple equipment requirement, short production period, lower cost and no environment pollution. The activity of the prepared recombinant holothurian lysozyme is increased by 30 percent compared with the lysozyme extracted from holothurian tissues; and compared with egg-white lysozyme, the recombinant holothurian lysozyme has far wider bacteriostatic spectrum range, and particularly has more remarkable bacteriostatic effect for Gram-negative bacteria which can not be acted by the egg-white lysozyme.

Description

technical field [0001] The invention relates to a method for producing recombinant sea cucumber lysozyme by using biotechnology, and belongs to the technical field of molecular biology and pharmacy. The invention also relates to the recombinant sea cucumber lysozyme produced by the method. Background technique [0002] Lysozyme is an alkaline enzyme capable of hydrolyzing mucopolysaccharides, which catalyzes the hydrolysis of the β-1,4 glycosidic bond between the mucopolysaccharides N-acetylmuramic acid and N-acetylglucosamine in the bacterial cell wall, resulting in cell lysis , the contents escape and the bacteria die. It widely exists in the tissues, body fluids and secretions of animals, plants and microorganisms in nature, and plays an important role in the immune defense system of biological organisms. For invertebrates lacking specific immunity, the antibacterial effect is particularly important, and the most widely distributed antibacterial protein in invertebrate ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/81C12N9/36C12N1/19C12R1/84
Inventor 丛丽娜朱蓓薇王秀霞谷跃峰杨冠科郝明董秀萍
Owner DALIAN POLYTECHNIC UNIVERSITY
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