Human enterovirus strain and application thereof
A technology of human enteroviruses and viruses, applied in the direction of viruses, antiviral agents, virus antigen components, etc., to achieve good immune protection effect and good immune protection effect
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Embodiment 1
[0022] Example 1 Isolation and cultivation of gerbil primary muscle cells
[0023] Select 10 gerbils aged 10-15 days, kill them under humane anesthesia, sterilize them with iodine and 75% alcohol, cut off the hindlimb muscles, break them up, and wash them with MEM culture medium containing 2 times double antibody (purchased from Gibco) for 3 Over time, 0.2% (w / v) collagenase and 0.25% (w / v) trypsin were mixed and digested overnight at 4°C. The digestive solution was repeatedly blown into a suspension with 10 times (w / v) MEM solution, passed through 100-mesh and 200-mesh molecular sieves successively, collected the filtrate, centrifuged at 1000×g for 5-10min, blown the cell pellets evenly, and adjusted the pH to 5- 7. Set at 37°C, 5% CO 2 In the incubator, culture in MEM containing 10% fetal bovine serum for 3 to 4 days, replace the culture medium, the adherent cells are the primary gerbil muscle cells, the micrograph of the isolated primary gerbil muscle cells is shown in fi...
Embodiment 2
[0024] Example 2 Isolation of attenuated strain 157-11-2
[0025] After the primary cells of the gerbil muscle in Example 1 were covered with the culture flask, the culture medium was discarded, and EV71 virus (Genbank ID: KX639820) was inoculated, and the culture bottle was gently shaken to make the virus contact with the primary muscle cells evenly, and then discarded after adsorption at 37°C for 2 hours. Virus liquid, add MEM culture medium containing 3% serum and cultivate for 72-96h, after more than 50% of the cells have pathological changes (expressed as cell shrinkage, rounding and shedding) ( figure 1 In middle B), collect the culture medium, freeze and thaw repeatedly 3 times, and centrifuge at 1000-2000×g for 20 minutes. The supernatant is the EV71 virus solution. The EV71 virus solution is inoculated by a 35°C low-temperature continuous culture method, and repeatedly passaged for 10-20 generations. The attenuated strain 157-11-2 was isolated by plaque method, and th...
Embodiment 3
[0030] Example 3 Determination of the Nucleotide Sequence of the Attenuated Strain 157-11-2 Structural Protein
[0031] The Qiagen RNeasy Mini Kit kit (purchased from QIAGEN Company) extracted the total RNA of the attenuated strain 157-11-2, and the experimental steps were as follows according to the method provided by the kit:
[0032] a) Take 200 μl of virus liquid, add 600 μl of RLT, and vibrate with an oscillator for 1 min.
[0033] b) Add 600 μl of pre-cooled -20°C absolute ethanol and mix well.
[0034] c) Add about 600 μl of the mixed solution to the separation column in two times, centrifuge at 12,000 rpm for 30 s, and discard the filtrate;
[0035] d) Wash the column with 700 μl RW1 washing solution, and centrifuge at 12000 rpm for 30 seconds;
[0036] e) Take 500 μl of RPE wash solution to wash the column, and centrifuge at 12000rpm for 30s;
[0037] f) Take 500 μl RPE and wash again, centrifuge at 12000 rpm for 30 seconds;
[0038] g) The column is vacated at 12...
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