Preparation method of recombinant human C-reactive protein

A reactive protein and reaction technology, applied in the field of preparation of recombinant human C-reactive protein, can solve the problems of difficulty in renaturation, inability to use clinical detection, inability to perform ELISA detection, etc., and achieve the beneficial effects of improving yield and purification process

Active Publication Date: 2020-04-07
润方(长春)生物科技有限公司 +1
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, C-reactive protein is mainly extracted directly from human blood. Since the content of C-reactive protein in normal human serum is extremely low, below 10ug/ml, and human blood is inconvenient to obtain, it cannot meet the needs of a large number of clinical and scientific research.
In the prior art, there are also reports of expressing C-reactive protein through proka...

Method used

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  • Preparation method of recombinant human C-reactive protein
  • Preparation method of recombinant human C-reactive protein
  • Preparation method of recombinant human C-reactive protein

Examples

Experimental program
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Embodiment 1

[0031] The prokaryotic expression and purification process of recombinant human C-reactive protein is as follows:

[0032] (1) Cloning of human C-reactive protein gene

[0033] The amino acid sequence of human C-reactive protein was obtained from NCBI, and the C-reactive protein coding gene was artificially optimized and synthesized according to the codon preference characteristics of E. (-10×His-) is used as a purification tag. The amino acid sequence of the specific protein is shown in SEQ ID NO: 1, and the nucleotide sequence of the protein-coding gene is shown in SEQ ID NO: 2.

[0034] Design primers for PCR reaction to amplify the synthetic gene, and introduce Nde I and Xho I restriction sites at both ends of the primers. See SEQ ID NO: 3 for the sequence of the forward primer and SEQ ID NO: for the sequence of the reverse primer. 4. The PCR conditions are as follows:

[0035] Pre-denaturation at 94°C for 5 minutes,

[0036]

[0037] Extension at 72°C for 10 min te...

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Abstract

The invention discloses a preparation method of recombinant human C-reactive protein. The method comprises the following steps: acquiring a protein sequence of the human C-reactive protein from NCBI;artificially optimizing and synthesizing a C-reactive protein encoding gene according to codon bias characteristics of escherichia coli; further constructing an expression vector of the human C-reactive protein; converting the expression vector into the escherichia coli for induced expression; and purifying an expression product to obtain pure recombinant protein. ELISA detection results show thatthe immunological characteristics of the expressed and purified recombinant human C-reactive protein are very similar to those of natural human C-reactive protein, and the detection requirements canbe met. In addition, the purification process of the recombinant human C-reactive protein is beneficial to quality control and the requirement of industrial production are met.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing recombinant human C-reactive protein. Background technique [0002] C-reactive protein (C-reactive protein, CRP) refers to some proteins (acute proteins) that rise sharply in plasma when the body is infected or tissue damaged. C-reactive protein can activate complement and strengthen the phagocytosis of phagocytic cells to play an opsonizing role, thereby removing pathogenic microorganisms invading the body and damaged, necrotic, and apoptotic tissue cells, and playing an important protective role in the body's natural immune process. [0003] At present, C-reactive protein is mainly extracted directly from human blood. Since the content of C-reactive protein in normal human serum is extremely low, below 10ug / ml, and human blood is inconvenient to obtain, it cannot meet the needs of a large number of clinical and scientific research. In the prior art, there ar...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/70C12R1/19
CPCC07K14/4737C12N15/70C12N2800/22Y02A50/30
Inventor 霍栋栋杨文魁黄磊夏志平游可为陈浩源
Owner 润方(长春)生物科技有限公司
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