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CN-122012567-A - Constitutive expression plasmid vector for high expression of xanthine oxidase and application thereof

CN122012567ACN 122012567 ACN122012567 ACN 122012567ACN-122012567-A

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a constitutive expression plasmid vector for high expression of xanthine oxidase and application thereof. The plasmid vector can be used as a heterologous expression system to efficiently express recombinant xanthine oxidase from Cellularomyces TH20 in Pseudomonas putida KT 2440. Specifically, by replacing the replication origin of pUCP18 with the replication origin of pSC101 having a low copy number, constitutive expression plasmid pPegP119,119 was constructed to reduce the metabolic burden. Cloning TH20 XOD gene cluster into the vector to construct recombinant strain, and can produce xanthine oxidase with low cost and high efficiency. The invention realizes the high-efficiency expression of xanthine oxidase in Pseudomonas putida KT2440 by a gene recombination technology, and the expression can realize the maximum expression quantity of xanthine oxidase without adding an inducer.

Inventors

  • GUO XIAOYAN
  • WANG JIANJUN
  • GAO YUAN

Assignees

  • 北京石油化工学院

Dates

Publication Date
20260512
Application Date
20260206

Claims (10)

  1. 1. A constitutive expression plasmid vector for high expression of xanthine oxidase is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1; the constitutive expression plasmid vector is constructed by replacing the replication origin of the basic skeleton plasmid pUCP18 with the replication origin of pSC101 with low copy number, and is named pPegP119,119.
  2. 2. The constitutive expression plasmid vector for high expression of xanthine oxidase according to claim 1, characterized in that it consists of pSC101 replication origin containing RepA protein coding sequence, J23119 constitutive promoter, RBS sequence, XOD gene cluster and terminator; The XOD gene cluster is derived from the genus cellomyces TH20, including XodBA gene cluster or XodCBA gene cluster; the XodBA gene cluster includes xodB and xodA genes; the XodCBA gene cluster includes xodC, xodB, and xodA genes.
  3. 3. The constitutive expression plasmid vector for high expression of xanthine oxidase according to claim 2, wherein the constitutive expression plasmid vector is specifically prepared by the following steps: S1, replacing an ampicillin resistance box (95-1062 site) in a basic skeleton plasmid pUCP18 with a P119-RBS-MCS fragment to obtain an intermediate plasmid pUCP18P119; S2, replacing the pBR322 replication origin sequence (1232-1860 locus) in the middle plasmid pUCP18P119 with the replication origin of the pSC101 plasmid containing RepA protein sequence with low copy number, and finally obtaining pPegP119,119 plasmid; The low copy number is 6 copies/cell.
  4. 4. A constitutive expression plasmid vector for high expression of xanthine oxidase according to claim 3, characterized in that the nucleotide sequence of said P119-RBS-MCS fragment is shown in SEQ ID No. 2; the nucleotide sequence of the replication origin of the pSC101 plasmid containing the RepA protein sequence is shown in SEQ ID NO. 3.
  5. 5. A recombinant expression vector for high expression of xanthine oxidase, wherein the recombinant expression vector is obtained by cloning at least one XOD gene cluster into the constitutive expression plasmid vector of claim 1, and then obtaining a plurality of recombinant expression vectors containing xanthine oxidase expression cassettes respectively; The xanthine oxidase expression frame consists of a promoter and an XOD gene cluster connected to the downstream of the promoter; The XOD gene cluster is XodBA or XodCBA.
  6. 6. The recombinant expression vector for high expression of xanthine oxidase of claim 5, wherein said recombinant expression vector is pPegP119, 119XodBA and pPegP119,119, 119XodCBA.
  7. 7. A recombinant strain for high expression of xanthine oxidase, characterized in that it is obtained by transforming the constitutive plasmid vector of claim 1 into a host strain and screening; The host bacterium is Pseudomonas putida KT2440.
  8. 8. Use of a recombinant strain for the high expression of xanthine oxidase according to claim 7 for the preparation of xanthine oxidase and/or hypoxanthine oxidase.
  9. 9. Use of a recombinant strain for high expression of xanthine oxidase according to claim 7 for the preparation of a reagent for detecting xanthine and/or hypoxanthine concentration.
  10. 10. A xanthine oxidase which is highly expressed, characterized in that it is expressed by the recombinant strain according to claim 7.

