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Schizosaccharomyces pombe engineering strain having defensins function and constructing method thereof

A technology of Schizosaccharomyces pombe and a construction method, which are applied in the field of Schizosaccharomyces pombe engineering bacteria and its construction, can solve the problems of few research reports and the like, and achieve the effects of high product expression level, improved screening efficiency and low cost

Inactive Publication Date: 2008-04-09
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of Schizosaccharomyces pombe as a host has not attracted attention until recently. There are many examples of foreign proteins being successfully expressed by using Schizosaccharomyces pombe, but there are very few domestic research reports

Method used

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  • Schizosaccharomyces pombe engineering strain having defensins function and constructing method thereof
  • Schizosaccharomyces pombe engineering strain having defensins function and constructing method thereof
  • Schizosaccharomyces pombe engineering strain having defensins function and constructing method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, the cloning of defensin gene

[0034] 1. Using northeast mung bean (Vigna radiata) as material, the genomic DNA was extracted by the improved CTAB method.

[0035] 2. Primer design

[0036] According to the DNA sequence of the defensin gene D1 (GenBank accession number AY437639) published by GenBank, a pair of specific primers were designed using PrimerPremier5.0 primer design software. In order to facilitate directional cloning and vector construction, the upstream primer and downstream primer Restriction endonuclease sites and protective bases were added to the 5' end of the DNA respectively (the underlined part is the enzyme cutting site added):

[0037] D1 upstream primer: 5′-TTT GCTAGC TTG GAT CCA CCT CAA CAA TTC ATC ACT C-3'(NheI)

[0038] Downstream primer: 5′-GGT AGATCT GGG AAT TCC GAAAAG GGT TCAACA G-3′ (BglII)

[0039] 3. PCR amplification of defensin gene

[0040] Using Northeast mung bean (Vigna radiata) DNA as a template, the DNA fr...

Embodiment 2

[0049] Example 2. Construction of Defensin Gene Recombinant Schizosaccharomyces pombe Expression Vector

[0050] The construction process of the recombinant Schizosaccharomyces pombe expression vector pESP-2-D1 containing the target gene is shown in Figure 2, and the specific steps are as follows:

[0051] 1. Construction of recombinant Schizosaccharomyces pombe expression vector

[0052] A large amount of recombinant cloning vector pMD18-T-D1 and expression vector pESP-2 (purchased from Stratagene, USA) plasmid DNA were extracted in large quantities by alkaline lysis, and then the recombinant cloning vector pMD18-T-D1 and the expression vector of Schizosaccharomyces pombe were double-digested respectively pESP-2. Digestion system 20μl, pMD18-T-D1 and pESP-2 were double-digested with restriction endonucleases NheI and BglII, and digested at 37°C for 3h. After the reaction, the whole amount of the reaction solution was electrophoresed on 1% agarose gel, and the DNA was recove...

Embodiment 3

[0058] Example 3, Transformation and identification of recombinant Schizosaccharomyces pombe expression vector

[0059] 1. A large amount of plasmid DNA of recombinant Schizosaccharomyces pombe expression vector pESP-2-D1 was extracted by alkaline lysis method.

[0060] 2. Electroporation conversion method

[0061] Host bacteria: SP-Q01

[0062] YCD medium: yeast extract powder 10g, glucose 20g, hydrolyzed casein 2g, sorbitol 218.6g dissolved in 1L distilled water

[0063] 1.2M Sorbitol: Dissolve 218.604g of sorbitol in 1L of distilled water.

[0064] EMM medium (L):

[0065] Sodium hydrogen phthalate 14.7mM Na 2 HPO 4 15.5mM

[0066] NH 4 Cl 93.5mM Glucose 111mM

[0067] Salts 2% (v / v) 50×stock Vitamins 0.1% (v / v) 1000×stock

[0068]Minerals 0.01% (v / v) 10000×stock

[0069] Salts(50×stock)(L)

[0070] MgCl 2 ·6H 2 O 0.26M CaCl 2 2H 2 O 4.99mM

[0071] KCl 0.67M Na 2 SO 4 14.1mM

[0072] Vitamins (1000×stock)

[0073] Pantothenic Acid 4.20mM Niac...

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Abstract

The invention provides an engineering bacteria of fission yeast schizosaccharomyces pombe which has a function of defensin and a construction method thereof. A constructed expression carrier of the fission yeast schizosaccharomyces pombe which contains defensin genes is led into the yeast schizosaccharomyces pombe and the fission yeast schizosaccharomyces pombe provided by the invention is obtained. The defensin genes are inserted in the polyclonal locus of the expression carrier of the fission yeast schizosaccharomyces pombe and the constructed expression carrier of the fission yeast schizosaccharomyces pombe is led into the fission yeast schizosaccharomyces pombe to gain recombinant fission yeast schizosaccharomyces pombe which is cultured to get the expression of defensin genes. The invention makes up the shortcomings of the traditional preparation method of defensin and the expression system of pronucleus, saccharomyces cerevisiae and pichia. The invention is applied to the production of industrial defensin and has advantages of large production scale, high activity of defensin, strong sterilizing ability, short growth cycle, and low cost of extraction.

Description

technical field [0001] The invention discloses a Schizosaccharomyces pombe engineering bacterium with defensin function and its construction method Background technique [0002] An antibacterial polypeptide widely exists in organisms in nature. In the past, it has not attracted attention for a long time because of the effective antigen-specific response in mammals. However, in recent years, due to various diseases caused by the absence or inactivation of antimicrobial peptides, especially the emergence of more and more resistance to classic antibiotics, antimicrobial peptides have been paid more attention by researchers. [0003] The real rise of antimicrobial peptide research began in the early 1980s. In 1980, Boman et al. isolated a polypeptide with antibacterial activity—ceropins—from the chrysalis of the American silkworm, and published its amino acid sequence in Nature the following year. In the same year, Hirsh et al. isolated another antibacterial polypeptide, defen...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/12C12N15/65C12N15/87C12N15/81
Inventor 王丕武王俊玲付永平
Owner JILIN AGRICULTURAL UNIV
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