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Design and synthesis method of a kind of spidroin protein and spinning

A technology of spidroin and protein, which is applied in the field of biomedical materials, can solve the problems of fiber inhomogeneity, genetic instability of large fragments, low availability of natural materials, etc., and achieve the effect of uniform thickness

Active Publication Date: 2021-12-07
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 and synthesis method of a kind of spidroin protein and spinning
  • Design and synthesis method of a kind of spidroin protein and spinning
  • Design and synthesis method of a kind of spidroin 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 present invention relates to a method for designing and synthesizing a spider silk protein and spinning; combining the high solubility and pH-sensitive N-terminal domain of E.australis spider MaSp1 protein, the high solubility and pH-sensitive C of A.ventricosus spider MiSp protein The terminal domain and the core domain module derived from C.moluccensis were used to assemble a highly soluble, pH-sensitive spider silk protein, whose amino acid sequence is shown in SEQ ID NO.4. The target protein is separated and purified by affinity chromatography or urea dissolution method, and a high-concentration, uniform and transparent spinning stock solution is prepared, and the spinning stock solution is injected into the coagulation bath by wet spinning method to directly form silk. The high pH sensitivity allows it to form silk in 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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Patent Type & Authority Patents(China)
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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