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Bombyx mori silk heart protein heavy-chain expression system with expression protein distributed on bombyx mori silk glue layer and preparation method and application thereof

An expression system, fibroin technology, applied in the biological field to achieve the effect of promoting long-term development and maintaining sustainable development

Active Publication Date: 2020-10-30
SOUTHWEST UNIV
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Problems solved by technology

In the research report on the microinjection transgenic technology of silkworm embryos using the transposon piggyBac, there is no report on the method of expressing foreign proteins by using the silk fibroin heavy chain expression system (N-terminal only contains signal peptide, no C-terminal). This will also further explore the role of the N and C terminals of the silk fibroin heavy chain element, and provide an experimental and theoretical basis for the study of silk silk formation mechanism

Method used

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  • Bombyx mori silk heart protein heavy-chain expression system with expression protein distributed on bombyx mori silk glue layer and preparation method and application thereof
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  • Bombyx mori silk heart protein heavy-chain expression system with expression protein distributed on bombyx mori silk glue layer and preparation method and application thereof

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Embodiment 1

[0029] Embodiment 1, the construction of carrier

[0030] The piggyBac-derived vector pBac[3×P3DsRedaf] obtained earlier was used as the injection transposon vector, and the recombinant pBac[3×P3DsRedaf] injection transposon vector was constructed using the pSLfa1180fa cloning shuttle vector using a two-step cloning method. First, the complex The construction was completed in the pSLfa1180fa cloning shuttle vector containing all possible restriction endonuclease recognition sites of 6 bases, and then, the constructed recombinant pSLfa1180fa cloning shuttle vector was used to recognize 10 bases in the restriction endonuclease Dicer AscI and FseI were digested, and the resulting recombinant target gene was finally cloned into the piggyBac derivative vector pBac[3×P3-DsRedaf] (inserted through the restriction sites AscI and FseI) to form the target recombinant injection vector.

[0031] The PCR amplification of the promoter element is to use the bacterial artificial chromosome (B...

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Abstract

The invention discloses a bombyx mori silk heart protein heavy-chain expression system with expression protein distributed on a bombyx mori silk glue layer and a preparation method and an applicationthereof, the bombyx mori silk heart protein heavy-chain expression system contains a target gene expression cassette, and the 5'end of the expression cassette is a signal peptide sequence of a bombyxmori silk heart protein heavy chain promoter 3; the 3'end is a heavy chain gene poly (A) sequence; when the system is used for expressing the target protein, the target protein only exists in a sericin layer and is not expressed in a silk fibroin layer, so that the system can be used for expressing the target protein in the sericin layer, the creation of a real and practical bombyx mori silk glandbioreactor is facilitated, and the long-term development of silkworm science and insect discipline in China is promoted; and experimental and theoretical bases are provided for the silk forming mechanism of the silk.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a silkworm fibroin heavy chain expression system expressing protein distributed in the silkworm sericin layer, and also to a preparation method and application of the expression system. Background technique [0002] The main components of the silkworm cocoon are two proteins, fibroin and sericin, among which silk is the main component of the cocoon, accounting for about 82% of the weight of the cocoon. 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. Both of them gather in the middle silk gland, and finally pass through the front silk gland (ASG) to be squeezed and spun out. Silk cocoons. Silk fibroin consists of three proteins: 350-kDa heavy chain protein (H-chain), 26-kDa light chain protein (L-chain) and 30-kDa fibrohexamerin / P25(fhx). These proteins firstly form silk fibroin bas...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/62
CPCC12N15/85C07K14/43595C07K2319/02Y02A50/30
Inventor 赵爱春郝占章龙定沛
Owner SOUTHWEST UNIV
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