Method for in vitro rapid proliferation of plasmodium mature schizonts
A technology for mature schizonts and malaria parasites, applied in the biological field, can solve the problems of cumbersome steps, time-consuming and laborious, etc., and achieve the effect of short time, high efficiency and large quantity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0022] The method for rapidly multiplying mature schizonts of Plasmodium in vitro comprises the following steps:
[0023] (1) Select liquid nitrogen with a parasite rate of 25-30% to freeze the malaria parasites, place them in a constant temperature water bath at 37°C to thaw quickly, then centrifuge at 2000rpm for 3min, remove the supernatant, and use 1mL of malaria parasite culture medium After resuspension, transfer to a 50mL centrifuge tube, add 40mL of malaria parasite medium to wash, and then centrifuge at 2000rpm for 3min; wherein the malaria parasite medium is RPMI1640 medium, and contains 20% fetal bovine serum by volume fraction. 10% red blood cells, 75000 units / L heparin, 10 5 Unit / L of penicillin and 100mg / L of streptomycin;
[0024] (2) Resuspend the pellet after centrifugation in step (1) with 1 mL of Plasmodium culture medium and transfer it to a 500 mL wide-mouth culture bottle, then add 100 mL of Plasmodium culture medium and mix well, then infuse N 2 , CO ...
Embodiment 2
[0029] Constructing a plasmid for gene knockout, comprising the following steps:
[0030] (1) According to the Plasmodium genome sequence, design primers for amplifying the Plasmodium gene UIS3, the upstream primer is: 5'-atgggtaccaacaccctcaatgtct-3' (SEQ ID NO.1), the boldface indicates the KpnI restriction site, and the downstream primer is: 5'-taaggcgcctataaaattataatat-3'(SEQ ID NO.2), the boldface indicates the NarI restriction site, and then use the Plasmodium genome as a template, and the nucleotides shown in SEQ ID NO.1 and SEQ ID NO.2 as primers PCR amplification, the PCR amplification conditions were pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 10 s, annealing at 54°C for 20 s, and extension at 68°C for 1 min, a total of 35 cycles; finally, extension at 68°C for 5 min to obtain the UIS3 gene;
[0031] (2) Design primers for amplifying GFP according to the nucleotide sequence of GFP on the pEGFP-N1 (GenBank: U55762.1) vector, the upstream primer is ...
Embodiment 3
[0035] The transfection of mature schizonts of Plasmodium comprises the following steps:
[0036] (1) Resuspend the Plasmodium mature schizont precipitate obtained in the example separation with 1 mL of Plasmodium medium, then add 9 mL of Plasmodium medium and mix well, then add 10 mL of Plasmodium mature schizont suspension to In 10 1.5mL EP tubes, each tube is 1mL, which can satisfy 10 knockouts at the same time;
[0037] (2) Centrifuge the EP tube containing schizonts subpacked in step (1) at 16,000 g for 5 s, remove the supernatant, and use 100 μL of parasite electrotransfer solution containing the linearized gene knockout plasmid prepared in Example 2 for precipitation Resuspend, then transfer the transfer suspension to the electroporation cup, select the program U33 for electroporation, inject the product into the mouse body through the tail vein after completion, and start feeding pyrimethamine syrup for screening for 5-6 days after 24 hours; B The preparation method o...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More