Fermentation method for production of recombination protein by lactose-induced pMFH carrier

A fermentation method and recombinant protein technology are applied in the field of genetic engineering drug preparation, and achieve the effects of simple post-processing, simple fermentation method and low production cost

Inactive Publication Date: 2010-12-15
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the recombinant vector const

Method used

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  • Fermentation method for production of recombination protein by lactose-induced pMFH carrier
  • Fermentation method for production of recombination protein by lactose-induced pMFH carrier
  • Fermentation method for production of recombination protein by lactose-induced pMFH carrier

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] (1) Construction of pMFH-GLP-1-DP6.2 prokaryotic expression vector

[0039] ① Construction of pMFH-GLP-1(7-37) recombinant plasmid

[0040] GLP-1(7-37) cDNA sequence is:

[0041] CAC GCT GAA GGT ACC TTC ACT TCC GAC GTT TCC TCT TAC CTG GAA GGG CAG GCT GCA AAAGAA TTT ATC GCT TGG CTG GTT AAA GGT CGT GGC TAA

[0042] Design 4 oligonucleotide fragments according to the sequence of GLP-1 (7-37) above, the 5' end of primer 1 introduces the EcoR Ⅰ sticky end; the 5' end of primer 4 contains the BamH Ⅰ sticky end, and make it There are 27 bases complementary to primer 1 at the 3' end, and the primer sequence is as follows:

[0043] EcoR Ⅰ

[0044] Primer 1 (P1): 5' CAC GCT GAA GGT ACC TTC ACT TCC GAC GTT TCC TCT TAC CTGGAA GGG CAG GCT GCA AAA-3’

[0045] Primer 2 (P2): 5'-GAA TTT ATC GCT TGG CTG GTT AAA GGT CGT GGC TAA G-3'

[0046] Primer 3 (P3): 5'-GGA AAC GTC GGA AGT GAA GGT ACC TTC AGC GTG G-3'

[0047] BamH Ⅰ

[0048] Primer 4 (P4): 5' TTA GCC ACG ACC TTT AAC CAG...

Embodiment 2

[0117] (1) The preparation of the secondary seeds of this embodiment is the same as that of Embodiment 1.

[0118] (2) Fermentation and induction: inoculate the secondary seeds in the fermentation medium, the fermenter culture conditions: 5L fermenter (Taiwan Bio-top company, model: BTF-A5L) working volume is 3.5L, inoculum size is 12% (volume percentage), the fermentation temperature is 37°C, the dissolved oxygen is controlled at 30% by adjusting the ventilation and stirring speed (0-1000rpm), and the pH value is controlled at 7.25. During the fermentation process, 2mol / L HCl and 2mol / L NaOH are added To adjust the pH value, the fermentation time is 13h; 4h after inoculation, start to add 200ml of lactose feed solution at a slow speed (1h after feeding), at this time, the pH value of the fermentation liquid will always drop, because glucose metabolism will produce acid , when the added glucose is consumed, the pH value will start to rise, and at this time it enters the first ...

Embodiment 3

[0122] (1) Activation of bacterial classification: Glycerol bacterium pMFH-GLP-1-DP6.2-BL21 (DE3) is carried out shaking flask culture, fills 15ml culture medium in 150ml Erlenmeyer flask, activates, obtains the activated bacterial classification; Shake The condition of bottle culture is 35 ℃, 250rpm cultivates 20h, and the inoculum size of glycerol bacteria is 0.5% (volume percentage), and culture medium is the LB culture medium that contains ampicillin, and the concentration of ampicillin is 100 μ g / ml; The group of LB culture medium The components are as follows: tryptone 10g / L, yeast powder 5g / L, NaCl 10g / L, prepared with deionized water, pH 6.8;

[0123] (2) Preparation of first-class seeds: inoculate the activated bacterial classification prepared in step (1) into the fermentation basal medium and carry out shake flask culture; the condition of shake flask culture is: the filling volume is 25ml fermentation base in 250ml shake flask Culture medium, cultivate overnight at...

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Abstract

The invention discloses a fermentation method for the production of recombination protein by lactose-induced pMFH carrier. According to the invention, expression of target protein is induced by successfully replacing IPTG with lactose as inducer mainly on the basis of formula of optimized fermentation culture medium, formula of feeding liquid prior to inducement, formula of feeding liquid subsequent to inducement and formula of lactose inducing liquid. The expression of the target protein is high, and level of the target protein in total protein of strain can reach 35 to 49%, which is equivalent to that IPTG inducing level. In addition, after lactose is added for inducement in the process of fermentation, glycerin is also added to serve as carbon source, which can not only raise biomass remarkably, but also reduce the generation of byproducts of metabolism, i.e. acetic acid, and avoid the influence of the presence of glucose in the fermentation liquid on lactose inducing effect. Obviously, the method according to the invention is not only simple and convenient, short in fermentation time and low in production cost, but also achieves no residual toxic substances in the resultant product.

Description

technical field [0001] The invention belongs to the field of genetic engineering medicine preparation, and more specifically, the invention relates to a fermentation method for producing recombinant protein by using lactose to induce pMFH vector. Background technique [0002] At present, most of the industrial production uses Escherichia coli as the engineering bacteria to produce the target protein. The high-density cultivation of the engineering bacteria is very important. The high-density cultivation technology is used to increase the fermentation density of the bacteria, and finally improve the specific productivity of the product (unit volume unit time It can not only reduce the culture volume, strengthen downstream separation and purification, but also shorten the fermentation cycle, reduce equipment investment, thereby reducing production costs, and greatly improve its competitiveness in the market. In order to achieve this goal, not only the expression properties of ...

Claims

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Application Information

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IPC IPC(8): C12P21/00C12R1/19
Inventor 李弘剑蒋德旗苏正定周天鸿冉艳红
Owner JINAN UNIVERSITY
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