Application of Ginkgolic Acid in Anti-Cryptosporidium
A technology of Cryptosporidium and Cryptosporidium anseri, which is applied in the direction of resistance to vector-borne diseases, anti-infective drugs, active ingredients of salicylic acid, etc., and can solve the problems of ineffectiveness in immunodeficiency patients
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Embodiment 1
[0060] Example 1: Construction of the Cryptosporidium ansei COWP gene plasmid
[0061] 1 reagent
[0062] RPMI1640 was purchased from Hyclone Inc., USA.;
[0063] Trypsin was purchased from GIBCO, USA;
[0064] Dimethylsulfoxide (DMSO) was purchased from Amresco Inc., USA.;
[0065] Penicillin–Streptomycin was purchased from Gibco, USA;
[0066] Cell counting kit (CCK-8) was purchased from Dojindo Molecular Technologies of Japan;
[0067] Human colon cells (HCT-8 cells) were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences;
[0068] The DNA extraction kit was purchased from Shanghai Jierui Bioengineering Co., Ltd.;
[0069] DNA gel recovery kit was purchased from Beijing Biotech;
[0070] Sodium taurocholate was purchased from Sigma.
[0071] 2 Experimental Cryptosporidium species
[0072] Bovine-derived Cryptosporidium oocysts were collected from cow feces, sieved through 60 meshes and stored in 2.5% potassium dichromate (K 2 Cr 2 o 7 ...
Embodiment 2
[0102] Embodiment 2: Determination of drug maximum safe concentration
[0103] 1. Prepare the stock solution of nitazoxanide (NTZ group), GA group, GB group and GA+GB group (mixture of GA and GB), etc., and the components and concentrations of the stock solution are shown in the table below.
[0104]
Composition (content ratio)
total concentration
NTZ group:
10mg / ml
[0105] GA group:
The content (wt%) ratio of C13:0:C15:1:C17:2 is 9.3:48.33:1.54
10mg / ml
GB group:
The content ratio (wt%) of C15:0:C17:1 is 3.42:37.42
10mg / ml
GA+GB group:
GA group and GB group are mixed at a volume ratio of 1:1
10mg / ml
[0106] Each group was diluted to the required experimental concentration with culture medium.
[0107] NTZ group (stock solution concentration 10mg / ml): experimental concentrations were 100μg / ml, 50μg / ml, 25μg / ml, 12.5μg / ml, 6.25μg / ml, 3.125μg / ml, 1.56μg / ml, 0.78μg / ml ml;
[010...
Embodiment 3
[0123] Example 3: Drugs inhibit the in vitro proliferation effect of Cryptosporidium ahnii
[0124] 3.1 Experimental method
[0125] 3.1.1 Cell culture is the same as in Example 1.
[0126] 3.1.2 Oocyst collection and aseptic treatment are the same as in Example 1.
[0127] 3.1.3 Cell infection is the same as that in Example 1.
[0128] 3.1.4 In vitro proliferation experiment of drugs inhibiting Cryptosporidium ahnii
[0129] Infect HCT-8 with Cryptosporidium amnesia, and inoculate 1×10 parasites in 6-well cell culture plate 3 Each well, add 5ml of Cryptosporidium ahnii culture solution, place at 37℃, 5%CO 2 Cultivate in the environment for 8 hours, discard the original culture solution, wash twice with sterile PBS buffer, and add the NTZ group, GA group, GB group and GA+GB group with three concentrations of high, medium and low diluted ratio:
[0130] NTZ group (stock solution concentration 10mg / ml):
[0131] The experimental concentrations were 3.125 μg / ml (high concen...
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