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Phage Phi X174 lytic cell protein E and application thereof

A bacteriophage Ф, X174 technology, applied in the field of genetic engineering, can solve the problems of increasing the cost of conjugated linoleic acid, etc., and achieve the effect of simple operation, good application prospect and easy use

Inactive Publication Date: 2018-10-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, when Escherichia coli is used as the host to construct engineering bacteria to produce linoleic acid isomerase, the recombinant linoleic acid isomerase is located in the cytoplasm of recombinant Escherichia coli. In order to obtain intracellular enzymes, the cells need to be broken, and this process greatly increases. The cost of industrial production of conjugated linoleic acid

Method used

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  • Phage Phi X174 lytic cell protein E and application thereof
  • Phage Phi X174 lytic cell protein E and application thereof
  • Phage Phi X174 lytic cell protein E and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Pick a single colony from a fresh plate containing E.coli (PAI / GroELS), and culture it in LB medium with kanamycin and chloramphenicol at a final concentration of 100 μg / ml, and culture at 37°C, 200rpm OD 600 = 0.4-0.6, take it out from the shaker, and centrifuge in ice bath for 30 minutes. With 0.1mol / L CaCl 2 Wash the cells 3 times. Add pBV220-E plasmid to competent cells, heat shock in 42°C water bath for 90s after ice bath for 30min, add 1ml of LB medium, incubate at 37°C for 1h, spread the corresponding resistance plate, and pick the correct one after it grows out. Transformants were obtained to obtain recombinant E. coli (PAI / GroELS / E) containing corresponding plasmids.

Embodiment 2

[0033] Pick a single colony of recombinant bacteria E.coli (PAI / GroELS / E) from the plate in the LB medium containing the corresponding antibiotics, cultivate it overnight as a seed solution, and inoculate it in 50 mL of fermentation medium with an inoculum of 2%. , while adding the final concentration of 4.0mg / ml L - Arabinose induces chaperone protein expression. Cultivate the bacteria concentration to OD 600 When =1.5, add IPTG to a final concentration of 0.1 mmol, lower the temperature to 20°C and culture at 200rpm for 20 hours, raise the temperature to 42°C and maintain for 3 hours to induce lysis of recombinant E. coli cells.

Embodiment 3

[0035]The induced lysed and intact cells were collected at 8000×g, 4°C, and centrifuged for 5 minutes, and the same amount of cells were suspended in hypertonic 30% glycerol solution, isotonic saline solution and hypotonic double-distilled solution, and placed in an ice-water bath Shake for 2h. The supernatant was collected by centrifugation at 8000×g at 4°C for 10 min, and the enzyme activity of the supernatant was determined. PAI release rate=released enzyme activity÷intracellular enzyme activity (measured value of enzyme activity after wall breaking by ultrasound)×100%. EDTA, Tween-20, sodium deoxycholate (DOC), and Triton X-100 were added to double-distilled water to a final concentration of 100 mM, 0.5%, 0.2%, and 0.5% to promote the release of PAI.

[0036] The DOC concentration was optimized, and the results showed that the addition of 0.005-0.1% DOC had little effect on the release rate of PAI, and the release rate was 35-39%. The PAI release rate increased significan...

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Abstract

The invention discloses a phage Phi X174 lytic cell protein E and application thereof and belongs to the field of genetic engineering. The method in the invention comprises the following steps: takingrecombinant Escherichia coli E. coli (PAI / GroELS) as an original strain, introducing a phage Phi X174 lytic cell protein E, producing holes in the surfaces of recombinant Escherichia coli cells, andreleasing linoleate isomerase from the cells by utilizing osmotic pressure differences inside and outside the cells. Finally, by optimizing the release conditions, 81% of linoleate isomerase in the cells is released out of the recombinant Escherichia coli cells.

Description

technical field [0001] The invention relates to a bacteriophage ФX174 cleavage protein E and an application thereof, belonging to the field of genetic engineering. Background technique [0002] Conjugated linoleic acid (CLA) is an isomer of linoleic acid (LA) with conjugated double bonds starting at carbons 9, 11 or 10, The 12th digit is a nutritional element commonly found in human or animal bodies. CLA is a newly discovered nutrient element. It has almost become a panacea for preventing modern diseases in the health food industry in Europe and America. It has been developed into food additives, health products and nutritional feed by these countries. Animal and human studies have shown that CLA has a variety of physiological functions, including scavenging free radicals, inhibiting tumor cell proliferation, preventing atherosclerosis, regulating the immune system, and controlling lipid accumulation. At present, conjugated linoleic acid is mainly synthesized by chemical m...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/90C12N15/33C12R1/19
CPCC07K14/005C12N9/90C12N2795/00022C12Y502/01005
Inventor 诸葛斌黄孟楠陆信曜宗红
Owner JIANGNAN UNIV
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