Preparation method and application of sarcosine oxidase

A technology encoding sarcosine oxidase and sarcosine oxidase, which is applied in the fields of genetic engineering and protein expression, can solve the problems of high manual operation, low detection cost, difficulty, etc., and achieve high yield of finished products, low preparation cost, Simple operation effect

Inactive Publication Date: 2013-04-03
BEIJING HOSPITAL
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Problems solved by technology

The detection cost of these two methods has been greatly reduced compared with the chromatography-mass spectrometry detection method, but it is still necessary to purchase special detection devices during detection, such as fluorescence detectors, capillary electrophoresis instruments and other special equipment
Moreover, the manual operation is very high, and it is still difficult to apply to routine clinical testing.

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  • Preparation method and application of sarcosine oxidase
  • Preparation method and application of sarcosine oxidase
  • Preparation method and application of sarcosine oxidase

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Embodiment Construction

[0053] The present invention will now be described more fully with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the exemplary embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0054] The invention establishes a method for preparing sarcosine oxidase with simple operation, rapid operation and low cost. The principle of this method mainly lies in: inserting the optimized sarcosine oxidase coding gene into the constructed expression vector, and inducing its high-level expression in Escherichia coli, the method is specifically as follows:

[0055] 1. Construction of sarcosine oxidase expression vector:

[0056] It was cloned into the PRSF Duet vector by genetic engineer...

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Abstract

The invention discloses a preparation method of sarcosine oxidase. The method comprises the following steps: transforming a whole-gene synthesis humanization sarcosine oxidase coding deoxyribonucleic acid sequence, and removing escherichia coli rare codon; inserting the transformed sarcosine oxidase coding deoxyribonucleic acid sequence into an expression carrier, and carrying out transformation and expression by using an escherichia coli strain; inoculating the strain subjected to transformation and expression into a luria-bertani (LB) culture medium, and carrying out isopropyl-beta-d-thiogalactoside (IPTG) induction; and breaking the obtained thallus, centrifugally separating and purifying through an Ni-nitrilotriacetic acid (NTA) column. The method is simple in sarcosine oxidase preparation operation, low in preparation cost, high in finished product yield, high in purity and high in biological activity, and can be used as a diagnostic reagent for sarcosine assay kit research.

Description

technical field [0001] The invention belongs to the field of genetic engineering and protein expression, and relates to a method for efficiently preparing sarcosine oxidase by using bioengineering technology, and the product can be used to produce sarcosine detection kits. Background technique [0002] A study published on February 12, 2009 in the journal Nature (Nature 2009, 457: 910) by Chinnaiyan of the University of Michigan, Ann Arbor, etc. stated that a kind of sarcosine (sarcosine, also N-methylglycine, a metabolite of glycine) can effectively reflect the aggressiveness of prostate cancer and distinguish the growth behavior of cancer cells (slow growth or highly invasive). The other two important findings of the study are: sarcosine is involved in the process of prostate carcinogenesis and is one of the culprits of cell carcinogenesis; urine sarcosine detection is more accurate in diagnosing prostate cancer than the current clinically used blood prostate-specific Ant...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12Q1/26
Inventor 刘明王萌张传宝肖飞王大光
Owner BEIJING HOSPITAL
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