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Recombinant human alanine aminotransferase protein standard, recombinant human aspartate aminotransferase protein standard, and preparation methods thereof

The technology of alanine aminotransferase protein and aminotransferase protein is applied in the fields of botanical equipment and method, biochemical equipment and method, transferase, etc., and can solve the problems of producing standard raw material of aminotransferase, and it is difficult to obtain serum with high level of aminotransferase.

Inactive Publication Date: 2013-09-25
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] At present, domestic research on transaminase standard products is mainly by collecting serum with different concentrations of transaminase, which is used as a quality control product. This method is difficult to obtain serum with high levels of transaminase. Up to now, there is no method of gene recombination in China. A report on raw materials for the production of transaminase standard products

Method used

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  • Recombinant human alanine aminotransferase protein standard, recombinant human aspartate aminotransferase protein standard, and preparation methods thereof
  • Recombinant human alanine aminotransferase protein standard, recombinant human aspartate aminotransferase protein standard, and preparation methods thereof
  • Recombinant human alanine aminotransferase protein standard, recombinant human aspartate aminotransferase protein standard, and preparation methods thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0106] Example 1: Acquisition of ALT target gene

[0107] Escherichia coli was used as expression vector and PRSF-Duet was used as expression plasmid to express human alanine aminotransferase gene. The steps of target gene acquisition and optimization are as follows:

[0108] 1. Retrieve the amino acid sequence of human alanine aminotransferase (ALT) protein and the gene sequence encoding this protein on the NCBI website, its amino acid sequence is shown in SEQ ID NO: 1, and its nucleotide sequence is shown in SEQ ID NO: 2 Shown:

[0109] 2. The retrieved gene sequence encoding human alanine aminotransferase (ALT) protein was analyzed by software for Escherichia coli preferred codons, and the usage frequency of the preferred codons in this sequence was analyzed. The obtained report is as follows: figure 1 Shown: figure 1 The usage frequency map of human natural alanine aminotransferase codons in the E. coli expression system, where note: the black bar graph in the table ind...

Embodiment 2

[0113] Embodiment 2: Obtaining of AST target gene

[0114] Escherichia coli was used as expression vector and PRSF-Duet was used as expression plasmid to express human aspartate aminotransferase gene. The steps of target gene acquisition and optimization are as follows:

[0115] 1. Retrieve the amino acid sequence of human aspartate aminotransferase (AST) protein and the gene sequence encoding this protein on the NCBI website: its amino acid sequence is shown in SEQ ID NO: 4, and its nucleotide sequence is shown in SEQ ID NO: 5 Show;

[0116] 2. The retrieved gene sequence encoding human aspartate aminotransferase (AST) protein was analyzed by software for Escherichia coli preferred codons, and the usage frequency of the preferred codons in this sequence was analyzed. The obtained report is as follows: image 3 Shown: image 3 The usage frequency map of human natural aspartate aminotransferase codons in the E. coli expression system, where note: the black bar graph in the t...

Embodiment 3

[0120] Embodiment 3: Construction of genetic engineering expression strain

[0121] In this example, the method of molecular biology is used to clone the target gene into the PRSF-Duet vector, construct a plasmid for recombinant expression of transaminase, and transform the successfully constructed recombinant expression plasmid into Escherichia coli competent cells to prepare human A genetically engineered strain expressing alanine aminotransferase protein and aspartate aminotransferase protein. According to the information of the target gene fragment and the carrier plasmid, the map of the recombinant transaminase expression plasmid is drawn. For the plasmid map of the recombinant alanine aminotransferase, see Figure 5 , recombinant aspartate aminotransferase plasmid map see Image 6 .

[0122] 1. Construction of recombinant transaminase expression plasmid

[0123] 1.1 Digestion: adopt the method of double enzyme digestion. Cut the target fragment with restriction endon...

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Abstract

The invention relates to preparation of standards, and particularly relates to a recombinant human alanine aminotransferase protein standard, a recombinant human aspartate aminotransferase protein standard, and preparation methods thereof. A nucleotide sequence of encoding the recombinant human alanine aminotransferase protein standard is shown in SEQ ID NO:3; the nucleotide sequence of encoding the recombinant human aspartate aminotransferase protein standard is shown in SEQ ID NO:6. The invention also relates to the preparation method of the recombinant human alanine aminotransferase protein standard or the recombinant human aspartate aminotransferase protein standard. The preparation method comprises the following steps: obtaining a target gene; building a gene expression strain; expressing and purifying target protein; processing matrix serum, evaluating the standard, and evaluating a constant value and the uncertainty of the standard. The recombinant human alanine aminotransferase protein standard and the recombinant human aspartate aminotransferase protein standard prepared by adopting a gene recombination technique disclosed by the invention can be applied to clinical test of a laboratory and mass control of a kit.

Description

technical field [0001] The invention relates to the preparation of standard products, in particular to a recombinant human alanine aminotransferase protein standard product, a recombinant human aspartate aminotransferase protein standard product and a preparation method thereof. Background technique [0002] Standard substance refers to a material or substance that has one or more sufficiently uniform and well-defined characteristic values ​​for calibrating measuring devices, evaluating measuring methods, or assigning values ​​to materials. The reference material of in vitro diagnostic reagents is the main tool to realize the accuracy and consistency of the data and results of the supervision and inspection of in vitro diagnostic reagents, and it is also the physical standard of measurement to ensure the effective transmission of values. In the complete traceability, value transmission and calibration process, the reference material plays the role of reproducing the value, t...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12R1/19
Inventor 徐超王玉梅黄杰高尚先
Owner NAT INST FOR FOOD & DRUG CONTROL
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