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
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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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