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Posterior silk gland gene expression unit and transgenic silkworm containing same

A technology of gene expression and silk gland, applied in animal/human peptides, animal husbandry, peptide sources, etc., can solve the problems of low expression and secretion efficiency

Inactive Publication Date: 2015-09-09
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, this method also has the problem of low expression level and secretion efficiency, although it can be expressed as the target protein, as in the above-mentioned method.

Method used

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  • Posterior silk gland gene expression unit and transgenic silkworm containing same
  • Posterior silk gland gene expression unit and transgenic silkworm containing same
  • Posterior silk gland gene expression unit and transgenic silkworm containing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0137]

[0138] Various expression vectors containing the posterior silk gland gene expression unit of the present invention were constructed. In this example, expression vectors such as the posterior silk gland gene expression unit composed of the first and second subunits were constructed.

[0139] (method)

[0140] 1. Construction of expression vector

[0141] (1) The first auxiliary unit for the middle silk gland gene expression unit: pBacSer-pro GAL4 / 3xP3DsRed2 ( Figure 4 (a))

[0142] The first auxiliary unit for the middle silk gland gene expression unit as a control, the promoter of the sericin 1 gene specifically expressed in the middle silk gland and the transcriptional regulator functionally linked downstream thereof were constructed GAL4 gene, and pBacSer-pro GAL4 / 3xP3DsRed2 with hsp70 polyA addition sequence linked downstream.

[0143] Using the primers containing the restriction enzyme AscI site shown in SEQ ID NO: 12 and the primer containing the BamHI si...

Embodiment 2

[0151]

[0152] Various transgenic silkworms were produced using each expression vector constructed in Example 1.

[0153] (Materials and methods)

[0154] (1) Silkworm strains

[0155]The w1-pnd strain of white eye, white egg, non-dormant strain maintained at the Institute of Agricultural Bioresources was used as a host strain.

[0156] (2) Feeding conditions

[0157] In a rearing room at 25 to 27° C., the whole instar larvae were reared with an artificial diet (SilkMate original species 1-3 instar S, Nippon Agricultural Industries). The artificial feed was replaced every 2-3 days (Uchino K. et al., 2006, J Insect Biotechnol Sericol, 75:89-97).

[0158] (3) Production of transgenic silkworm

[0159] Transgenic silkworms were produced according to the method of Tamura et al. (Tamura T. et al., 2000, Nature Biotechnology, 18, 81-84).

[0160] The posterior silk gland gene expression unit constructed in embodiment 1 is used respectively with the auxiliary plasmid pHA3PIG ...

Embodiment 3

[0161]

[0162] (method)

[0163] Each of the transgenic silkworms produced in Example 2 was reared in the same manner as in Example 2. Before silk spinning on the 5th instar and 6th day, mochi was applied on ice, and the back side was incised, and the middle and rear parts were not damaged with tweezers. The method of silk gland removal (refer to Yasushi Mori, カイコによるNew Biology 実験, Sanseido, 1970, pp.249-255). It was observed with a fluorescence microscope (OLYMPUS SZX16, GFP filter) without fixing it.

[0164] (result)

[0165] Figure 5 Indicates the result. In the silk gland of the recombinant silkworm having the middle silk gland gene expression unit, fluorescence was only observed in the middle silk gland. On the other hand, in the silk gland of the recombinant silkworm having the posterior silk gland gene expression unit, fluorescence was observed in both the posterior silk gland and the middle silk gland. This result shows that after EGFP is expressed in the pos...

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Abstract

The present invention addresses the problem of providing: a posterior silk gland gene expression unit, whereby a recombinant peptide, in particular a water-soluble peptide, can be expressed in a large amount in the posterior silk gland of a silkworm such as Bombyx mori; and a transgenic silkworm into which the aforesaid gene expression unit has been transferred. Provided is a posterior silk gland gene expression unit which comprises: a promoter of a gene encoding a protein, said protein being expressed specifically in the posterior silk gland; a DNA encoding a signal peptide originating in a protein, said protein being expressed specifically in the middle silk gland, functionally bound to the downstream of the promoter; and a DNA encoding a target peptide, said peptide being free from proteins secreted as constituents of fibroin, ligated to the downstream thereof.

Description

technical field [0001] The present invention relates to a posterior silk gland gene expression unit capable of expressing a target peptide in large quantities in posterior silk glands of silkworms such as silkworms, and transgenic silkworms having the same. Background technique [0002] The silk gland of the silkworm (Bombyx mori) has the ability to synthesize a large amount of protein in a short period of time. In addition, since the silk gland of the silkworm is a large organ, it is easy to remove, and it also has the advantage of being easy to recover since the synthesized protein is stored in the lumen of the silk gland. Therefore, the transgenic silkworm expressing the target protein in the silk gland has a prospect as a protein mass production system. [0003] Morphologically, the silk glands of the silkworm are figure 1 The left and right pairs of organs shown are composed of three regions: the anterior silk gland, the middle silk gland, and the posterior silk gland...

Claims

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Application Information

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IPC IPC(8): C12N15/09A01K67/033C07K14/435
CPCC07K14/43536A01K2217/052A01K67/0333C12N15/85A01K67/04A01K2227/706A01K2267/02
Inventor 立松谦一郎瀬筒秀树内野恵郎饭塚哲也
Owner 国立研究开发法人农业生物资源研究所
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