A kind of mrrp protein, gene, carrier, engineering bacteria and application thereof

A technology of genetically engineering bacteria and proteins, applied in the field of protein biopolymers, to achieve the effects of high yield, uniform length and no chemical pollution

Active Publication Date: 2018-10-16
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 MRRP proteins of the present invention may exist in any known organism

Method used

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  • A kind of mrrp protein, gene, carrier, engineering bacteria and application thereof
  • A kind of mrrp protein, gene, carrier, engineering bacteria and application thereof
  • A kind of mrrp protein, gene, carrier, engineering bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0024]Step 2: Inoculate the MRRP expressing strain clone into 1000mL self-induction medium, put it in a Erlenmeyer flask at a loading rate of 200mL / L, ferment and culture it at 16°C at a shaking speed of 220 rpm for 12h, and then press 35g Ratio of / 100mL fermentation broth Add ammonium sulfate gradually to the fermentation broth, precipitate in ice bath for 30min, then centrifuge at 12000×g, 4°C for 10min to collect the precipitate. The self-induction...

Embodiment 2

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

[0033] Step 2: Inoculate MRRP-expressing strain clones into 1000mL autoinduction medium, put them in a Erlenmeyer flask at a loading rate of 200mL / L, and culture at 40°C for 24h at a shaking speed of 220 rpm, and then press 35g / L Proportion of 100mL fermentation broth Add ammonium sulfate gradually to the fermentation broth, precipitate in ice bath for 30min, then centrifuge at 12000×g, 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 chloride 7.0mg / L, zinc chloride 2.5mg / L, glycerin 10.0g / L, se...

Embodiment 3

[0040] Step 1: The MRRP expression strain is the same as in Example 1.

[0041] Step 2: Inoculate MRRP-expressing strain clones into 1000mL autoinduction medium, place them in a Erlenmeyer flask at a loading rate of 200mL / L, and culture at 28°C for 18h at a shaking speed of 220 rpm, and then press 35g / L Proportion of 100mL fermentation broth Add ammonium sulfate gradually to the fermentation broth, precipitate in ice bath for 30min, then centrifuge at 12000×g, 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 chloride 4.0mg / L, zinc chloride 1.5mg / L, glycerin 5.5g / L, serine 2.25g / L, gl...

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Abstract

The invention discloses MRRP protein, a gene, a carrier, engineering bacteria and application of the MRRP protein, the gene, the carrier and the engineering bacteria to preparation of wound healing medicine. The MRRP protein is formed by sequentially connecting mussel attachment protein Ma1, elastic drosophila melanogaster protein Res1, a spider silk protein nitrogen end sequence Nt1, a spider silk protein middle-piece repetitive sequence SSR, a spider silk protein carbon end sequence Ct1, elastic drosophila melanogaster protein Res2 and mussel attachment protein Ma2. The MRRP protein integrates self-assembling performance of spider silk protein, high elasticity of the elastic drosophila melanogaster protein and high viscosity of the mussel attachment protein, comprehensive performance is excellent, lengths are uniform, the MRRP protein can be biodegraded completely, the yield is high by adopting a microorganism method for preparing, and chemical pollution is avoided; the MRRP protein has high elasticity, and can bond wounds, so that the wounds are bonded without using lines.

Description

(1) Technical field [0001] The invention relates to a protein biopolymer MRRP 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 single-len...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K38/17A61P17/02
Inventor 孙燕卢陈杰刘欢欢李玉儒
Owner 山东润土节能环保工程有限公司
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