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Method for preparing isotope-labeled recombinant C reactive protein

A technology of isotope labeling and reactive protein, which is applied in the preparation of recombinant protein and recombinant C-reactive protein, can solve problems such as unreachable, inaccurate measurement results, incomplete protein digestion, etc., to reduce uncertainty and improve Measurement accuracy, resource saving effect

Active Publication Date: 2011-02-16
NAT INST OF METROLOGY CHINA
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CRP (C-reactive protein-C-reactive protein) recombinant expression technology is a basis of this invention, and some laboratories have explored it, but none of them can achieve the results we require
In 2002, Toshio et al. used plasmids pTZ18U and pTZ19U as gene carriers, and obtained recombinant human C-reactive protein secreted to the outside of cells by co-expressing kil gene and CRP gene. Purification is difficult, and the purity required by the protein cannot be reached
Isotope dilution mass spectrometry is recognized as the authoritative method for absolute quantification, but its direct use to quantify protein macromolecules is problematic because inaccurate measurements may be caused by incomplete protein digestion or other variables introduced during sample processing

Method used

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  • Method for preparing isotope-labeled recombinant C reactive protein
  • Method for preparing isotope-labeled recombinant C reactive protein
  • Method for preparing isotope-labeled recombinant C reactive protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Construction of recombinant vector-pET11a-CRP

[0025] Design primers:

[0026] The upstream primer is R1: AGAAGGAGATATA CATATG CAAACGGACATGTCTC, where the underlined parts in italics are restriction enzymes Nde Ⅰ recognition site;

[0027] The intermediate primer is F1: GTGGTGGTGGTG ACTACCGCG TGGCCACAGCTGCGG, where the underlined part is the nucleotide complementary sequence of 6 histidines, and the blackened part is the recognition site for thrombin digestion; the downstream primer is F2: CTTTGTTAGCAGCC GGATCC TTA GTGGTGGTGGTGGTGGTG ACTA, where the underlined parts in italics are restriction endonucleases Bam The recognition site of HI, in which the nucleotide complementary sequence of 6 histidines in the underlined part, is then amplified by PCR twice to obtain the target gene of C-reactive protein, and its sequence is the core of SEQ ID NO:1 in the sequence table Nucleotide sequence, the coding sequence is in SEQ ID NO: 1, the 7-616 nucl...

Embodiment 2

[0049] Example 2 Small-scale expression of recombinant C-reactive protein

[0050] a. Transform the recombinant vector-pET11a-CRP into the host cell BL21, verify that the host cells containing the recombinant vector are obtained, and then streak the host cells containing the recombinant vector.

[0051] b. Pick a healthy single clone and inoculate it in 5mL LB or TB (Amp: Ampicillin 100μg / mL) at 37°C, 250rpm and culture overnight.

[0052] c. Expand the culture to 50mL LB (Amp100μg / mL) at a ratio of 1:100 (v / v), a total of 4 bottles, shake at 37°C and 250rpm until OD600=0.5-0.6, and add IPTG to two of the bottles until the end Concentration of 1mM was then placed at 20°C and 37°C for shaking culture, and the other two bottles were added with 1% galactose (lacose) for induction and placed at 20°C and 37°C for shaking culture respectively.

[0053] d. After induction at 37°C for 5 hours and overnight at 20°C, take 1-2 mL of bacterial solution, centrifuge at 8000 rpm, 4°C for 5...

Embodiment 3

[0060] Example 3 Massive expression and affinity chromatography purification of recombinant C-reactive protein

[0061] The buffer used in the following test steps is as follows: Lysis buffer: 10mM tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-Cl) pH8.0, 1mM ethylenediaminetetraacetic acid (EDTA: Ethylene Diamine Tetraacetic Acid ), 0.5% polyethylene glycol octylphenyl ether (triton), 1mM phenylmethylsulfonyl fluoride (PMSF); buffer A: 50mM Tris-ClpH8.0, 500mM NaCl, 8M urea (Urea); buffer B: 50mM Tris-Cl PH8.0, 500mM NaCl, 0.5%triton; Buffer C: 50mM Tris-Cl PH8.0, 150mM NaCl, 400mM imidazole; Dialysis buffer: 50mM Tris-Cl PH8.0, 150mM NaCl, 1mM EDTA, 1mM MDTT, 10% glycerol.

[0062] 1) Pick the healthy single clone obtained in step a of Example 2 above and inoculate in 10mL LB Amp (100μg / mL) at 37°C, 250rpm and culture overnight;

[0063] 2) 1:100 (v / v) expanded culture to 250mL LB (Amp100μg / mL), a total of 2 bottles, 37 ° C, 250rpm shaking culture to OD600 = 0.5 ~...

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Abstract

The invention relates to a method for preparing an isotope-labeled recombinant C reactive protein, which comprises the following steps of: constructing a C reactive protein gene-containing recombinant vector; transferring the recombinant vector into a host cell for recombinant expression, wherein a stable isotope is added into a culture medium for recombinant expression; and purifying the expressed recombinant vector and measuring the purity thereof. The method has the advantages that: the isotope 15N-labeled recombinant C reactive protein is designed, an N source in the culture medium is replaced, the expressed and purified product is taken as an interior label and subjected to enzyme cutting with a target C reactive protein, and mass-spectrometer measurement is preformed, so the uncertainty of incomplete enzyme cutting on the measured result can be eliminated, more accurate quantification on the protein is achieved, the measurement error caused by incomplete enzymolysis and hydrolysis of the protein in the prior art is avoided, the uncertainty in the measurement process is greatly reduced and the measurement accuracy is improved.

Description

technical field [0001] The invention relates to a method for preparing recombinant protein, in particular to a method for preparing isotope-labeled recombinant C-reactive protein, and belongs to the technical field of recombinant protein. Background technique [0002] Proteins play a very important role in clinical health. Many disease markers are composed of proteins, such as tumors and cardiovascular and cerebrovascular diseases. Isotope dilution mass spectrometry is a widely used method in the determination of standard substances. Due to the complex structure and large molecular weight of proteins, direct measurement of intact protein molecules by mass spectrometry is problematic. Generally, the protein is digested into peptides with enzymes before analysis, and then a known amount of isotope-labeled peptide is added as an internal standard for quantification. However, this approach is also not ideal, as inaccurate measurements may result from incomplete protein digest...

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K14/47C07K1/22
Inventor 宋德伟徐蓓戴新华李红梅何亚娟
Owner NAT INST OF METROLOGY CHINA
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