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Exogenous gene expression enhancer

A gene expression, exogenous technology, applied in genetic engineering, introduction of foreign genetic material using vectors, biochemical equipment and methods, etc., can solve the problem of low gene expression inhibition effect, and achieve the effect of improving production efficiency and recovery rate

Inactive Publication Date: 2016-06-15
国立研究开发法人农业生物资源研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has been reported that the effect of gene expression suppression by asRNA in the silk gland of silkworm silkworm is also very low, only about a few hundredths compared with double-stranded RNA (Non-Patent Document 6)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154]

[0155] (Purpose)

[0156] It was demonstrated that the gene expression of sericin 1 (Ser1), which is a protein specific to the middle silk gland, can be suppressed by asRNA directed against the Ser1 gene.

[0157] (method)

[0158] 1. Production of a specific expression vector for the silk gland in the middle of the silkworm

[0159]As the expression vector that silk gland in the silkworm middle part can specifically express, make the Ser1asRNA expression vector expressing the asRNA (referred to as " Ser1asRNA ") to Ser1 gene and express the shRNA (represented as " Ser1shRNA " to Ser1 gene. It should be explained, The shRNA in this specification also includes the Ser1 shRNA expression vector having a longdsRNA with a hairpin structure.

[0160] (1) Preparation of UAS backbone plasmid

[0161] PCR amplification was performed using pAmCyan1-N1 (Takara) as a template and AmCyankozakU (SEQ ID NO: 23) and SV40PCRL (SEQ ID NO: 24) as a primer pair, and inserted into pZ...

Embodiment 2

[0190]

[0191] (Purpose)

[0192] Example 1 demonstrates the expression suppression effect of the asRNA for the gene of the middle part silk gland specific protein Ser1. Therefore, this Example proves that the expression of the FibH gene can also be suppressed by using asRNA directed against the repeat region of the FibH gene for the silk fibroin H (FibH) chain which is the posterior silk gland-specific protein.

[0193] (method)

[0194] 1. Production of specific expression vectors for the posterior silk gland of the silkworm

[0195] A Fib-HasRNA expression vector expressing asRNA for the FibH chain gene was prepared as an expression vector capable of specific expression in the posterior silk gland of the silkworm.

[0196] (1) Preparation of UAS backbone plasmid

[0197] BsmBIadapU (SEQ ID NO: 40) and BsmBIadapL (SEQ ID NO: 41) were annealed to make a BsmBI linker. A BsmBI linker was inserted into the BlnI site of pBac[SerUAS / 3xP3AmCyan] produced in Example 1. The o...

Embodiment 3

[0215]

[0216] (Purpose)

[0217] It was proved that the combination of asRNA targeting the repeat sequence region of each gene of Ser1 and FibH can simultaneously inhibit the expression of Ser1 gene and FibH chain gene when expressed simultaneously.

[0218] (method)

[0219] 1. Production of expression vectors for the middle silk gland and posterior silk gland of the silkworm

[0220] (1) Production of Fib-HasRNA and Ser1asRNA simultaneous expression UAS vector (UAS-3'Ser1-FibH1asRNA expression vector)

[0221] The 3'Ser1SB prepared in Example 1 was inserted into the BlnI site of pBac[SerUAS-asFibH1 / 3xP3AmCyan] to make pBac[SerUAS-3'asSer1_asFibH1 / 3xP3AmCyan]( Figure 12 A). The plasmid is an expression vector (UAS-3'Ser1-FibH1asRNA expression vector) in which 3'Ser1asRNA-encoded DNA and Fib-HasRNA-encoded DNA are configured in series downstream of the UAS promoter, which is equivalent to containing the exogenous gene expression enhancer of the present invention The s...

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Abstract

The purpose is to develop and provide a method for easily and efficiently suppressing gene expression of sericin and / or fibroin H-chain, which are major silk gland-specific proteins, to efficiently produce a target protein in the silk glands as a protein mass production system in genetically-modified silkworms. Provided is a silkworm line capable of expressing within the silkworm antisense RNA having as a target site a repetitive sequence region of sericin and / or fibroin H-chain.

Description

technical field [0001] The present invention relates to an exogenous gene expression enhancer which uses an antisense RNA expression vector as an active ingredient in a protein expression system utilizing the silk gland of silkworm, and a genetically recombined silkworm having the antisense RNA expression vector. Background technique [0002] The silk gland of the silkworm (Bombyxmori) has the ability to synthesize a large amount of protein in a short time. In addition, since the silk gland of the silkworm is a large organ, it is easy to extract, and the synthesized protein is stored in the lumen of the silk gland, so it also has the advantage of being easy to recycle. Therefore, the genetically recombined silkworm that expresses the target protein using the silk gland is considered promising as a mass production system for proteins with high added value such as useful proteins or high-performance silk. [0003] The silk gland of silkworm silkworm is morphologically as foll...

Claims

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Application Information

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IPC IPC(8): C12N15/09A01K67/033C12N15/113C12P21/02
CPCA01K67/033A01K2217/058A01K2217/20A01K2227/706A01K2267/01C12N15/113C12N15/85C12N15/8509C12N2310/111C12P21/02
Inventor 立松谦一郎濑筒秀树内野惠郎小岛桂
Owner 国立研究开发法人农业生物资源研究所
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