Construction method for recombinant baculovirus used for expressing L-amino acid oxidase of Siganus oramin

Abstract

The invention discloses a method for constructing a recombinant baculovirus for expressing L-amino acid oxidase of the macula macula. The gene whose DNA sequence is shown in SEQ ID NO: 1 is cloned into the vector pMD18-T to construct a recombinant plasmid; Restriction endonucleases EcoRI and XhoI cut the recombinant plasmid and the donor plasmid respectively, and ligated the digested products to obtain the recombinant donor plasmid; the recombinant donor plasmid was transformed into E. coli, so that the target gene and the bacillus contained in E. The virus shuttle vector is specifically transposed, and the recombinant bacmid is screened and extracted; the recombinant bacmid is transfected into insect cells by liposome transfection method, and the recombinant baculovirus is harvested after the insect cells are pathological.

Description

technical field [0001] The invention relates to the technical field of L-amino acid oxidase (LAAO), in particular to a method for constructing a recombinant baculovirus for expressing the L-amino acid oxidase of macular bluefish. Background technique [0002] L-amino acid oxidase (LAAO) is a class of flavin-like proteases with flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as coenzymes. It can stereospecifically catalyze the oxidative deamination of L-amino acids to generate ل-keto acids, ammonia and H 2 O 2 . A large number of studies have shown that the main biological functions of LAAO have many practical values, such as anti-virus, anti-bacterial, anti-fungal, anti-tumor cell, anti-parasitic and so on. The enzyme is widely distributed in nature, such as bacteria, fungi, algae, snakes and mammals have been reported, of which the snake venom source LAAO is the most in-depth study because of its high content and strong enzyme activity. However, the res...

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Problems solved by technology

[0004] Li et al. showed that SR-LAAO recombinantly expressed in Escherichia coli can induce and stimulate the lysis and death of Cryptocaryon larvae after renaturation in vitro, and has the biological function of killing parasites similar to that of the natural protein, but the activity of the recombinant protein is relatively low. Low

In 2014, the researchers selected the Pichia pastoris expression system to realize the eukaryotic expression of SR-LAAO. The recombinant protein has inhibitory effects on a variety of Gram-negative bacteria and Gram-positive bacteria, which can lead to rough surfaces and particles attached to the bacteria , the cell wall shrinks and the cell membrane ruptures, but the expression level is low

However, up to now, the recombinant expression of L-amino acid oxidase of the macula macula using the baculovirus insect cell expression system has not been reported yet.

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