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Method for synthesizing and secreting black widow spider traction silk protein 2 through silkworm silkgland 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 2, can solve the problems of lack of spider silk and no expression of spider pulling silk protein 2, etc., and achieve the effect of improving mechanical properties

Active Publication Date: 2016-04-06
浙江超丝生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the various heterologous expression systems used so far have not expressed spider dragline protein 2, and the prior art lacks a method that can secrete as long as possible a spider silk with a single spider dragline protein 2 molecule

Method used

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  • Method for synthesizing and secreting black widow spider traction silk protein 2 through silkworm silkgland bioreactor
  • Method for synthesizing and secreting black widow spider traction silk protein 2 through silkworm silkgland bioreactor
  • Method for synthesizing and secreting black widow spider traction silk protein 2 through silkworm silkgland bioreactor

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Experimental program
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Embodiment 1

[0043] The above constructed pBac[3xP3-DsRed]-MaSp2×24 plasmid ( figure 1 ) and the helper plasmid pHA3PIG plasmid that can provide piggyBac transposase ( figure 2 ) 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, a transgenic silkworm moth expressing the DsRed marker gene was observed and obtained through a fluorescence microscope (Olympus, SZX12, Japan). The transgenic silkworms from the G2 generation onwards are all reared with single moths, and the trans...

Embodiment 2

[0050] The above constructed pBac[3xP3-DsRed]-MaSp2×24 plasmid ( figure 1 ) and the helper plasmid pHA3PIG plasmid that can provide piggyBac transposase ( figure 2 ) 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...

Embodiment 3

[0057] The above constructed pBac[3xP3-DsRed]-MaSp2×24 plasmid ( figure 1 ) and the helper plasmid pHA3PIG plasmid that can provide piggyBac transposase ( figure 2 ) 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, a transgenic silkworm moth expressing the DsRed marker gene was observed and obtained through a fluorescence microscope (Olympus, SZX12, Japan). The transgenic silkworms from the G2 generation onwards are all reared with single moths, and the trans...

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Abstract

The invention discloses a method for synthesizing and secreting black widow spider traction silk protein 2 through a silkworm silkgland bioreactor. The method comprises the steps that a pBac[3xP3-DsRed]-MaSp2 plasmid carrier of the black widow spider traction silk protein 2 synthesized and secreted by silkworms is built, then the plasmid and auxiliary plasmid are introduced into silkworm fertilized ova through micro-injection, red fluorescence protein genes and black widow spider traction silk protein 2 are introduced into silkworm gene groups through transposition characteristics of a piggyBac transposon, inheritance and expression are stabilized, transgenic silkworms are made, silkworm moth selfing is carried out to achieve homozygosis of black widow spider traction silk protein 2 genes, and the transgenic silkworms for secreting black widow spider traction silk protein 2 are bred. The transgenic silkworms are screened by means of fluorescence marker genes, the black widow spider traction silk protein 2 is specifically synthesized through the silkworm silkgland cells for spider silk development and utilization, and meanwhile the mechanical performance of silk is improved.

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 2 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): C12N15/866C07K14/435A01K67/04
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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