Candida albicans attenuated live vaccine as well as preparation method and application thereof
A technology of Candida albicans and live bacteria, applied in the field of biomedicine, can solve the problems of high mortality rate of deep fungal infection, increase of fungal infection rate, loss of body flora, etc., and achieve good protection effect, short cycle and low cost Effect
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Embodiment 1
[0032] Example 1. Deletion bacteria gpi7 / gpi7 The build:
[0033](1) Materials: Genomic DNA extraction kits, plasmid extraction kits, and DNA purification kits were purchased from Beijing Tiangen Gene Technology Co., Ltd. rTaq, exTaq, dNTP, etc. were purchased from Takara Company. The 96wells PCR instrument was purchased from BIO-RAD Company.
[0034] (2) Preparation of experimental reagents:
[0035] 1. PBS buffer
[0036] NaCl 8.0g, KCl 0.4g, NaCl 2 HPO 4 12H 2 O 3.48g, KH 2 PO 4 0.2g, adjust the pH to 7.4, add triple distilled water to 1000ml, dissolve and dispense, autoclave and store at 4°C for later use.
[0037] 2. Sandcastle Dextrose Agar Solid Medium (SDA)
[0038] Add 10g of peptone, 40g of glucose, and 18g of agar, add 900ml of triple-distilled water and stir to dissolve, dilute to 1000ml, autoclave (121°C, 15min) and store at 4°C for later use.
[0039] 3. Synthetic complete medium (synthetic complete medium, SC)
Embodiment 2
[0088] Example 2. Virulence detection of deletion bacteria gpi7 / gpi7
[0089] Experimental animals: 6-8 week old C57 female mice, from Shanghai Slack Company. Certificate number: SCXK-(military) 2009-0004.
[0090] Experimental method and results:
[0091] C57BL / c female mice were equally divided into two groups, 12 in each group. Injected into tail vein of mice respectively gpi7 / gpi7 Deletion bacteria and parent bacteria SN152 each 5 × 10 5 CFU / only, causing systemic fungal infection. 15 rats (5 rats / group) were sacrificed 24 hours after the administration of bacteria, and both kidneys were taken, weighed, cut open, homogenized with 5ml of 0.9% normal saline, and then diluted with 0.9% normal saline for 10 2 、10 3The number of homogenates obtained by diluting multiples of different orders of magnitude was obtained, and 100ul each was spread on an SDA plate, cultured in a fungal incubator at 30°C for 48 hours, and the number of colonies was counted, with the number of ...
Embodiment 3
[0093] Example 3. Observation of deleted bacteria under electron microscope gpi7 / gpi7 cell wall:
[0094] Take a small amount of each strain from the glycerol frozen bacteria at -80°C, and inoculate it into YPD solid culture based on 30°C for 48 hours; pick a single colony from it and shake it in 1ml YPD liquid medium for 16 hours to activate it, so that the fungus is in the index Late growth period. Take 1ml of each of the above bacterial solutions in a 250ml Erlenmeyer flask, add 100ml of YEPD culture solution, incubate at 30°C and 200rpm for 14h, centrifuge at 1000×g for 10min, wash with PBS 3 times until the supernatant is colorless, remove the supernatant, add 3 % Glutaraldehyde 4ml was fixed overnight at 4°C. After washing with PBS for 3 times, it was fixed with osmic acid, dehydrated with ethanol and acetone gradient, and then mixed with acetone and embedding agent in different proportions to soak the sample. Finally, the sample was embedded, polymerized, positioned...
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