Bombyx mori posterior silk gland bioreactor dual-promoter universal plasmid for expressing T4 ligase and application and method thereof

A bioreactor, dual-promoter technology, applied in biochemical equipment and methods, ligase, introduction of foreign genetic material using vectors, etc. in reaching etc.

Inactive Publication Date: 2016-08-31
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, looking at the research results of silkworm bioreactors at home and abroad for more than ten years, the expression efficiency of exogenous proteins in transgenic silkworm silk gland bioreactors is very low. Most o

Method used

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  • Bombyx mori posterior silk gland bioreactor dual-promoter universal plasmid for expressing T4 ligase and application and method thereof
  • Bombyx mori posterior silk gland bioreactor dual-promoter universal plasmid for expressing T4 ligase and application and method thereof
  • Bombyx mori posterior silk gland bioreactor dual-promoter universal plasmid for expressing T4 ligase and application and method thereof

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Embodiment Construction

[0031] The present invention will be further described below in conjunction with drawings and embodiments.

[0032] Embodiments of the present invention are as follows:

[0033] A) preparation of the plasmid of the present invention:

[0034]Use the 5' end sequence of T4 ligase with ApaI restriction site sequence and the 3' end sequence of T4 ligase with NheI restriction site sequence as primers to contain A3 gene promoter-green fluorescent protein gene-SV40 - sericin gene promoter-T4 ligase gene-SV40 ([A3-EGFP-SV40]-[Serpromoter-T4Ligase-SV40]) piggy-10522 plasmid (its base sequence such as SEQ ID NO.2) is used as a template to obtain A T4 ligase gene with a length of 1536bp (its base sequence is as shown in SEQ ID NO.3), and the 5' end of the sequence contains an ApaI restriction site, and the 3' end contains an NheI restriction site.

[0035] Then use ApaI and NheI to double-digest the double-promoter plasmid piggy-8480 plasmid (its base sequence is as SEQ ID NO.4) in the...

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Abstract

The invention discloses a Bombyx mori posterior silk gland bioreactor dual-promoter universal plasmid for expressing T4 ligase and application and a method thereof. The plasmid takes a piggyBac transposon as a basis, carries an Amp resistant gene and contains a T4 ligase gene serving as an exogenous gene and a function expression box of a green fluorescent EGFP gene serving as a marker gene. The plasmid is constructed by using a molecular biology method, and two restriction enzyme cutting sites unique to ApaL and NheI are contained between DDDDK and a fibroin light chain gene poly. After the ApaL and NheI double restriction enzyme cutting universal plasmid is connected with the T4 ligase gene, the plasmid and an auxiliary plasmid are jointly injected into Bombyx mori zygote, and characteristics of the transposon are utilized to guide the green fluorescent protein gene and the T4 ligase gene into a Bombyx mori genome and to be stably inherited and expressed to obtain transgenic Bombyx mori. The transgenic Bombyx mori is screened with the help of the fluorescent marker gene, and a Bombyx mori silk gland cell is utilized to specifically synthesize T4 ligase protein.

Description

technical field [0001] The invention relates to a plasmid and its application and method, in particular to a double-promoter universal plasmid for silkworm posterior silk gland bioreactor for expressing T4 ligase by using transgenic technology and its application and method. Background technique [0002] The piggyBac transposon was originally isolated from the genome of Trichoplusia ni TN-368 cell line, and it is the DNA transposon with the highest transposition activity found so far. The piggyBac transposition system is a non-viral vector with high transposition efficiency. Compared with sleeping beauty, the piggyBac vector has a larger capacity, can carry 18kb foreign genes, and can realize the co-expression of multiple genes, and the transposition fragments will not leave footprints at the original site after being excised, and the genome can realize Precise repair after excision plays an important role in the application of reversible genes. [0003] PiggyBac transposo...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12N15/65A01K67/04
CPCA01K67/0339A01K2207/00A01K2217/072A01K2227/706A01K2267/01C07K14/43595C12N9/93C12N15/65C12N15/66C12N15/8509C12N2800/105C12N2800/60C12N2800/90C12N2830/008C12Y605/01001
Inventor 钟伯雄张玉玉叶露鹏
Owner ZHEJIANG UNIV
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