Artificial spnthesized American pokeberry root antivirus protein gene PAPs, expression carrier and its recombination engineering bacteria

A technology for artificial synthesis and expression vectors, applied in the fields of biogenetic engineering and biotechnology, can solve the problems of limited PAP content and difficult extraction, and achieve high-efficiency expression

Inactive Publication Date: 2008-11-12
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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Problems solved by technology

However, due to the limited content of PAP in plants and the difficulty of extraction, coupled with the high stability of PAP in vitro, it is possible to operate in vitro. Therefore, the method of genetic engineering is used to construct a PAP expression vector, and then a large amount of PAP is produced through eukaryotic microbial fermentation. , is a feasible method

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  • Artificial spnthesized American pokeberry root antivirus protein gene PAPs, expression carrier and its recombination engineering bacteria
  • Artificial spnthesized American pokeberry root antivirus protein gene PAPs, expression carrier and its recombination engineering bacteria
  • Artificial spnthesized American pokeberry root antivirus protein gene PAPs, expression carrier and its recombination engineering bacteria

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Embodiment Construction

[0052] The present invention will be described in further detail below in conjunction with embodiment.

[0053] All methods in the following examples are conventional methods unless otherwise specified.

[0054] 1. Materials selected for the experiment:

[0055] 1.1 Strains and vectors: Escherichia coli JM110 strain was preserved in our laboratory; Pichia pastoris GS115 strain and expression vector pPIC9K were purchased from Invitogen; T-easy vector was purchased from Promaga.

[0056] 1.2 Main reagents: Pyrobest Taq DNA polymerase and DNA standard molecular weight were purchased from Takara; T4 ligase was purchased from Promaga; restriction enzymes Not I, SnaB I, SaII were purchased from New England Biolabs (NEB); blunt-ended DNA Fragment Timing A kit was purchased from Beijing Tianwei Times Company; 5'AOX1 and 3'AOX1 primers were synthesized by Beijing Aoke Biological Company, and the PAP rabbit antiserum was preserved in our laboratory and the public can obtain it through ...

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Abstract

The present invention relates to an artificial synthetic pokeweed antiviral protein gene PAPs, expression vector and its recombinant engineering bacterium. Said artificial synthetic pokeweed antiviral protein gene PAPs is characterized by that it has the nucleotide sequence described by SEQ ID NOI, constructs recombinant expression vector p PIC9K-PAPs, and utilizes electric conversion of Pasteur Pichia yeast GS115 strain to obtain PAP recombinant engineering bacterium. The results obtained by making the expression product undergo the process of SDS-PAGE and Westem-blotting detection show that the expression quantity of PAPs gene optimized by codon can be up to 180-200 mg / L. It can be used for controlling plant viral diseases.

Description

technical field [0001] The patent of the invention belongs to the technical fields of biotechnology and biogenetic engineering, and in particular relates to an artificially synthesized pokeweed antiviral protein gene PAPs. Background technique [0002] Virus disease is a kind of important disease of crops, which causes crop yield reduction and quality decline, and brings huge economic losses to agricultural production. Pokeweed antiviral protein (PAP) is a single-chain basic protein isolated from Pokeweed (Phytolacca americana), with a molecular weight of 29kD (Irvin, J.D.1975). The mature PAP protein consists of 262 amino acid composition. This protein belongs to type I ribosome-inactive protein (Ribosome-inactive Protein, RIP), has the activity of RNA N-glycosidase, and can specifically cut from the 28S rRNA special site of the 60S large subunit of the eukaryotic ribosome The next adenine makes it difficult to combine with the elongation factor EF-2 of protein synthesis,...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C07H21/04C12N15/63C12N1/19C12N15/82C12N15/29
Inventor 王锡锋李莉周广和刘艳杨宇
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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