Hantavirus-like particles fused with RGD for tumor gene therapy and preparation method of hantavirus-like particles fused with RGD
A hantavirus and virus-like technology, applied in the field of RGD-fused hantavirus-like particles and their preparation, can solve the problems of serious harm, high fatality rate, no specific and effective therapeutic drugs, etc., to reduce damage and reduce operation. Difficulty, effect suitable for industrial production
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[0039] Example 1. Extraction of mRNA
[0040] Follow the instructions of the mRNA miniprep kit, and the specific operations are as follows:
[0041] 1) Add 750 μL Tripure LS Reagent to 250 μL of anticoagulated serum samples, repeatedly pipette and shake to mix, and lyse at room temperature for 5 minutes;
[0042] 2) Add chloroform into the lysis solution at a rate of 0.2 mL per mL, and shake the sample to mix well;
[0043] 3) The mixture was centrifuged at 12 000 r / min for 15 min at 4°C to separate the mixture into two phases, DNA and protein were extracted into the organic phase, and RNA remained in the aqueous phase. Transfer the upper aqueous phase to a new centrifuge tube;
[0044] 4) Add isopropanol and RNA precipitation solution to the water phase, after fully mixing, place at room temperature for 25min to precipitate RNA;
[0045] 5) Centrifuge at 12 000 r / min at 4°C for 15 min, remove the supernatant, and collect the precipitated RNA;
[0046] 6) Fully wash the pr...
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[0050] Example 2. Synthesis of cDNA by reverse transcription reaction
[0051] According to the instructions of the RT-PCR kit, the reverse transcription reaction system was prepared on ice, and the operation was performed in a 0.2 mL microcentrifuge tube. The specific ingredients are as follows:
[0052]
[0053] In the PCR instrument, put the microcentrifuge tubes into the corresponding wells, react at 42°C for 1 hour, and then inactivate the RT enzyme in a water bath at 70°C for 10 minutes, and place the obtained RT product cDNA at -80°C for 1 hour. Save for backup.
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[0054] Example 3. PCR amplification of HFRS target fragment
[0055] According to the instructions of the PCR kit, prepare the PCR reaction system in a 0.5 mL microcentrifuge tube on ice. in,
[0056] Upstream primer P1: 5'-ATGGACATCGACCACTAC-3';
[0057] Downstream primer P2: 5'-TTAGTGGTGGTGGTGGTGGTGCGGCAGAGTGG-3'.
[0058] The specific ingredients are as follows:
[0059]
[0060] After the system is prepared, cover the centrifuge tube, invert it up and down several times to mix, and immediately place it on the PCR machine to perform amplification. The reaction conditions are as follows:
[0061]
[0062] After finishing the reaction, place the PCR product at 4°C for electrophoresis detection or at -20°C for long-term storage.
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