Method for synthesizing and secreting black widow spider dragline silk protein 1 through bombyx mori silk gland bioreactor

A technology of pulling silk protein and bioreactor, which is applied in the field of synthesizing and secreting black widow spider pulling silk protein 1, can solve the problems of lack of protein motif in recombinant protein, small molecular weight of spider silk protein, affecting the mechanical properties of exogenous protein, etc. The effect of improving mechanical properties

Active Publication Date: 2016-03-16
浙江超丝生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, the molecular weight of the spider silk proteins expressed by various heterologous expression systems used so far is small, and many recombinant proteins lack some key protein motifs, and many proteins are not pure spider silk protein molecules, but Fusion proteins with molecules such as fluorescent proteins that ultimately affect the mechanical properties of the foreign protein
Therefore, the prior art lacks a method to secrete as long as possible a single spider silk protein molecule spider silk

Method used

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  • Method for synthesizing and secreting black widow spider dragline silk protein 1 through bombyx mori silk gland bioreactor
  • Method for synthesizing and secreting black widow spider dragline silk protein 1 through bombyx mori silk gland bioreactor
  • Method for synthesizing and secreting black widow spider dragline silk protein 1 through bombyx mori silk gland bioreactor

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Embodiment 1

[0044] The pBac[3xP3-DsRed]-MaSp1×2 plasmid ( figure 1 ) and the helper plasmid pHA3PIG plasmid that can provide piggyBac transposase ( Figure 4 ) were mixed at a ratio of 1:1, the total concentration of the two plasmids was 0.4 μg / μl, the plasmids were dissolved in pH=7, 0.5 mM phosphate buffer, and then introduced into the silkworm within 6 hours after laying eggs by microinjection. In fertilized eggs, the total volume of introduction is 10nl. The microinjected silkworm eggs are reared to adults under the condition of 25° C. and 85% humidity, and crossed with non-transgenic silkworms for passage, which is the G1 generation. During the turning green stage of the G1 generation eggs in the transgenic experiment, the transgenic silkworm 2 moths expressing the DsRed marker gene were obtained by observation with a fluorescence microscope (Olympus, SZX12, Japan), and the silkworms were reared until the adults were hybridized with non-transgenic silkworms, which was called G2. T...

Embodiment 2

[0054] The pBac[3xP3-DsRed]-MaSp1×12 plasmid ( figure 2 ) and the helper plasmid pHA3PIG plasmid that can provide piggyBac transposase ( Figure 4 ) were mixed at a ratio of 1:1, the total concentration of the two plasmids was 0.4 μg / μl, the plasmids were dissolved in pH=7, 0.5 mM phosphate buffer, and then introduced into the silkworm within 6 hours after laying eggs by microinjection. In fertilized eggs, the total volume of introduction is 10nl. The microinjected silkworm eggs are reared to adults under the condition of 25° C. and 85% humidity, and crossed with non-transgenic silkworms for passage, which is the G1 generation. During the turning green stage of the G1 generation eggs in the transgenic experiment, 10 transgenic silkworms expressing the DsRed marker gene were obtained through fluorescence microscopy (Olympus, SZX12, Japan), and the silkworms were reared until the adults were hybridized with non-transgenic silkworms, which was G2. Transgenic silkworms from the...

Embodiment 3

[0063] The pBac[3xP3-DsRed]-MaSp1×16 plasmid constructed above ( image 3 ) and the helper plasmid pHA3PIG plasmid that can provide piggyBac transposase ( Figure 4 ) were mixed at a ratio of 1:1, the total concentration of the two plasmids was 0.4 μg / μl, the plasmids were dissolved in pH=7, 0.5 mM phosphate buffer, and then introduced into the silkworm within 6 hours after laying eggs by microinjection. In fertilized eggs, the total volume of introduction is 10nl. The microinjected silkworm eggs are reared to adults under the condition of 25° C. and 85% humidity, and crossed with non-transgenic silkworms for passage, which is the G1 generation. During the turning green stage of G1 eggs in the transgenic experiment, 16 transgenic silkworm moths expressing the DsRed marker gene were obtained by observation with a fluorescence microscope (Olympus, SZX12, Japan), and the silkworms were reared until the adults were hybridized with non-transgenic silkworms, which was called G2. T...

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Abstract

The invention discloses a method for synthesizing and secreting black widow spider dragline silk protein 1 through a bombyx mori silk gland bioreactor. The method includes the steps that firstly, pBac[3xP3-DsRed]-MaSp1 plasmids serving as a carrier for bombyx mori to synthesize and secrete black widow spider dragline silk protein 1 are established; then, plasmids and assistant plasmids are introduced into bombyx mori zygotes through microinjection, red fluorescence protein genes and black widow spider dragline silk protein 1 genes are introduced into bombyx mori genomes through the transposition characteristic of piggyBac transposons, stable inheritance and expression are performed, transgenic bombyx mori is obtained, mori moth selfing is performed to achieve homozygosis of black widow spider dragline silk protein 1 genes, and transgenic bombyx mori secreting black widow spider dragline silk protein 1 is bred. According to the method, transgenic bombyx mori is screened through fluorescence marker genes, and black widow spider dragline silk protein 1 is specifically synthesized and secreted through bombyx mori silk gland cells; the method is used for development and utilization of spider silk and can also be used for improving the mechanical performance of silk.

Description

technical field [0001] The invention relates to a method for synthesizing and secreting exogenous protein by silkworm, in particular to a method for synthesizing and secreting black widow spider traction silk protein 1 by using a silk gland bioreactor of silkworm using transgenic technology. Background technique [0002] There are many kinds of spiders. So far, there are 112 families, 3905 genera and more than 44000 species of spiders named in the world. Spider silk is a natural polymer protein fiber secreted by spider silk glands. It has excellent mechanical properties, such as good elasticity, high strength, strong toughness, high and low temperature resistance, impact resistance, small specific gravity, good biocompatibility, As well as excellent characteristics such as biodegradability, its unique comprehensive performance is unmatched by other natural fibers and synthetic fibers. There are seven types of typical spider silk, which are composed of different protein mole...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K67/04
Inventor 钟伯雄尤征英叶露鹏钱秋杰
Owner 浙江超丝生物科技有限公司
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