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A fusion protein sacp, encoding gene, engineering bacteria and application thereof

A fusion protein and coding gene technology, applied in the field of protein biopolymer fusion, to achieve the effect of outstanding application performance, excellent ductility, and uniform length

Active Publication Date: 2018-05-04
山东润土节能环保工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the existence of natural reproductive isolation, there is no theoretical support for the possibility that the SACP protein of the present invention may exist in any known organism

Method used

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  • A fusion protein sacp, encoding gene, engineering bacteria and application thereof
  • A fusion protein sacp, encoding gene, engineering bacteria and application thereof
  • A fusion protein sacp, encoding gene, engineering bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Step 1: entrust the fusion protein SACP expression cassette sequence to Biotechnology Co., Ltd. to carry out the whole gene synthesis (the nucleotide sequence is shown in SEQ ID NO.2, the amino acid sequence is shown in SEQ ID NO.1), and use restriction enzymes The cut ligation method was inserted into the EcoR V site on PUC57, and then the E. coli DH5α strain was introduced by chemical method or electric shock method, and the SACP expression strain was obtained by screening on carbenicillin-resistant medium and DNA sequencing verification.

[0024]Step 2: inoculate the fusion protein SACP expression strain clone into 1000mL self-inducing medium, put it in a Erlenmeyer flask at a loading rate of 200mL / L, and ferment and cultivate at 16°C at a shaking speed of 220 rpm for 12h, and then Ammonium sulfate was gradually added to the fermentation broth at a ratio of 35g / 100mL of fermentation broth, precipitated in an ice bath for 30min, and then centrifuged at 12000×g at 4°C f...

Embodiment 2

[0032] Step 1: The preparation of the fusion protein SACP expression strain is the same as in Example 1.

[0033] Step 2: Inoculate the fusion protein SACP expression strain clone into 1000mL self-induction medium, put it in a Erlenmeyer flask with a filling volume of 200mL / L, and culture it at 40°C for 24h at a shaking speed of 220 rpm, and then press The ratio of 35g / 100mL fermentation broth was gradually added to the fermentation broth with ammonium sulfate, precipitated in ice bath for 30min, and then centrifuged at 12000×g at 4°C for 10min to collect the precipitate. The self-induction medium formula is: yeast extract 6.0g / L, tryptone 10.0g / L, casein peptone 10.0g / L, glucose 2.0g / L, dipotassium hydrogen phosphate 8.0g / L, potassium dihydrogen phosphate 9.0g / L, ammonium phosphate 5.0g / L, magnesium sulfate 1.5g / L, calcium chloride 6.0mg / L, cobalt chloride 4.0mg / L, copper chloride 2.0mg / L, manganese sulfate 8.0mg / L, Sodium molybdate 7.0mg / L, boric acid 0.6mg / L, ferric chlori...

Embodiment 3

[0039] Step 1: The fusion protein SACP expression strain is the same as that in Example 1.

[0040] Step 2: Inoculate the fusion protein SACP expression strain clone into 1000mL self-induction medium, put it in a Erlenmeyer flask according to the filling amount of 200mL / L, and cultivate it at 28°C for 18h at a shaking speed of 220 rpm, and then press The ratio of 35g / 100mL fermentation broth was gradually added to the fermentation broth with ammonium sulfate, precipitated in ice bath for 30min, and then centrifuged at 12000×g at 4°C for 10min to collect the precipitate. The self-induction medium formula is: yeast extract 4.0g / L, tryptone 7.5g / L, casein peptone 7.5g / L, glucose 1.0g / L, dipotassium hydrogen phosphate 6.0g / L, potassium dihydrogen phosphate 6.0g / L, ammonium phosphate 3.5g / L, magnesium sulfate 0.85g / L, calcium chloride 4.0mg / L, cobalt chloride 2.25mg / L, copper chloride 2.25mg / L, manganese sulfate 4.5mg / L, Sodium molybdate 5.0mg / L, boric acid 0.35mg / L, ferric chlori...

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Abstract

The invention discloses a fusion protein SACP, an encoding gene, an engineering bacterium and an application thereof in preparing wound dressing medicines. The fusion protein is composed of a spider silk protein nitrogen terminal sequence Nt1, fruit fly elastin Res1, Protein Ma1, Drosophila elastin Res2, mussel adhesion protein Ma2, Drosophila elastin Res3 and spider silk protein nitrogen-terminal sequence Nt2 are sequentially connected to form a total of 7 blocks; the novel biopolymer-fusion protein SACP of the present invention, Integrating the self-assembly performance of spider silk protein, the high elastic performance of Drosophila elastin and the high viscosity performance of mussel adhesion protein, the comprehensive performance is excellent, and the length is uniform, and it can be completely biodegraded. Pollution; Wound dressing drugs made of SACP can adhere to skin and wounds efficiently, and have excellent ductility, and have outstanding application performance in wound dressing.

Description

(1) Technical field [0001] The invention relates to a protein biopolymer fusion (SACP) and its application. (2) Background technology [0002] In recent years, a variety of biologically derived peptides, long peptides and proteins with excellent properties have been discovered by researchers. A large number of new protein materials with excellent properties, such as silicon deposition peptides, calcium-binding peptides, chitosan-binding peptides, spider silk proteins, spider silk mucins, arthropod elastin, and shell adhesion proteins, have attracted the attention of materials science research. Due to the limitations of chemical synthesis methods, the current research on protein biomaterials mostly adopts the research idea of ​​synthesizing small peptides first, and then forming larger drug-loaded complexes through multi-stage self-assembly of small peptides. Genetic engineering is a common method for exogenous expression of proteins. The target protein produced by it is a s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N15/72C12N1/21C12P21/02A61L15/32A61L15/42C12R1/19
Inventor 孙燕卢陈杰李玉儒刘欢欢
Owner 山东润土节能环保工程有限公司
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