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Preparation method of traceable enzyme calibration substance

A substance and matrix technology, applied in the field of preparation of traceable enzyme calibration substances, can solve problems such as product valuation contradictions, lack of traceability analysis, etc., and achieve good interchangeability and traceability, wide application, and reliable methods.

Inactive Publication Date: 2009-09-16
嘉兴博泰生物科技发展有限公司
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Problems solved by technology

At present, the international reference materials come from various animal or human tissues, and the calibration materials of its next-level products are mainly produced by large foreign companies according to their own product characteristics. There is a lack of traceability analysis, and there are contradictions in the valuation of different products.

Method used

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  • Preparation method of traceable enzyme calibration substance
  • Preparation method of traceable enzyme calibration substance
  • Preparation method of traceable enzyme calibration substance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0020] Example: Take creatine kinase as an example

[0021] 1. Human-derived serum enzyme gene cloning and expression

[0022] 1. The specific construction process of creatine kinase gene expression vector pET21b-HMCK: using the full-length human creatine kinase cDNA (Genbank: NM-001824) as a template, design the creatine kinase upstream and downstream primers respectively,

[0023] The upstream primer is: GATTATTCATATGCCATTCGGTAACACC;

[0024] The downstream primer is: AAGGATCCTACTTCTGGGCGGG; after the target fragment is amplified with the corresponding primer, it is digested with NdeI and BamHI and then cloned into the pET21b plasmid to construct the expression vector pET21b-HMCK. It was identified by single restriction digestion and multiple double restriction digestion, and sequenced, which was consistent with the expected results.

[0025] 2. Optimal expression of protein: transfer the constructed expression vector to BL21 strain, culture in LB liquid medium containing ampici...

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Abstract

A preparation method of traceable enzyme calibration substance includes the followings: firstly, human-derived seroenzyme gene clone and expression; secondly, purification of high-purity gene recombination seroenzyme; thirdly, preparation of stroma ground substance with fine interchangeability; fourthly, interchangeability verification of calibration substance candidate; fifthly, homogeneity determination after subpackage; sixthly, freeze drying: the human-derived seroenzyme gene clone and expression are as follows: enzyme gene expression carrier Pet21b-HMCK specific construction process and protein optimization expression; the purification of high-purity gene recombination seroenzyme finishes high-purity protein of enzyme mainly by three reaction steps such as the first stage separation, anion displacement chromatography and the third molecular sieve gel chromatography; and then hybrid enzyme is determined and eliminated; the preparation method has the characteristics of reliable method, wide use, fine interchangeability and traceable performance, and the like.

Description

Technical field [0001] The invention relates to a method for preparing a traceable enzyme calibration substance with good interchangeability for laboratory medicine, and belongs to the technical field of in vitro diagnostic reagents for medical tests. Background technique: [0002] The measurement of enzyme activity is the most frequent measurement item in clinical testing, which is of great significance. However, due to the characteristics of the existing clinical testing system and enzyme reaction, there are many problems in actual work. Firstly, the determination of enzyme activity concentration is obtained by indirect calculation; secondly, there are many factors that affect the correct determination of enzyme concentration; in addition, due to the small sampling volume of test samples in clinical testing, multiple tests may be required for each sample; Timely reporting and other reasons have sacrificed method specificity and accuracy to a certain extent. As a result, there a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/25C12Q1/50
Inventor 胡卫江周海梦李海龙孟凡国
Owner 嘉兴博泰生物科技发展有限公司
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