Biological sample structure hydrogen-deuterium substitution verification and deuterium percentage content determining method
A technology for biological samples and percentage content, applied in biological testing, separation methods, chemical instruments and methods, etc., can solve the problems of sample processing and testing and analysis techniques that are cumbersome and time-consuming, expensive testing equipment, and high synthesis prices.
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Embodiment 1
[0041] Demonstration of hydrogen-deuterium displacement of JEV whole virions Primary chicken embryo cells (CEC) were used to propagate JEV. CEC first without heavy water (D 2 O) culture medium for 24 h, and then cultured at 37° C. for 2 days in a maintenance solution containing 36% heavy water. The maintenance solution containing heavy water was discarded, and JEV was added to the cells and adsorbed for 2h. After the adsorption was completed, the adsorption solution was discarded, the cells were washed twice with PBS, fresh maintenance solution containing heavy water was added, and harvested three days later (V D stands for deuterated JEV, V H stands for undeuterated JEV, C D represents a deuterated virus-free cell control). All viruses and corresponding control samples were purified by polyethylene glycol (PEG) precipitation, 0.45mm filter filtration, 40% sucrose bottom ultracentrifugation and other methods. After the virus sample was inactivated by formaldehyde, its tit...
Embodiment 2
[0054] Confirmation of hydrogen-deuterium replacement in HAV whole virions and determination of the percentage of deuterium Hepatitis A virus vaccine strain (H2 strain) was obtained by propagating in human lung diploid cells. Cells infected with the H2 strain or cells not infected with the virus were harvested, resuspended in PBS, frozen-thawed and ultrasonicated three times, and centrifuged at 8000g for 30 minutes to remove cell debris. Harvested supernatants were extracted three times with chloroform, followed by ultracentrifugation to harvest viruses or their cell controls. The following method was used for the deuteration of the sample: the sample was resuspended in the minimum essential medium (MEM) containing 87% heavy water, and incubated at 36° C. for one week. The deuterated samples were subjected to ultracentrifugation to discard the MEM containing heavy water, and the precipitate was fully washed with distilled water, and then ultracentrifuged again to harvest the d...
Embodiment 3
[0076] Confirmation of hydrogen-deuterium replacement in ribonucleic acid (RNA) of deuterated HAV virus and determination of deuterium percentage content The fully washed deuterated HAV or cell control is used to prepare deuterated RNA, the method refers to guanidine isothiocyanate- One-step extraction of phenols. RNA samples were quantified spectrophotometrically and their OD was calculated 260 / OD 280 The ratio is used to characterize the purity of RNA.
[0077] Select bovine serum albumin (BSA) as the matrix, dilute the RNA extracted from deuterated HAV, non-deuterated HAV and deuterated cell control with PBS to the required concentration, the final volume is 0.1ml, and then add 0.5ml containing 5mgBSA PBS solution. Then put the mixed sample into the GEL-1 type freeze dryer to freeze-dry. After freeze-drying, the water content of the mixed sample is less than 3%. D , RNA-V H and RNA-C D express. Freeze-dried mixed samples were mixed with oxidants CuO (500mg) and V 2...
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