Preparation and application of hemagglutinin peptide mark recombinant protein immunoaffinity purification enriching column

A technology of recombinant protein and hemagglutinin, which is applied in the preparation method of peptides, peptides, organic chemistry, etc., can solve the problems that limit the application, detection, non-specific adsorption, and metal ion shedding of target proteins, and achieve accurate separation and elution The effect of mild conditions and high coupling density

Inactive Publication Date: 2013-05-22
NANNING LANGUANG BLUE LIGHT BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Since the affinity chromatography columns currently used for separation, purification and enrichment of HA recombinant proteins mainly include metal chelate affinity columns and affinity columns using enzyme substrates and reactive fuels as ligands, these affinity columns have non-specific Significant shortcomings such as heterosexual adsorption, severe elution conditions, and poor purification effects, and the metal chelate affinity column also has the defect that metal ions fall off into the eluent, which will limit the subsequent application of the separated and purified target protein. detection

Method used

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  • Preparation and application of hemagglutinin peptide mark recombinant protein immunoaffinity purification enriching column
  • Preparation and application of hemagglutinin peptide mark recombinant protein immunoaffinity purification enriching column
  • Preparation and application of hemagglutinin peptide mark recombinant protein immunoaffinity purification enriching column

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Preparation of HA specific polyclonal antibody purified by immunoaffinity

[0044] 1.1 HA short peptide hapten design

[0045] Using a short peptide consisting of 9 amino acid residues, namely tyrosyl-prolyl-tyrosyl-aspartyl-valine-prolyl-aspartyl-tyrosyl-alanine, These 9 amino acid residues were divided into two groups to introduce the cysteine ​​sulfhydryl SH group in opposite directions to prepare the thiolated HA short peptide.

[0046] 1.2 Preparation of HA short peptide immunogen

[0047] Take 5 mg / mL KLH 10 mL, add 100 μL saturated Na 2 CO 3 , Then add 1 mL of 10% SDS, mix well, boil for 5 minutes to denature, and then react with iodoacetyl-NHS to generate iodoacetylated KLH. After removing excess iodoacetic acid by G25 Sephadex, add 55 mg of HA short peptide hapten. Adjust the pH to 8.5 and react at room temperature for 60 minutes to obtain the HA-KLH covalent bond complex. The immune antigen is obtained after desalting by G25 Sephadex. Use OVA as the carrie...

Embodiment 2

[0061] Example 2 Preparation of a high-density HA tag recombinant protein immunoaffinity purification enrichment column.

[0062] 2.1 Take 3 mL of Agarose activated by sodium iodate and transfer it to the column, wash with 100 mL of distilled water, drip dry, and then pass 2 times the volume of Agarose 0.1 mol / L Na 2 CO 3 , Drip dry and reserve;

[0063] 2.2 Use 300 mg of immunoaffinity purified HA specific polyclonal antibody with 0.1 mol / L Na 2 CO 3 Dissolve to reach a concentration of 20 mg / mL, and centrifuge to take the supernatant after complete dissolution;

[0064] 2.3 Add the supernatant to the affinity column of step 2.1, collect the effluent and pass it through the column again, repeat 3-5 times;

[0065] 2.4 Wash out unbound antibodies with 20 mL of distilled water and collect them together;

[0066] 2.5 Prepare 1.2 mL of 10 mg / mL sodium borohydride solution with distilled water, add it to the column, shake and mix, and react at 4°C for 30 minutes;

[0067] 2.6 Wash the filler...

Embodiment 3

[0070] Example 3 HA-specific antibody labeling horseradish peroxidase

[0071] 3.1 Weigh 5 mg immunoaffinity purified HA specific polyclonal antibody, dissolved in 100 μL 0.1 mol / L Na 2 CO 3 , React for 10 minutes, according to the amount of antibody: horseradish peroxidase=1:1, add 5 mg of horseradish peroxidase that has been activated, and mix;

[0072] 3.2 Add 1 mg of sodium borohydride, mix well and exhaust, place at -20°C for 1 minute, then move to 4°C and stand for 2 hours;

[0073] 3.3 Desalting via G25 Sephadex desalting column;

[0074] 3.4 Add BSA to make the final concentration of BSA 1mg / mL, then add 50% glycerol, and use ELISA to detect the titer of the labeled antibody.

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Abstract

The invention provides a high-efficiency specificity enriched hemagglutinin peptide HA mark recombinant protein immunoaffinity purification enriching column as well as a preparation method and an application thereof. The immunoaffinity purification enriching column provided by the invention comprises an activated agarose filler coupled with HA specificity polyclonal antibody which is subjected to immunoaffinity purification and a plastic column carrying the immunoaffinity filler. The immunoaffinity purification filler coupled HA specificity polyclonal antibody is extracted by using an immunoaffinity method; and the immunoaffinity purification enrichment column is obtained by carrying the immunoaffinity filler into a specific plastic column. The immunoaffinity purification enrichment column enriched HA mark recombinant protein is high in efficiency, strong in specificity and temperate in elution condition, can be repeatedly utilized and is an ideal material for separating, purifying and enriching the HA mark recombinant protein at present.

Description

Technical field [0001] The present invention involves a kind of biological separation and purification enrichment device, especially a preparation method and application of efficient separation and purification and purification, enriched hemagglutonin peptide HA label reorganization protein. Background technique [0002] Molecular biology and protein groups are popular disciplines in the field of life science research. The use of recombinant protein has increased significantly in recent years.In order to facilitate the labeling, tracking, identification and purification of genetic restructuring proteins, a reorganized protein will be artificially introduced in target protein expression.Therefore, the technology of separation and purification, enrichment of reorganization is increasingly displaying its importance.The separation and purification of the complicated expression system and the rich target protein are a difficult and heavy task, and the affinity layer method is the most...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/22B01D15/38
Inventor 潘丽金谢体三宁欢欢张芬萧浩
Owner NANNING LANGUANG BLUE LIGHT BIOTECH
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