Lysis reagent, purification reagent and application of bacterial cells after armored RNA virus-like particle expression
A technology for expressing RNA viruses and bacteria, which is applied in the field of lysing reagents and purification reagents for bacteria after the expression of armored RNA virus-like particles, and can solve the problems of difficult removal of nucleic acids
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Embodiment 1
[0088] The expression of embodiment 1 armored RNA virus-like particle
[0089] The glycerol bacteria of the armored RNA virus-like particle expression strain CBC NO.150601 of influenza A virus preserved by Boao Bio Group Co., Ltd. were activated by streaking on the plate, and the single colony after activation was picked and inoculated in 10ml containing Kan and Cm resistance LB liquid medium, 37 ° C, 220 rpm, constant temperature shaking incubator for overnight culture. Inoculate the cultured bacteria solution as the seed bacteria into 200ml of LB liquid medium containing Kan and Cm resistance at a ratio of 1% (v / v), 37°C, 220rpm, constant temperature shaking culture value OD600=0.4-0.6, add the final IPTG with a concentration of 1M was used to induce expression. The induction expression conditions were: 22°C, 200rpm culture for 6h, the expressed bacterial liquid was divided into 20ml bacterial liquid per tube, centrifuged at 8000rpm for 5min, the supernatant was discarded, t...
Embodiment 2～6
[0090] Examples 2-6 Ultrasonic disruption of expression cells and purification of armored RNA virus-like particles
[0091] The settings of ultrasonic reagents and purification reagents used in Examples 2-6 are shown in Table 1:
[0092] Table 1 Embodiment 2~6
[0093] ultrasonic reagent Purification Reagent Example 2 1 1 Example 3 2 1 Example 4 3 1 Example 5 1 2 Example 6 1 3
[0094] Wherein, each reagent component is as follows:
[0095] Ultrasonic reagent 1: 35mM Tris-HCl (pH8.0), 0.5M Na 2 SO4, 15 mM KCl, 0.5 mM EDTA, 10% glycerol, 0.5 mM DTT, 0.5% Triton X-100.
[0096] Ultrasonic reagent 2: 50mM Tris-HCl (pH8.0), 1.0M Na 2 SO4, 20 mM KCl, 1 mM EDTA, 20% glycerol, 1 mM DTT, 2% Triton X-100.
[0097] Ultrasonic reagent 3: 100mM Tris-HCl (pH8.0), 1.5M Na 2 SO4, 25 mM KCl, 1.5 mM EDTA, 30% glycerol, 2 mM DTT, 5% Triton X-100.
[0098] Purification reagent 1: 25mM Tris-HCl (pH8.0), 20mM NaCl, 10mM MgCl 2 , 0.5mM DT...
Embodiment 7
[0120] Example 7 Purification Effect Verification
[0121] One, extract the nucleic acid of embodiment 2~6 preparation armored RNA virus-like particles
[0122] The armored RNA of purified gained is carried out nucleic acid extraction (extracted by Tiangen virus RNA extraction kit), extraction steps are as follows:
[0123] 1. Take 140 μl of the virus-like particle solution of the to-be-extracted solution.
[0124] 2. Add 560 μl AVL buffer, vortex and mix for 15 seconds to fully lyse the virus particles.
[0125] 3. Incubate at room temperature for 10 minutes and centrifuge briefly.
[0126] 4. Add 560 μl of absolute ethanol, vortex and mix for 15 seconds, and centrifuge briefly.
[0127] 5. Carefully pipette 630 μl of the above mixed solution into the adsorption column. Do not let the liquid stick to the edge of the adsorption column. Place it at room temperature for 2 minutes, centrifuge at 8000 rpm for 1 minute, and transfer the adsorption column to a new collection tube...
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