Polycistron, vector for specific expression of polycistron through salivary glands and construction method thereof

Abstract

The invention discloses polycistron, a vector for specific expression of the polycistron through salivary glands and a construction method thereof. The multi gene co-expressed polycistron has a base sequence shown as SEQ ID No:1; the vector for specific expression of the polycistron through salivary glands has a nucleotide sequence shown as SEQ ID No:3, and is obtained by constructing with an eukaryotic expression vector of the polycistron, namely pCD-PXAT, and an pPB-mPSP-neoGFP vector as raw materials. The constructed polycistron by the invention can co-express cellulase, xylanase, dextranase, pectase and phytase, includes all the major members of the non-starch polysaccharide enzyme (NSP Enzyme) and phytase in the conventional feed, these enzymes are well compatible with the pH environment of the gastrointestinal tract in animals, have good pepsin tolerance and trypsin tolerance, and always maintain high activity, the defects and problems of the traditional feed which uses an enzyme preparation in the processing, production and use process are overcome, and because of animal's own secretion, there is almost no cost problem.

Description

technical field [0001] The invention belongs to the field of biotechnology, more specifically, the invention relates to a polycistronic and polycistronic enzyme that can co-express cellulase, xylanase, glucanase, pectinase and phytase in pig cells Its construction method and application, as well as the vector for specifically expressing the polycistronic in salivary glands and its construction method. Background technique [0002] Non-starch polysaccharides (NSP) are the main anti-nutritional factors in feed. Non-starch polysaccharides are composed of three major types of substances: cellulose, non-fibrous polysaccharides (hemicellulose polymers) and pectin polysaccharides. Among them, cellulose constitutes the "cell wall" The “sandwich” framework structure, while the matrix polysaccharides such as hemicellulose and pectin mainly composed of xylan and β-glucan are the main components in the cellulose framework of the cell wall, which play the role of filling and connecting (...

AI Technical Summary

Problems solved by technology

[0006] However, NSP enzymes and phytases have many shortcomings in fermentation production and use: 1. The production of enzymes such as phytase, cellulase, xylanase, dextranase, and pectinase in the feed industry is all through Produced by microbial fermentation, the strains used mainly include Pichia pastoris, Trichoderma, Penicillium, Escherichia coli, Bacillus, etc. Due to the relatively stable production of strains, often a high-yield strain can only produce one enzyme , the production of all the above-mentioned enzymes requires multiple fermentation production lines to complete, which increases the production cost of enzyme preparations; 2. NSP enzymes have a high degree of specificity when selecting substrates, and each enzyme is only for one substrate, so In production, the feed formula is frequently changed. In order to reduce feed cost, it is necessary to explore and study different NSP enzyme combinations according to the characteristics of NSP in feed, which causes inconvenience to production.

3. The essence of NSP enzyme is protein, which is easily affected by factors such as light, humidity, microorganisms, storage time, feed crushing, premixing, and granulation during use, which will cause great uncertainty to the effect of the enzyme preparation. The effect is not ideal; 4. Due to the difference in the cell structure of the prokaryotic expression system or the expression system of lower organisms and the expression system of higher animals, their respective protein secretion signal guide peptides are quite different, and the proteins from lower organisms are expressed and secreted in the cells of higher animals There are system obstacles; 5. Due to the preference of lower organisms and higher organisms for codon usage, the expression efficiency of lower biological proteins in higher organism cells is low, and pig-derived genetic modification is required; 6. It is used in different organisms Due to differences in protein post-translational modification systems, various NSP enzymes and phytases from microbial sources are directly used in animal cell expression systems, and there is a greater risk of expression failure, which must be verified by animal cell expression; 7. Multi-gene co-expression technology involves There are many genes, and after recombination, each gene is greatly disturbed by connecting peptides and other sequences, requiring a large number of sequence optimization and recombination experiments to explore its best combination, and repeated sequences pose a certain threat to the genetic stability of genes; 8. Due to the NSP enzyme and phytase There are many types of genes and enzymes involved, the traditional single-gene transgenic technology has a large workload, and the transgenic inheritance is complex, so it is difficult to aggregate and apply

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