Application of an eu-containing polymetallic oxygen cluster in the detection of human papillomavirus early oncoprotein e6 in vitro

A technology of human papillomavirus and polymetallic oxygen clusters, which can be used in measuring devices, biological tests, material inspection products, etc., and can solve the problem of lack of HPV16E6 effective and commercially available mAbs

Active Publication Date: 2019-08-30
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In summary, the lack of effective, commercially available mAbs for HPV16 E6 is a dilemma facing researchers

Method used

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  • Application of an eu-containing polymetallic oxygen cluster in the detection of human papillomavirus early oncoprotein e6 in vitro
  • Application of an eu-containing polymetallic oxygen cluster in the detection of human papillomavirus early oncoprotein e6 in vitro
  • Application of an eu-containing polymetallic oxygen cluster in the detection of human papillomavirus early oncoprotein e6 in vitro

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Embodiment 1

[0037] 将编码151个氨基酸的HPV16 E6 4C / 4S突变体(MFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYSYSLYGTTLEQQYNKPLSDLLIRCINCQKPLSPEEKQRHLDKKQRFHNIRGRWTGRCMSCSRSSRTRRETQL)基因按照基因工程的方法克隆(Nature,2016,529,541-545.)到质粒pGEX-6p-1载体,并将0.5μL此质粒转化到 In 50 μL of competent cells of Escherichia coli XA90, use LB solid medium containing 50 mg / mL Amp (ratio: 100 mL of water plus 1 g of peptone, 0.5 g of yeast powder, 1 g of sodium chloride, and 1.5 g of agar powder) to perform monoclonal colonization and cultured overnight in a 37°C incubator. Pick a single clone colony and add it to 5mL LB medium containing 50mg / mL Amp (ratio: 100mL water plus 1g peptone, 0.5g yeast powder, 1g sodium chloride), in a constant temperature culture shaker at 37°C at a speed of 220rpm Shake culture for 14h. Take 1 mL of the above bacterial solution and add it to 1 L of new LB medium containing 50 mg / mL Amp, continue to shake and culture at 37 °C in a constant temperature culture shaker at a speed...

Embodiment 2

[0040] 8.3g Na 2 WO 4 2H 2 O was dissolved in 20 mL of water, with CH 3 COOH adjusted the pH of the solution to 7.0. Subsequently, 2 mL containing 1.1 g Eu(NO 3 ) 3 ·6H 2 The aqueous solution of O was added dropwise to the above solution with stirring at 90°C. Colorless crystals of Na can be precipitated after cooling at room temperature 9 [EuW 10 o 36 ]·32H 2 O(EuW 10 ), the mass is 6.58g.

Embodiment 3

[0042] Get the colorless crystal EuW that 3.39mg embodiment 2 obtains 10 Dissolve in 1mL Wahaha purified water to prepare a stock solution with a concentration of 2mmol / L, and 10μL EuW 10The stock solution was added to 1990 μL MES-NaOH buffer and mixed well. The HPV16 E6 small peptide stock solution was gradually added dropwise to the mixed solution so that the final concentrations were 0-26 μmol / L (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 22, 26 μmol / L), after each concentration was added, use a vortex shaker to fully shake the sample, and place it at room temperature for 5 minutes. Afterwards, the polymetallic oxygen cluster EuW was recorded using a fluorescence spectrometer 10 Fluorescence emission spectra (excitation wavelength 265nm) of the solution in response to different concentrations of HPV16 E6 small peptides. Such as figure 1 As shown, with the increase of HPV16 E6 small peptide concentration, EuW 10 The fluorescence emission peak intensity at 593nm and ...

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Abstract

The invention relates to application of Eu-containing polyoxometalate in the in-vitro assay of the early pathogenic protein E6 of human papillomavirus, which belongs to the technical field of assaying the early pathogenic protein E6 of human papillomavirus by means of polyoxometalate. The invention utilizes the strong recognition interaction between basic amino acid-rich small peptides on EuW10 and HPV E6 proteins to assay the HPV E6 protein. A research discovers that the E6 small peptide and protein can be assembled with EuW10 into spheres to promote the remarkable enhancement of fluorescence. The invention is the first example of utilizing polyoxometalate for in-vitro E6 protein assay. The polyoxometalate EuW10 synthesis method utilized by the invention is simple and easy to obtain. The assay method disclosed by the invention has the advantages of high assay speed, simplicity and convenience in operation, simple system, stable signals and high sensitivity, and does not need any pretreatment and a complex assay instrument. Through the invention, the application of polyoxometalate as a biological probe is expanded, which is of a great significance to the pre-cancer early assay of cervical cancer.

Description

technical field [0001] The invention belongs to the technical field of detection of human papillomavirus early oncogenic protein E6 by polymetallic oxygen clusters, and specifically relates to a method using polymetallic oxygen clusters Na 9 [EuW 10 o 36 ]·32H 2 O(EuW 10 ) and a small peptide rich in basic amino acids on the E6 protein as a platform for the in vitro detection of HPV early oncogenic protein E6. Background technique [0002] Human papillomavirus (HPV) is a small, double-stranded DNA virus that infects squamous epithelial cells of the human skin and mucous membrane basal layer. HPV can cause a variety of cancers in women (cervix, vagina, vulva, anus, and oropharynx) and men (oropharynx, anus, and penis), as well as benign diseases such as genital warts. Worldwide, about 500,000 cases of cervical cancer occur each year, resulting in more than 250,000 deaths, which makes cervical cancer the second largest killer of women's health (The Lancet, 2007, 370, 890-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/574G01N33/68
Inventor 吴玉清刘玉雪李洪伟远新新
Owner JILIN UNIV
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