Tetrahymena expression vector of chitinases and its application in expressing chitinases

Abstract

The invention discloses a Tetrahymena expression vector of chitinases and its application in expressing chitinases. The invention provides an expression cassette, which includes an HSP70-2gene promoter as shown in the 9th-1126th nucleotides from the 5' terminal of sequence 1, an encoding gene of chitinases in sequence 2 and an HSP70-1 gene termination signal as shown in the 3835th-4312th nucleotides from the 5' terminal of sequence 1. The invention also provides a recombinant plasmid containing the expression cassette. When transferred to a Tetrahymena through electroporation, the recombinantplasmid can, under the drug screening effect of paromomycin, replace most of the original rDNA in the Tetrahymena, and multiply an exogenous gene in the Tetrahymena massively, thus realizing genetic transformation of a target gene. Under heat shock conditions, the HSP70-2 strong promoter can start the high level expression of CBD3 gene, and the expressed chitinases have bioactivity (able to hydrolyze chitin). In conclusion, the expression vector of the invention has wide application prospects.

Description

technical field [0001] The invention relates to a Tetrahymena expression vector of chitinase and its application in expressing chitinase. Background technique [0002] Chitin, also known as chitin or chitin, is a biopolymer made of N-acetyl-D-glucosamine (GlcNAc) as a monomer connected by β-1,4 glycosidic bonds. The biological polysaccharide with the largest reserve except cellulose in China has an annual biosynthesis capacity of 10 billion tons. Chitin has been found in microorganisms, plants, and animals, especially in fungal cell walls, arthropod exoskeletons, and shells of crustaceans. Structural matter, accounting for 40%-60% of the dry weight of the cell wall. [0003] Chitin has good biocompatibility and biodegradability, and has broad application prospects in various fields such as biochemical industry, food, environmental protection, and pesticides. Chitin also has antibacterial, cholesterol-lowering and anti-tumor activities. Chitin and related materials are al...

AI Technical Summary

Problems solved by technology

The application of animal cell expression system is limited due to the high cost

Among the commonly used Escherichia coli expression system and yeast expression system, although the Escherichia coli expression system has the advantages of high expression level of exogenous genes, but because of the lack of eukaryotic post-transcriptional processing and post-translational processing functions, the expressed products are often not active. High or inactive, etc., especially when used to express eukaryotic proteins

Yeast expression system, like animal cells, has a variety of eukaryotic transcription and post-translation processes, and can express a variety of active foreign proteins, which has attracted much attention, but there are also obvious limitations, such as Saccharomyces cerevisiae The expression system of the main body lacks a strong promoter, the secretion efficiency is poor, the plasmid is unstable, it is difficult to achieve a high fermentation density, only a small amount of protein can be secreted, the expressed protein is easily degraded, and its post-translational processing is somewhat different from that of higher eukaryotes. not equal

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