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Silkworm fibroin heavy-chain expression system and preparation method and application thereof

An expression system, fibroin technology, applied in silkworm fibroin heavy chain expression system, the field of preparation of the system

Active Publication Date: 2020-10-20
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the research report on silkworm embryo microinjection transgenic technology using the transposon piggyBac, the method of expressing foreign proteins by using the silk fibroin heavy chain expression system (N-terminal only contains signal peptide, C-terminal contains truncated LBS) has not been reported. According to the report, this will further explore the role of the N and C ends of the silk fibroin heavy chain elements, and provide an experimental and theoretical basis for the study of the silk formation mechanism of silk

Method used

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  • Silkworm fibroin heavy-chain expression system and preparation method and application thereof
  • Silkworm fibroin heavy-chain expression system and preparation method and application thereof
  • Silkworm fibroin heavy-chain expression system and preparation method and application thereof

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Embodiment 1

[0030] Example 1. Construction of vector

[0031] The piggyBac-derived vector pBac[3×P3DsRedaf] (Aichun zhao.et al2010) obtained earlier was used as the transposon vector for injection, and the recombinant pBac[3×P3DsRedaf] was used to clone the shuttle vector using pSLfa1180fa using a two-step cloning method. First of all, the complex construction is completed in the pSLfa1180fa cloning shuttle vector containing all the multiple cloning sites that can recognize 6-base restriction enzymes. Then, the constructed recombinant pSLfa1180fa cloning shuttle vector is identified by After digestion with 10 base restriction enzymes AscI and FseI, the recombinant target gene was finally cloned into piggyBac-derived vector pBac[3×P3-DsRedaf] (inserted by restriction enzyme sites AscI and FseI). Purpose recombinant injection vector;

[0032] The PCR amplification of the promoter element uses the bacterial artificial chromosome (BAC) clone (hereinafter referred to as H-chain BAC) containing the...

Embodiment 2

[0040] Example 2. Obtaining of transgenic silkworm (transgenic silkworm named TS-H-GC)

[0041] pHA3PIG (Tamura et al., 2000) was used as a helper plasmid to produce transposase, and the injected plasmid DNA was purified with QIAGEN Plasmid Midi kit (Qiagen) kit, followed by the method described by Kanda & Tamura (1991) for silkworm embryo microinjection. The silkworm eggs are sealed with non-toxic glue. At 25℃, they are allowed to incubate until they hatch. The hatched silkworms are reared, and the contemporary (G0) silkworm moths are selfed or backcrossed to obtain G1 silkworm eggs. Scan G1 Generation of silkworm eggs to obtain transgenic individuals; finally, the obtained transgenic individuals were reared, passaged, and tested for EGFP expression and fluorescence observation. The results are as follows figure 2 Shown. The results showed that TS-H-GC silkworm moths showed red eyes under red fluorescence, and silkworm cocoons showed green under green fluorescence conditions. ...

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Abstract

The invention discloses a silkworm fibroin heavy-chain expression system and a preparation method and application thereof. The silkworm fibroin heavy-chain expression system comprises a target gene expression frame, wherein a 5' terminal of the expression frame is a fibroin heavy-chain promoter signal peptide sequence, and a 3' terminal is a truncated light-chain binding site; when the expressionframe is used for expressing a target protein, it is found that the target protein can be expressed in fibroin layers of silk gland and cocoon filaments, and the expression and secretion of the targetprotein are not affected after an N terminal and a C terminal containing a signal peptide are truncated; and through analysis of the N terminal and the C terminal of fibroin, an experimental and theoretical basis is provided for a silk forming mechanism of silk, a truly practical silkworm silk gland bioreactor is created, sustainable development of the silk industry is kept, and long-term development of sericology and entomology in China is promoted.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a silkworm fibroin heavy chain expression system (the N-terminus contains only signal peptide and the C-terminus contains truncated LBS), as well as the preparation method and application of the system. Background technique [0002] The main components of silkworm cocoon silk are fibroin and sericin. Among them, silk fibroin is the main component of cocoon silk, accounting for about 82% of the weight of cocoon silk. Silk fibroin is synthesized by the posterior silk gland (PSG) cells of the silkworm, and sericin is synthesized by the middle silk gland (MSG) cells. They are all gathered in the middle silk gland, and finally passed through the anterior silk gland (ASG) after being squeezed and spit out. Silky cocoon. Silk fibroin is composed of three proteins: 350-kDa heavy chain protein (H-chain), 26-kDa light chain protein (L-chain) and 30-kDa fibrohexamerin / P25 (fhx). These prot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N15/62A01K67/04
CPCC12N15/8509C12N15/66C07K14/43586A01K67/0339C12N2800/105C12N2830/008C07K2319/02A01K2207/05A01K2227/706A01K2267/01Y02A50/30
Inventor 赵爱春郝占章龙定沛
Owner SOUTHWEST UNIV