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Platelet-derived growth factor modifying gene suitable for silkworm expression and expression vector and application thereof

A platelet-derived, growth factor technology, applied in the direction of platelet-derived growth factors, growth factors/inducible factors, using vectors to introduce foreign genetic material, etc., can solve the problem of destroying the Ser1 promoter tissue specificity, transgenic silkworm death, ectopic expression, etc. question

Active Publication Date: 2018-09-28
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, hr3 / IE1 will destroy the tissue specificity of the Ser1 promoter, leading to ectopic expression of the marker gene and the foreign gene, and then causing the death of the transgenic silkworm

Method used

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  • Platelet-derived growth factor modifying gene suitable for silkworm expression and expression vector and application thereof
  • Platelet-derived growth factor modifying gene suitable for silkworm expression and expression vector and application thereof
  • Platelet-derived growth factor modifying gene suitable for silkworm expression and expression vector and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1, the synthesis of gene

[0029] Download the human platelet-derived growth factor protein (HumanPlatelet Derived GrowthFactor subunit B, hPDGFB, GenBank: NM_002608.3) mature peptide amino acid sequence from NCBI, and optimize the coding sequence according to the silkworm codon usage preference. The nucleotides are as shown in SEQ ID NO .1 Positions 7-399 indicate BamHI upstream and NotI restriction sites downstream. The encoded amino acid sequence is shown in SEQ ID NO.2, and the gene sequence shown in SEQ ID NO.1 was synthesized by the company.

Embodiment 2

[0030] Embodiment 2, the construction of transgenic vector

[0031] The commercially synthesized PDGF gene coding sequence was constructed into psl1180[hr3CQSer1spDsRedSerPA] (SEQ ID NO.3) through the BamHI and NotI restriction sites to form psl1180[hr3CQSer1spPDGFSer1], and then constructed into the pBac{3xp3EGFPaf} vector through the AscI site In the AscI site, a transgene expression vector pBac{3xp3EGFP, hSPDGF-BBSer1PA} was formed, named phPDGFSer1, with the structure as figure 1 Shown in A.

Embodiment 3

[0032] Embodiment 3, microinjection and fluorescence screening

[0033] The transgene expression vector phPDGFSer1 and the auxiliary carrier pHA3PIG plasmid were extracted using the QIAGEN Plasimd Mini Kit plasmid extraction kit, and the concentration of each plasmid was diluted to 400ng / μl, and mixed with the auxiliary carrier pHA3PIG plasmid at a molar ratio of 1:1. Inject the mixed plasmids into Dazao early embryos that have been released from diapause (2-5 hours after oviposition), then seal the injection hole with non-toxic glue, sterilize with 35% formaldehyde steam for 5 minutes, and place at 25°C. Hatched in an environment with a relative humidity of 85%, the hatched larvae (G0 generation) were reared with artificial feed, and self-crossed or backcrossed to produce seeds after adulthood, and the obtained G1 generation silkworm eggs (7th day) were examined under a macroscopic stereofluorescence microscope. (Olypus MVX10, Japan), the green fluorescence observation adopts...

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Abstract

The invention relates to a platelet-derived growth factor modifying gene suitable for silkworm expression and an expression vector and application thereof. The nucleotide sequence of the platelet-derived growth factor modifying gene is shown as 7th-399th bots of SEQ ID NO.1, encoded amino acid is shown as SEQ ID NO.2, an expression box is formed by the platelet-derived growth factor modifying gene, a secretory sericin 1 gene promoter and a secretory sericin 1 gene terminator, the expression is enhanced by an enhanser hr3, and meanwhile a piggyBac transposition arm and a fluorescent screening marker gene are connected to form an expression system, a recombined platelet-derived growth factor is efficiently expressed by the expression system in a silkworm silk gland, and the expression systemhas biological activity and can be used for producing recombined platelet-derived growth factors in a large scale and has an excellent market prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a modified platelet-derived growth factor gene suitable for expression in silkworms, and also relates to an expression vector and application for expressing the factor. Background technique [0002] With the development of society, the demand for the production of recombinant proteins is getting higher and higher. However, for the current large demand for recombinant proteins, the yield of recombinant proteins that can be provided is far from enough. The transgenic silk gland bioreactor of silkworm is a transgenic animal expression system that uses the silk gland of silkworm to express recombinant protein. [0003] The silkworm belongs to the Lepidoptera Bombyx mothidae. After 5,000 years of artificial domestication and breeding, it has lost the ability to fly and escape. It has strong ability in synthesis and secretion. Within 5-6 days of the 5th instar silkworm, the b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/85A01K67/04C07K14/49C07K1/36C07K1/34C07K1/22C07K1/18
CPCA01K67/0339A01K2207/05A01K2227/706A01K2267/01C07K14/49C12N15/8509C12N2800/105
Inventor 夏庆友王峰陈文静王元成赵萍
Owner SOUTHWEST UNIV
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