Bombyx mori fibroin heavy chain expression system for expressing target protein distributed on fibroin and sericin, and preparation method and application

An expression system, fibroin technology, applied in the field of Bombyx mori fibroin heavy chain expression system

Pending Publication Date: 2020-10-20
重庆西蚕生物技术研究院有限公司 +1
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  • 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, there have been reports on the complete N-terminal and complete C-terminal (LBS) of the silk fibroin heavy chain, and the use of silk fibroin heavy chain expression system ( The method of expressing exogenous protein with complete N-terminus and no C-terminus has not been reported, which will further explore the role of N and C-termini of silk fibroin heavy chain elements, and provide experimental and theoretical basis for the study of silk silk formation mechanism

Method used

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  • Bombyx mori fibroin heavy chain expression system for expressing target protein distributed on fibroin and sericin, and preparation method and application
  • Bombyx mori fibroin heavy chain expression system for expressing target protein distributed on fibroin and sericin, and preparation method and application
  • Bombyx mori fibroin heavy chain expression system for expressing target protein distributed on fibroin and sericin, and preparation method and application

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Experimental program
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Embodiment 1

[0030] Embodiment 1, the construction of carrier

[0031] 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 is completed in the pSLfa1180fa cloning shuttle vector containing all possible restriction endonucleases that recognize 6 bases, and then the constructed recombinant pSLfa1180fa cloning shuttle vector uses a restriction endonuclease that recognizes 10 bases Digested with enzymes AscI and FseI, the part of the recombinant target gene obtained 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.

[0032] The PCR amplification of the promoter element is to use the bacterial artificial chromosome (BAC) ...

Embodiment 2

[0037]Embodiment 2, the acquisition of transgenic silkworm (transgenic silkworm named after TS-H-NG)

[0038] pHA3PIG (Tamura et al., 2000) was used as an auxiliary plasmid to produce translocase, and the injection plasmid DNA was purified with the QIAGEN PlasmidMidi kit (Qiagen) kit, and silkworm embryos were microinjected according to the method described by Kanda & Tamura (1991). After injection, The silkworm eggs were sealed with non-toxic glue, under the condition of 25 ℃, they were accelerated until they hatched, and the hatched silkworms were reared, and the contemporary (G0) silkworm moths were selfed or backcrossed to obtain the G1 generation silkworm eggs, and G1 was scanned Transgenic individuals were obtained from silkworm eggs; finally, the obtained transgenic individuals were reared, passaged, and EGFP expression detection and fluorescence observation were performed.

[0039] The detection of the specific expression of the promoter and the observation of the pres...

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Abstract

The invention discloses a bombyx mori fibroin heavy chain expression system for fibroin and sericin expressing a target protein, and a preparation method and application of the bombyx mori fibroin heavy chain expression system. The expression system contains a fibroin heavy chain promoter 3 at the 5' end and an expression cassette of a heavy chain gene poly(A) sequence at the 3' end. The system isused to express a foreign protein, through the identification and detection analysis of transgenic bombyx mori, it is found that the presence of EGFP is detected in both the silk gland and the silk,and EGFP is also distributed in the sericin layer in the silk, so that the result indicates that in the absence of the C-terminal, the N-terminal is not an essential factor for the formation of the silk, and the absence of the C-terminal can make the protein be distributed in the sericin layer. The analysis of the N-terminal of fibroin provides an experimental and theoretical basis for the silk formation mechanism of the silk, and is conducive to creating a really practical bombyx mori silk gland bioreactor, maintaining the sustainable development of the silk industry, and promoting the long-term development of sericulture and insect science in China.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Bombyx mori fibroin heavy chain expression system expressing target protein distributed in silk fibroin and sericin; and also to a preparation method and application of the 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 basic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66A01K67/04
CPCC12N15/8509C12N15/66A01K67/0339C12N2800/105C12N2830/008C12N2830/50A01K2207/05A01K2227/706A01K2267/01
Inventor 赵爱春郝占章龙定沛
Owner 重庆西蚕生物技术研究院有限公司
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