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High-stability recombinant procalcitonin and preparation method and application thereof

A procalcitonin, high stability technology, applied in the direction of calcitonin, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of easy degradation, instability, affecting the accuracy of test results, etc. Efficient expression and the effect of maintaining spatial conformation

Active Publication Date: 2015-05-13
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the PCT antigen contains degradation sites, it is extremely unstable under normal conditions and easily degraded, which greatly affects the accuracy of the test results

Method used

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  • High-stability recombinant procalcitonin and preparation method and application thereof
  • High-stability recombinant procalcitonin and preparation method and application thereof
  • High-stability recombinant procalcitonin and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Design of High Stability PCT Antigen Amino Acid Sequence

[0035] Download the natural PCT amino acid sequence GI:76880474 from the NCBI website, and replace PCT at the 58-59aa hydrolysis site (-Lys -Arg-) and the hydrolysis site (-Gly-Lys-Lys-Arg-) at 92-95aa to obtain the amino acid sequence of PCT with high stability (such as 1 in the sequence listing). The PCT amino acid sequence after replacement is compared with the original sequence as figure 1 Shown, where different amino acids are shown with "*" and identical amino acids with "=".

Embodiment 2

[0036] Example 2 Codon optimization and acquisition of high stability PCT antigen gene

[0037] Sequence 1 was reverse-translated into a nucleotide sequence using dominant codons of Escherichia coli, namely the gene sequence of high-stability PCT (such as 2 in the sequence listing).

[0038] Considering the correct rate of current domestic gene primer synthesis, the present invention designs PCT gene into two sections A and B, and 6 primers are designed for each section of gene to be synthesized respectively, each primer end has a matching sequence of 16 nucleotides, each The sequences of primers synthesized by paragraph 3-14 are shown in the sequence listing. In order to describe the preparation method conveniently, the corresponding primer sequences are named, and SEQ ID NO:3 to 8 are respectively named as AF1, AR1, AF2, AR2, AF3, AR3; SEQ ID NO:9 to 14 are respectively named as BF1, BR1, BF2, BR2, BF3, BR3;

[0039] The method is as follows:

[0040] The synthesis of the...

Embodiment 3

[0041] Example 3 Cloning, expression and labeling of highly stable PCT antigen gene

[0042] 1. Construction of highly stable PCT antigen expression plasmid

[0043] 1.1 PCR product and expression vector pBVIL-1 plasmid double digestion

[0044] Take 30 μl of the above gene products and pBVIL-1 expression vector and place them in Eeppendorf centrifuge tubes, add 4 μl of 10×buffer (D), 1 μl of XhoI (10u / μl) and XbaI (12u / μl), add sterilized distilled water to 40 μl, and placed in a 37°C water bath for enzyme digestion overnight.

[0045] Agarose gel electrophoresis purification and recovery of enzyme-cleaved products: PCR product and carrier pBVIL-1 are purified with 1.2% agarose gel after double digestion, and the specific method is according to "Molecular Cloning" (Science Press, second version) method. The purified gene is then recovered with a small amount of gel recovery kit produced by Shanghai Huashun Bioengineering Co., Ltd.: cut out the agarose containing the plasmi...

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Abstract

The invention provides a high-stability recombinant procalcitonin. The structure of protein is PCT amino polypeptide-joint arm 1-calcitonin-joint arm 2-katacalein, wherein an amino acid sequence is as shown in SEQ ID NO:1; two parts of degradable peptide chains, namely 58-59aa and 92-95aa in PCT are replaced with flexible peptide chain joint arms; an amino acid sequence of the antigen is reversely translated by virtue of optimal codons of escherichia coli so as to obtain a high-stability PCT antigen gene composed of the optimal codons of escherichia coli; and finally cloning expression is carried out to obtain a high-stability PCT antigen. Compared with a wild PCT, the antigen also keeps the antigenicity of the wild PCT except for higher stability. The high-stability PCT protein gene provided by the invention is composed of the optimal codons of escherichia coli, has relatively high expression quantity in escherichia coli, and is suitable for large-scale preparation. Meanwhile, a recombinant PCT can be applied to preparation of standard substances in immunodetection and drawing of standard curves, so that accurate quantification of the PCT content is achieved.

Description

technical field [0001] The invention relates to procalcitonin (Procalcitonin, PCT) high-stability transformation, codon-optimized gene, its recombinant protein, and also relates to the evaluation of the protein stability and its application in standard product curve drawing. Background technique [0002] Procalcitonin (PCT) is the precursor of human calcitonin (CT), composed of 116 amino acids, with a relative molecular mass of 13KD. PCT is produced and secreted by thyroid parafollicular C cells, and is decomposed into active calcitonin by special intracellular proteases. The content of PCT in normal human serum is very low, but it will rise sharply in the case of bacterial infection or sepsis, sepsis, etc. ), and C-reactive protein (CRP), these inflammatory factors have higher sensitivity and specificity. Therefore, the preliminary diagnosis of bacterial infection can be made according to the concentration of PCT, and whether to use antibiotics can be selected. It is usu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/585C12N15/16C12N15/70G01N33/74
CPCC07K14/585C07K2319/00G01N33/54326G01N33/74
Inventor 修冰水张贺秋阙海萍王晓丹冯晓燕杨锡琴张旭辉刘志强段翠密
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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