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Fusion protein SACP, coding gene, engineering bacterium and application of fusion protein SACP

A technology of fusion protein and coding gene, which is applied in the field of protein biopolymer fusion to achieve the effects of excellent comprehensive performance, outstanding application performance and high yield

Active Publication Date: 2015-11-11
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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  • Fusion protein SACP, coding gene, engineering bacterium and application of fusion protein SACP
  • Fusion protein SACP, coding gene, engineering bacterium and application of fusion protein SACP
  • Fusion protein SACP, coding gene, engineering bacterium and application of fusion protein SACP

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Step 1: entrust Biotechnology Co., Ltd. with the fusion protein SACP expression cassette sequence to carry out the whole gene synthesis (the nucleotide sequence is shown in SEQIDNO.2, the amino acid sequence is shown in SEQIDNO.1), and insert it by restriction enzyme ligation method The EcoRⅤ site on PUC57 was introduced into the Escherichia coli DH5α strain by chemical method or electric shock method, and the SACP expression strain was obtained by screening the carbenicillin-resistant medium and verifying by DNA sequencing.

[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 for 10min to coll...

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, a coding gene, an engineering bacterium and application of the fusion protein SACP to preparation of wound dressing medicines. The fusion protein is formed by sequentially connecting 7 blocks, namely spider silk protein N terminal sequence Nt1, drosophila elastin Res1, mussel adhesive protein Ma1, drosophila elastin Res2, mussel adhesive protein Ma2, drosophila elastin Res3 and spider silk protein N terminal sequence Nt2. The novel biomacromolecule-fusion protein SACP integrates the self-assembly property of spider silk protein, the high elasticity property of drosophila elastin and the high viscosity property of mussel adhesive protein, is excellent in comprehensive performance and equal in length, can be completely biodegraded, and is high in yield and free of chemical pollution due to the microbiological preparation method; the wound dressing medicine prepared from SACP can be efficiently adhered to the skin and a wound, meanwhile, has favorable ductility and has outstanding application performance for 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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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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