DNA (Deoxyribose Nucleic Acid) molecule, pichia pastoris recombinant plasmid and pichia pastoris recombinant bacterium for efficiently expressing PprI protein of deinococcus radiodurans

Abstract

The invention relates to the field of biotechnology, and discloses a DNA (Deoxyribose Nucleic Acid) molecule containing a sequence shown as SEQ ID NO:1, a pichia pastoris recombinant plasmid inserted into the DNA molecule and a pichia pastoris recombinant bacterium which is used for converting the pichia pastoris recombinant plasmid into a pichia pastoris competent cell and efficiently expressing the PprI protein of deinococcus radiodurans. The pprI gene sequence of the deinococcus radiodurans is optimized and modified on the premise that an amino acid sequence of the PprI protein is not changed, so that a new Pi-pprI gene is synthesized in a coding manner; and a recombinant plasmid pHBM-905A-Pi-pprI of the pichia pastoris Pi-pprI gene and a pichia pastoris recombinant engineering bacterium GS115-Pi-pprI for the secretory expression of the PprI protein are successfully constructed for the first time, so that the efficiently expressed and purified PprI protein is obtained, thereby laying a solid foundation for the development and the application of protein radiation resisting medicines independently developed in China in the field of radiation injury prevention and treatment.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a DNA molecule, a Pichia recombinant plasmid and a Pichia recombinant bacterium that highly expresses the PprI protein of radiodurans. Background technique [0002] In nuclear emergencies such as nuclear accidents, nuclear terrorism, and nuclear war, ionizing radiation can cause severe acute radiation injury (ARI) to the human body. The treatment and protection of radiation damage is related to the peaceful use of atomic energy and national nuclear safety. Therefore, research in this field has become a research field that governments and scholars around the world pay close attention to and focus on. [0003] Deinococcus radiodurans (DR) is the most radiation-resistant prokaryotic bacterium found on the earth so far. The strong radiation resistance of this bacterium is related to its own perfect and efficient DNA repair system. A variety of DNA repair genes and its protein play a cru...

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Problems solved by technology

[0005] The application of prokaryotic expression system Escherichia coli can efficiently express and purify PprI protein [Zhang Yongqin, Zhou Hui, Chen Jie, Yang Zhanshan. Journal of Radiation Research and Radiation Technology, 2011,29(2):117-122.] However, this technology exists Many defects: 1. The expressed protein has not undergone post-translational modification such as glycosylation, resulting in reduced activity; 2. The expressed protein exists in the form of inclusion bodies, and denatured extraction will also lead to reduced protein activity; 3. Escherichia coli itself endotoxin and toxic proteins can be mixed in the target protein product, resulting in limited application

However, Radiodurans belongs to prokaryotes, and there are huge differences in phylogenetic evolution between it and the eukaryotic Pichia pastoris, such as gene and protein composition and function, protein amino acid codon preference, etc.

Therefore, if the pprI gene of radiodurans is directly constructed into the expression system of Pichia pastoris by genetic engineering, preliminary experimental studies have proved that it is impossible and not conducive to the high-efficiency expression of radiodurans PprI protein, which is difficult according to the general genetic modification technology in the field successfully solved the problem

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