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Binding protein combination for detecting plasmodium falciparum HRP2 and plasmodium vivax LDH and preparation method and application thereof

A Plasmodium falciparum and binding protein technology, applied in the field of phage display, can solve the problems of high detection cost, impossibility of popularization and application, and expensive raw materials, and achieve the effect of low production cost, high clinical application value, and low requirements for storage conditions

Active Publication Date: 2019-10-18
JINAN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, microscopic examination and PCR nucleic acid amplification have problems such as low efficiency and high detection cost; immunological detection has problems such as product preservation difficulties and expensive raw materials, and cannot be quickly popularized and applied in developing countries where malaria is seriously spreading in Southeast Asia and Africa.

Method used

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  • Binding protein combination for detecting plasmodium falciparum HRP2 and plasmodium vivax LDH and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Phage display library panning process for protein reagents targeting P. falciparum L-lactate dehydrogenase and histidine-rich protein 2:

[0042] (1) First use NHS-EZ coupling reagent (purchased from Pierce) to label the target protein with biotin. The effect of biotin labeling was confirmed by detecting the presence of biotin by ELISA method. The biotin-labeled protein was determined by adsorbing the target protein on a 96-well plate (purchased from Nunc Company), and then using streptavidin-coupled horseradish peroxidase (HRP) for biotin detection. The panning process of the phage display library is as follows: Take a 5 μL aliquot of the phage display library containing complexity 10 12 Phage plasmids were mixed with 95 μL of phosphate buffer containing 0.1% Tween 20, and then pre-panned 3 times on a high-binding streptavidin-coated plate (purchased from Pierce Company). 1 hour. Add 100 μL of 100 nM biotinylated protein to the panning wells coated with streptavidin...

Embodiment 2

[0046] The presence of phage of the target protein reagent is detected by ELISA method, and the verification result is further confirmed by sequencing. The process is as follows:

[0047] The phages were recovered from the wells containing the target protein, and the quality control well was used to judge the amplification in the target well. The quality control well indicated that the reagents that bind the target protein were amplified efficiently. To test proteins purchased from display libraries, bound target phage were detected by ELISA. Single ER2738 colonies were purchased from infected cells from the last round of panning and cultured in 96-deep well plates in 100 μL of 2TY containing 100 μg / mL carbenicillin at 37 °C (900 rpm). The cells were picked out after 6 hours of culture in medium. Add a 25 μL aliquot of the culture solution to 200 μL 2TY medium containing carbenicillin, and incubate at 37° C. (900 rpm) for 1 hour. Then add helper phage (10 μL of 1011 / mL), and...

Embodiment 3

[0049] Introduce the specific DNA sequence for expressing the target protein into the target plasmid and transfer it into Escherichia coli for amplification and culture. After extraction and purification, the protein expression is tested by ELISA method. The steps are as follows:

[0050] All specific reagents were then introduced into protein expression vectors for cloning, and the products were determined as capture reagents in ELISA. The DNA coding sequence (SEQ ID NO: 1-4) of the binding protein that reacts with the target protein is amplified by PCR, and the product is treated with Nhel and Pstl restriction endonucleases, and the digested fragment is introduced into the structure containing the expression binding protein and pET11a plasmid with restriction sites. The clones were picked and cultured in 5 ml of LB medium at 37°C and 225 rpm overnight, and the plasmid DNA was extracted with a minipreps (from Qiagen) plasmid extraction kit, followed by sequencing to further c...

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Abstract

The invention discloses a binding protein combination for detecting plasmodium falciparum HRP2 and plasmodium vivax LDH and a preparation method and application thereof. The binding protein combination is composed of a binding protein 1 and a binding protein 2 which detect the plasmodium falciparum HRP2 and a binding protein 3 and a binding protein 4 which detect the plasmodium vivax LDH, and amino acid sequences of the binding protein 1, binding protein 2, binding protein 3 and binding protein 4 are shown in SEQ ID NO:5-8. According to the binding protein combination and the preparation method and application thereof, a phage display technology is adopted, the plasmodium falciparum HRP2 and the plasmodium vivax LDH are taken as target antigens, and the binding protein combination with high affinity and good specificity is selected from a phage display protein library. A genetic engineering technology is used for recombinant expression and purification, a binding protein combination reagent replaces an antibody reagent for malaria immunodetection, a colloidal gold chromatography rapid detection method is established, the storage stability of an obtained kit is higher than that of an existing clinical detection product, and the binding protein combination has the advantages of being reliable, accurate, safe, simple, convenient and stable.

Description

technical field [0001] The invention relates to the technical field of phage display, in particular to a binding protein combination for detecting Plasmodium falciparum HRP2 and Plasmodium vivax LDH, a preparation method and application thereof. Background technique [0002] Malaria is one of the three most serious infectious diseases in the world. About half of the world's population is at risk of malaria. In my country, there are still cases of malaria infection in some areas. Rapid diagnosis is very important in malaria detection because of the special modes of malaria transmission, carriage, and latent disease. [0003] Since the development of malaria detection technology, clinical detection methods mainly include microscopic examination, immunoassay (immunochromatography, enzyme-linked immunoassay) and PCR nucleic acid amplification test. Among them, microscopic examination and PCR nucleic acid amplification have problems such as low efficiency and high testing costs;...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/445C12N15/30C12N15/70G01N33/569G01N33/558G01N33/545
CPCC07K14/445C12N15/70G01N33/545G01N33/558G01N33/56905G01N2333/445Y02A50/30
Inventor 薛巍牟善松吴庆金
Owner JINAN UNIVERSITY
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