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Design synthesis method of spider silk protein and spinning

A technology of spidroin and protein, applied in the field of biomedical materials, can solve the problems of fiber inhomogeneity, low solubility of recombinant spidroin, difficulty in spinning, etc., and achieve the effect of uniform thickness

Active Publication Date: 2019-06-21
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The main disadvantage of natural spider silk is the non-uniformity of the fibers, as differences in silk properties can occur between individual spiders and even among individuals with varying environments; another disadvantage is due to agricultural problems based on the spider's cannibalistic behavior Low availability of natural materials
Therefore, in recent years, the use of microbial hosts to produce artificial spider silk has become a research hotspot, and the main bottlenecks of this method are: the genetic instability of large fragments of genes; the difficulty of subsequent spinning caused by the low solubility of recombinant spider silk proteins

Method used

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  • Design synthesis method of spider silk protein and spinning
  • Design synthesis method of spider silk protein and spinning
  • Design synthesis method of spider silk protein and spinning

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Spinning spider silk in a pH 2 coagulation bath

[0062] 1. Construction of recombinant expression vectors and strains

[0063] ①Construction of recombinant expression vector

[0064]The N-terminal domain (SEQ ID NO.1) of E.australis spider MaSp1 protein, the C-terminal domain (SEQ ID NO.2) of A.ventricosus spider and the core domain (SEQ ID NO.3) of C.moluccensis origin ) is fused by genetic engineering technology, and codon optimization is performed on the gene according to the expression preference of Escherichia coli without changing its amino acid sequence (SEQ ID NO.4). A NdeI restriction site was added at the 5' end of the gene sequence and a HindIII restriction site was added at the 3' end of the gene. The gene sequence (SEQ ID NO.5) was synthesized and cloned into the Escherichia coli expression vector pET-28a to obtain a recombinant expression vector pET-28a-NMC (SEQ ID NO.6), which contained a strong T7 promoter , lca lactose operon, kanamycin r...

Embodiment 2

[0085] Example 2: Spinning spider silk in pH 3 coagulation bath

[0086] 1. Construction of recombinant expression vectors and strains

[0087] ①Construction of recombinant expression vector

[0088] The N-terminal domain (SEQ ID NO.1) of E.australis spider MaSp1 protein, the C-terminal domain (SEQ ID NO.2) of A.ventricosus spider and the core domain (SEQ ID NO.3) of C.moluccensis origin ) is fused by genetic engineering technology, and codon optimization is performed on the gene according to the expression preference of Escherichia coli without changing its amino acid sequence (SEQ ID NO.4). A NdeI restriction site was added at the 5' end of the gene sequence and a HindIII restriction site was added at the 3' end of the gene. The gene sequence (SEQ ID NO.5) was synthesized and cloned into the Escherichia coli expression vector pET-28a to obtain a recombinant expression vector pET-28a-NMC (SEQ ID NO.6), which contained a strong T7 promoter , lca lactose operon, kanamycin re...

Embodiment 3

[0109] Example 3: Spinning spider silk in pH 4 coagulation bath

[0110] 1. Construction of recombinant expression vectors and strains

[0111] ①Construction of recombinant expression vector

[0112] The N-terminal domain (SEQ ID NO.1) of E.australis spider MaSp1 protein, the C-terminal domain (SEQ ID NO.2) of A.ventricosus spider and the core domain (SEQ ID NO.3) of C.moluccensis origin ) is fused by genetic engineering technology, and codon optimization is performed on the gene according to the expression preference of Escherichia coli without changing its amino acid sequence (SEQ ID NO.4). A NdeI restriction site was added at the 5' end of the gene sequence and a HindIII restriction site was added at the 3' end of the gene. The gene sequence (SEQ ID NO.5) was synthesized and cloned into the Escherichia coli expression vector pET-28a to obtain a recombinant expression vector pET-28a-NMC (SEQ ID NO.6), which contained a strong T7 promoter , lca lactose operon, kanamycin re...

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Abstract

The invention relates to a design synthesis method of spider silk protein and spinning. The method is characterized in that high solubility and ph-sensitive N-terminal domain of E. australis spider MaSP1 protein, high solubility and pH-sensitive C-terminal structure domain of A. ventricosus spider MiSP protein and high solubility and pH-sensitive spider protein assembled by C. moluccensis sourcedcore domain modules are constructed with the amino acid sequence of SEQ ID NO. 4. The target protein is obtained and purified by affinity chromatography or urea dissolution method to prepare a high concentration and uniformly transparent spinning solution, and the spinning solution is injected into the coagulation bath through a wet spinning method to directly form silk; due to the high pH sensitivity, the spider silk protein can be prepared at a wide range of pH2-pH11.

Description

technical field [0001] The invention belongs to the field of biomedical materials, and relates to a method for designing and synthesizing a spidroin protein and spinning; in particular, it relates to the gene design of a spidroin protein, the construction of a recombinant expression vector containing a fusion protein gene fragment, and its fermentation expression in Escherichia coli , the separation and purification method of fusion protein and the preparation of spider silk by wet spinning. Background technique [0002] A typical web-spinning spider secretes seven different types of silk fibers (tractor silks, capture silks, flagellar silks, encased silks, packing silks, fixative silks, and cohesive silks). Spider silk is biocompatible, biodegradable, and hypoallergenic suitable for biomedical applications. Spidroin proteins can be processed into coatings for improving the biocompatibility and surface properties of biomaterials, such as medical-grade silicone implants; mes...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/22C07K1/34C12N15/62C12N15/70D01D5/06D01F4/00
Inventor 张雷齐海山张晨
Owner TIANJIN UNIV
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