Recombinant organophosphate degrading enzyme gene and its expression vector and prepn process

An expression vector and organophosphorus technology, applied in the field of genetic engineering, can solve the problem of low expression efficiency, and achieve the effects of high enzymatic activity, broad degradation spectrum, and excellent enzymatic properties.

Active Publication Date: 2006-05-17
北京森根比亚生物工程技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The first technical problem to be solved by the present invention is to overcome the defect of low expression efficiency of the original organophosphate degrading enzyme gene (the gene's accession number in GenBank is AJ605330), carry out molecular transformation and modification of the original organophosphate degrading enzyme gene, and obtain a An artificial gene capable of highly expressing organophosphate degrading enzymes in yeast cells

Method used

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  • Recombinant organophosphate degrading enzyme gene and its expression vector and prepn process
  • Recombinant organophosphate degrading enzyme gene and its expression vector and prepn process
  • Recombinant organophosphate degrading enzyme gene and its expression vector and prepn process

Examples

Experimental program
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Effect test

Embodiment 1

[0055] [Example 1] Synthesis and cloning of recombinant organophosphate degrading enzyme gene

[0056] 1. According to the codon selection bias of Pichia pastoris, without changing the original organophosphate degrading enzyme gene [see: Wu Ningfeng, Deng Minjie, et al: Cloning and expression ofophc2, a new organophosphorus hydrolase gene.Chinese for the gene sequence Science Bulletin, 2004, 49 (12): 1245-1249] on the premise of the encoded amino acid sequence, the codons of the mature protein coding sequence of the organophosphate degrading enzyme gene were optimized.

[0057] Compared with the original gene, the modified organophosphate degrading enzyme gene has changed 154 bases, involving 135 amino acids, and the G+C content has changed from 62.89% to 50.27%, which is suitable for expression in Pichia pastoris.

[0058] Divide the modified whole gene sequence into three segments A, B, and C, and then further divide the large segment into small segments of about 50bp in siz...

Embodiment 2

[0062] [Example 2] Construction of yeast recombinant plasmid

[0063] The correctly detected recombinant plasmid pBS-ophc2-m and plasmid pPIC9 were treated with SnaBI / NotI for double enzyme digestion, and after electrophoresis recovery, they were washed with T 4 DNA ligase (Promega company) ligation. In this way, the gene of interest is inserted between the SnaBI and NotI sites on pPIC9 by using the restriction site to form the recombinant pPIC9-ophc2-m. For the identification of the recombinant, see figure 1 . Thus, the target gene was cloned downstream of the AOX1 promoter, and formed a correct reading frame with the signal peptide coding sequence.

Embodiment 3

[0064] [Example 3] Transformation, detection and screening of highly expressed organophosphate degrading enzyme engineering bacteria

[0065] 1. Transformation of recipient yeast: Extract a large amount of the yeast recombinant expression plasmid pPIC9-ophc2-m prepared in Example 2, take 10 μg and use a slightly excessive amount of BglII for linearization treatment, electrophoresis to detect whether the enzyme digestion is complete, and the linearized plasmid The DNA was extracted once with phenol-chloroform and chloroform respectively, precipitated with ethanol, centrifuged, discarded the supernatant, washed twice with 70% ethanol, and dissolved in sterile water. Then, mix 1-5 μg of linear DNA with 80 μL of yeast GS115 competent cells, pour it into a pre-cooled sterile electric shock cup (0.2 cm, BioRad), tap the electric shock cup to make the mixture fall into the bottom of the electric shock cup, (BioRad) set the voltage at 2.5 kV, the capacitance at 25 μF, and the resistan...

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Abstract

The present invention discloses one new kind of artificially synthesized organophosphate degrading enzyme gene suitable for expressing in eukaryotic cell and possessing the nucleotide sequence of SEQ ID No. 1. The gene of the present invention is prepared through molecular reformation and modification on available organophosphate degrading enzyme gene and subsequent artificial synthesis. Compared with original gene, the reformed recombinant gene has 154 altered bases and 135 amino acids, and G+C content changed from original 62.89 % to 50.27 % and is suitable for expressing in eukaryotic cell in high efficiency. The present invention also provides recombinant expression vector containing the above gene, host cell containing the recombinant expression vector and the method of preparing recombinant organophosphate degrading enzyme.

Description

technical field [0001] The present invention relates to an artificial gene, in particular to an artificially synthesized gene encoding an organophosphate degrading enzyme, a recombinant yeast expression vector containing the gene, a host cell transformed by the recombinant yeast expression vector, and preparation of the recombinant organophosphate degrading enzyme The method belongs to the field of genetic engineering. Background technique [0002] According to the China Statistical Yearbook, the annual output of chemical pesticides in my country is about 1 million tons, ranking second in the world. Among them, the output of highly toxic pesticides ranks first among pesticides in my country, accounting for 70% of the total output of pesticides. my country is also a big country in the use of pesticides. The area of ​​chemical control is about 4.4 billion mu per year. For vegetables alone, 48 million tons of losses caused by pests and diseases can be recovered. Including grai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N1/19C12N9/00C12N15/55C12N15/81C12P21/02C12R1/84
Inventor 伍宁丰姚斌范云六初晓宇邓敏捷柏映国
Owner 北京森根比亚生物工程技术有限公司
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