Description

Constitutive expression plasmid vector for high expression of xanthine oxidase and application thereof Technical Field The invention belongs to the technical field of genetic engineering, and particularly relates to a constitutive expression plasmid vector for high expression of xanthine oxidase and application thereof. Background Xanthine oxidase (Xanthineoxidase, XOD), which is a key enzyme in the metabolic pathway of purines, is widely present in organisms and is capable of catalyzing the oxidation of hypoxanthine to xanthine and further to uric acid. Currently, XOD has a deep application mainly in the field of medical diagnosis, and is mainly used for detecting purine compounds such as xanthine, hypoxanthine, inosine (inosine) and the like, and assisting in detecting guanine and guanosine, and can also be used for measuring the activity of 5' -nucleoside phosphorylase, so that the XOD is also suitable for detecting liver and gall tumors. Meanwhile, the XOD is also widely applied to the detection of the inorganic phosphorus level (hyperphosphatemia) of serum and the SOD activity of superoxide dismutase. In addition, xanthine oxidase is also used in a variety of fields such as disease treatment, food detection, industrial catalysis, and environmental protection, and is one of important biocatalysts. The natural xanthine oxidase is mainly derived from milk, but the quality of the xanthine oxidase product extracted by the milk is unstable, the yield is low, and the market demand can not be met. The XOD products mainly used in the market at present are mainly recombinant xanthine oxidase products derived from rhodococcus (r. Erythropolis), which have long growth cycle and expensive inducer, resulting in high production cost. The reason for the difficulty in heterologous expression of the protein is mainly that XOD gene clusters are obviously different among different species, and the prokaryotic heterologous expression of the XOD protein is difficult due to different microorganism assembly processes and different coenzyme molecule integration mechanisms. Therefore, developing a more efficient and economical XOD heterologous expression system has great market value. Disclosure of Invention Aiming at the problems of heavy metabolic burden, high induction cost, low heterologous expression efficiency, insufficient product activity and the like in the production of xanthine oxidase in the prior art, the invention provides an XOD preparation scheme without an inducer, high activity and low cost through the combined design of low-copy plasmid transformation, constitutive promoter optimization and specific gene cluster and host adaptation, simplifies the purification process, ensures the purity of the product and meets the severe requirements of medical diagnosis scenes such as uric acid detection and the like. The first object of the present invention is to provide a constitutive expression plasmid vector for high expression of xanthine oxidase, which is capable of expressing recombinant xanthine oxidase derived from Cellularomyces TH20 efficiently in Pseudomonas putida KT2440 as a heterologous expression system. Specifically, a constitutive expression plasmid pPegP119,119 was constructed by replacing the pUCP18 origin of replication with a low copy number pSC101 origin of replication to reduce the metabolic burden. Cloning TH20 XOD gene cluster into the vector to construct recombinant strain, and can produce xanthine oxidase with low cost and high efficiency. The invention realizes the high-efficiency expression of xanthine oxidase in Pseudomonas putida KT2440 by a gene recombination technology, and the expression can realize the maximum expression quantity of xanthine oxidase without adding an inducer. The second object of the present invention is to provide a recombinant strain for high expression of xanthine oxidase, which has the advantages of no need of induction, high expression level, simple affinity purification process flow, high purification yield, low production cost, and similar specific activity to the current commercial xanthine oxidase, and is suitable for various applications. In order to achieve the above purpose, the present invention adopts the following technical scheme: a constitutive expression plasmid vector for high expression xanthine oxidase has a nucleotide sequence shown in SEQ ID NO. 1; the constitutive expression plasmid vector is constructed by replacing the replication origin of the basic skeleton plasmid pUCP18 with the replication origin of pSC101 with low copy number, and is named pPegP119,119. Preferably, the constitutive expression plasmid vector consists of pSC101 replication origin containing RepA protein coding sequence, J23119 constitutive promoter, RBS sequence, XOD gene cluster and terminator; The XOD gene cluster is derived from the genus cellomyces TH20, including XodBA gene cluster or XodCBA gene cluster); The XodBA gene cluster includes xodB and xodA genes; the XodCBA