Coxsackie group A type-2 virus mutant strain and application thereof
A type 2 virus and mutant strain technology, applied in the direction of positive-sense single-stranded RNA viruses, viruses, antiviral agents, etc., can solve the problems of lack of screening and evaluation of CVA2-infected mouse models, and achieve strong growth ability and obvious infection effect
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Embodiment 1
[0032] Embodiment 1: Breeding of CVA2-M14 mouse adapted strain
[0033] (1) CVA2-1388R4 / XY / CHN / 2017 virus strain was cultivated in RD cells, and the virus titer was determined to be 3x10 on RD cells according to the Reed-Muench method 7 TCID 50 / ml.
[0034] (2) Infect 10 one-day-old SPF Kunming rat suckling mice with the virus strain by intracranial injection, 30 μL / mouse. Take the mice that are on the verge of death and carry out experimental operations in a biological safety cabinet. Dissect the mice and take out the mouse brain and other tissues and organs to weigh. Use a homogenizer to grind the tissue and finally prepare a homogenate, with a mass-volume ratio of 10%. Add MEM maintenance solution, centrifuge at 12000×g for 10 minutes, and take the supernatant and store it at -20°C.
[0035] (3) Continue to infect one-day-old suckling mice with the harvested virus according to the same infection method, and repeat three times.
[0036] (4) The same method was used to i...
Embodiment 2
[0039] Embodiment 2: CVA2-M14 virus strain purification
[0040] 1. Plaque purification of CVA2-M14 virus clone
[0041] (1) Perform a 10-fold serial dilution of the CVA2-M14 virus solution, and the dilution range is 10 -1 ~10 -6 .
[0042] (2) Take a 6-well plate with RD cell confluence of 90%-95%, add 200 μL / well of each gradient of diluted virus suspension, and incubate for 2 hours.
[0043] (3) After incubation, discard the virus solution. After the previously configured 2X low-melting point agarose is melted, mix it with 2X MEM virus maintenance solution 1:1 and spread it in a 6-well plate, 2ml / well.
[0044] (4) Microscopically check the cytopathic effect (CPE) of each well, pick the CPE-positive clones from the well corresponding to the maximum dilution, suspend the virus in 300 μL MEM maintenance solution, and blow and mix well.
[0045] (5) Take 100 μL of the virus mixed suspension in (4) above and perform a ten-fold dilution, with a dilution range of 10 -1 ~10 ...
Embodiment 3
[0051] Example 3: CVA2-M14-411 and CVA2-M14-111 Whole Genome Sequence Determination
[0052] 1. Viral RNA extraction
[0053] Use the Sangon Bioengineering (Shanghai) Co., Ltd. Column Viral RNA Extraction and Purification Kit (Product No.: B518667) to extract and purify viral RNA.
[0054] 2. cDNA synthesis by reverse transcription
[0055] Reverse transcription synthesis cDNA reaction system is shown in Table 6 and Table 7
[0056] (1) Add the reagents in the PCR tube as shown in Table 6, mix well, place in the PCR instrument, react at 65°C for 5min, and then quickly place it on ice to quench.
[0057] Table 6
[0058]
[0059] (2) Add reagents as shown in Table 7 to the above reaction solution, mix well and place in PCR instrument, 30°C, 15min; 42°C, 60min; 70°C, 15min.
[0060] Table 7
[0061]
[0062] 3. PCR amplification
[0063] (1) Amplification primers: See Table 8 for the full sequence determination and extension primers of CVA2.
[0064] (2) PCR amplifi...
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