Dual-promoter universal plasmid for expressing T4 ligase of domestic silkworm middle silk gland bioreactor as well as application and method of dual-promoter universal plasmid

Abstract

The invention discloses a dual-promoter universal plasmid for expressing T4 ligase of a domestic silkworm middle silk gland bioreactor as well as application and a method of the dual-promoter universal plasmid. The plasmid takes piggy Bac transposons as a foundation and carries an Amp resistance gene; the plasmid comprises function expression frames of a T4 ligase gene used as an exogenous gene and a green fluorescence EGFP (Enhanced Green Fluorescent Protein) gene used as a marker gene; the plasmid is constructed by utilizing a molecular biology method and two special restriction enzyme cutting sites containing ApaI and NheI are formed between DDDDK and a fibroin light-chain gene polyA; the universal plasmid is subjected to dual-enzyme digestion by adopting the ApaI and the NheI; after the universal plasmid is connected with the T4 ligase gene, the universal plasmid and an auxiliary plasmid are commonly injected into a domestic silkworm fertilized ovum; the green fluorescence protein gene and the T4 ligase gene are transferred into a domestic silkworm genome through utilizing properties of the transposons and are stably inherited and expressed to obtain transgenic domestic silkworms. The transgenic domestic silkworms are screened with the help of a fluorescence marker gene and domestic silkworm silk gland cells are used for specifically synthesizing and secreting T4 ligase protein.

Description

technical field [0001] The invention relates to a plasmid and its application and method, in particular to a dual-promoter universal plasmid used for expressing T4 ligase in silkworm middle silk gland bioreactor 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. 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-mediated transgenic silkworm silk gland bioreactor res...

AI Technical Summary

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 of the exogenous genes that drive the expression of the cocoon have not reached about 1% of the cocoon layer, which is far from the high-efficiency expression level expected by scientists like the expression of silk protein.

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