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Composite automatic induction culture medium for expressing exogenous protein by pronucleus expression system

A technology for inducing medium and exogenous protein, applied in the field of compound automatic induction medium, can solve the problems of hindering the expression of exogenous protein and affecting the bacterial density, and achieve the goals of saving medium cost, increasing protein production, and facilitating practical operation Effect

Inactive Publication Date: 2008-08-06
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can inhibit early expression, glucose will make the medium acidic, which affects the bacterial density in the stationary phase and hinders the expression of foreign proteins

Method used

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  • Composite automatic induction culture medium for expressing exogenous protein by pronucleus expression system
  • Composite automatic induction culture medium for expressing exogenous protein by pronucleus expression system
  • Composite automatic induction culture medium for expressing exogenous protein by pronucleus expression system

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Effect test

Embodiment 1

[0062] Embodiment 1 The preparation of the compound culture medium of the present invention

[0063] 1) Basal medium (2XZY):

[0064] 20g tryptone

[0065] 10g yeast extract

[0066] to 1L with H 2 O;

[0067] 2) 50X compound phosphate buffer solution:

[0068] 17.75g disodium hydrogen phosphate

[0069] 17.0g potassium dihydrogen phosphate

[0070] 13.4g ammonium chloride

[0071] 3.55g sodium sulfate

[0072] to 100ml with H 2 O;

[0073] 3) 100X organic acid buffer solution

[0074] 54g sodium succinate hexahydrate

[0075] 3g sodium citrate dihydrate

[0076] to 100ml with H 2 O;

[0077] 4) 500X magnesium sulfate storage solution:

[0078] 25g magnesium sulfate heptahydrate

[0079] to 100ml with H 2 O;

[0080] The above storage solution was sterilized at 0.1Mpa at 121.5°C for 20min.

[0081] 5) 1000X ferric chloride storage solution:

[0082] 3g ferric chloride hexahydrate

[0083] 1ml concentrated hydrochloric acid (pH~12M)

[0084] 99ml sterilize...

Embodiment 2

[0089] Construction and expression of embodiment 2 recombinant plasmid p-1, p-2, p-3, p-4, p-5, p-6 and p-7

[0090] 1. The construction process of the three recombinant plasmids p-1, p-2 and p-3 is as follows:

[0091] According to the coding sequences of the antimicrobial peptides CAP and IB, two oligonucleotide chains were artificially synthesized. After PCR, the gene fragments of CAP, CAP transformation and IB were respectively obtained. These three gene fragments have KpnI and EcoRI recognition at both ends. sites; connected to the vector pET-32a treated with the same two enzymes, and obtained recombinant plasmids p-1, p-2, p-3 after enzyme digestion and PCR identification.

[0092] 2. The construction process of p-4, p-5, p-6 and p-7 is as follows:

[0093] DNASIS and GENERUNNER software were used to analyze the antigenicity of gC, gD, gE, and gJ proteins of infectious laryngotracheitis virus, select regions with strong antigenicity, and design four pairs of different s...

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Abstract

The invention discloses a complex autoinduction culture medium which utilizes a prokaryotic expression system to express heterologous protein, each liter culture medium comprises the following components in weight such as tryptone 10-40g, yeast extract 5-20g, six hydrate sodium succinate 0-8.1g, two hydrate sodium citrate 0-1.5g, glycerin 15-50g, glucose 0.5g, milk sugar 2g, monobasic sodium phosphate 3.55g, monopotassium phosphate 3.40g, ammonium chloride 2.68g, sodium sulfate 0.71g, magnesium sulfate heptahydrate 0.50g and iron chloride hexahydrate 0.03g. Compared with normal culture medium LB, the yield of expression level of heterologous protein improves more than 8 times through utilizing complex autoinduction culture medium of the invention to express heterologous protein, compared with the same type complex autoinduction culture medium, the yield improves more than 2 times.

Description

technical field [0001] The invention relates to a culture medium, in particular to a compound auto-induction culture medium for expressing foreign proteins in a prokaryotic expression system controlled by a lac promoter and a T7lac promoter, and belongs to the field of biotechnology. Background technique [0002] The maturity of DNA sequencing technology can accurately provide the coding sequences of tens of thousands of proteins from different species. These coding sequences can be cloned into expression vectors by using DNA recombination technology to realize the expression of the target protein. In recent years, the use of genetic engineering to produce commercial proteins has increased dramatically, and these expressed recombinant proteins have been widely used in industry and medicine. Escherichia coli has the advantages of clear genetic background, easy operation, rapid growth, high expression level, and low culture cost, plus years of experience in exogenous gene exp...

Claims

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Application Information

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IPC IPC(8): C12N1/21
Inventor 王云峰童光志徐灵龙石星明王玫
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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