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Bombyx mori middle silkgland bioreactor universal plasmid for expressing T4 ligase as well as application and method of universal plasmid

A technology of bioreactor and ligase, which is applied in the field of silkworm synthesis and secretion of exogenous proteins, can solve the problems of low work efficiency, time-consuming and labor-intensive problems

Inactive Publication Date: 2016-09-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Since most of the PiggyBac vectors contain two expression cassettes, more elements and the same restriction site, each time a different exogenous gene is expressed, it is necessary to construct a plasmid from scratch, assemble the promoter, signal peptide, and exogenous gene , polyA and other structures, time-consuming and labor-intensive, low work efficiency

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  • Bombyx mori middle silkgland bioreactor universal plasmid for expressing T4 ligase as well as application and method of universal plasmid
  • Bombyx mori middle silkgland bioreactor universal plasmid for expressing T4 ligase as well as application and method of universal plasmid
  • Bombyx mori middle silkgland bioreactor universal plasmid for expressing T4 ligase as well as application and method of universal plasmid

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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 T4 ligase with NheI restriction site sequence as primers to contain the A3 gene promoter-green fluorescent protein Gene-SV40-sericin 1 gene promoter-T4 Ligase gene-SV40 ([A3-EGFP-SV40]-[Ser promoter-T4 Ligase-SV40]) plasmid piggy-10522 (its base sequence is as SEQ ID NO. 2) as a template, obtain a T4 ligase gene with a length of 1536 bp (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]Use ApaI and NheI to double-digest piggy-7192 (its base sequence as SEQ ID NO.4) to obtain [A3-EGFP-SV40]-[Ser1 promoter--FLS...

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Abstract

The invention discloses a bombyx mori middle silkgland bioreactor universal plasmid for expressing T4 ligase as well as an application and a method of the universal plasmid. The plasmid takes a piggyBac transposon as a base and contains an Amp resistance gene, and the plasmid comprises functional expression cassettes of the T4 ligase which serves as an exogenous gene and a green fluorescent EGFP gene which is used as a marker gene; the plasmid is constructed by virtue of a molecular biological method, and two specific restriction enzyme cutting sites, namely ApaI and NheI, exist between DDDDK and a light-chain fibroin gene polyA; the double-enzyme-digestion universal plasmid of the ApaI and NheI, after linked to the T4 ligase gene, is injected into fertilized eggs of bombyx mori together with an auxiliary plasmid; on the basis of the property of the transposon, the green fluorescent protein gene and the T4 ligase gene are delivered into a bombyx mori genome and are stably inherited and expressed, so that transgenic bombyx mori is obtained. According to the universal plasmid disclosed by the invention, the transgenic bombyx mori is screened by virtue of the fluorescent marker gene, and T4 ligase protein is specifically synthesized and secreted by virtue of bombyx mori gland cells.

Description

technical field [0001] The invention relates to a method for synthesizing and secreting exogenous proteins in silkworms, in particular to a general-purpose plasmid for expressing T4 ligase in silkworm middle silk gland bioreactors and its application and method using transgenic technology. 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 and can carry 18kb. It can realize the co-expression of multiple genes, and the transposition fragment will not leave a footprint in the original site after being excised, and the genome can be accurately excised. Repair plays an important role in the application of reversible genes. [0003] PiggyBac transposon-med...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12N9/00A01K67/04
CPCC12N15/8509A01K67/04A01K2217/206A01K2227/706A01K2267/01C12N9/93C12N15/66C12N2800/105C12N2800/90C12Y605/01001
Inventor 钟伯雄张玉玉叶露鹏
Owner ZHEJIANG UNIV
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