Mutant strain of coxsackie group a virus type 4 and its application
A technology of coxsackie virus and mutant strains, applied in the field of medicine and biology, to achieve the effect of strong growth ability
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Embodiment 1
[0042] Embodiment 1: Breeding of CV-A4-M14 mouse adapted strain
[0043] 1. Cultivate the CV-A4-3179R4 / XY / CHN / 2017 virus strain on RD cells, and adjust the virus titer on RD cells to 1x10 according to the Reed-Muench method 6 TCID 50 / mL (unless otherwise specified, the viral load during the infection process is consistent with this below).
[0044] 2. Infect 10 3-day-old SPF Kunming mice with the virus strain by intracranial injection, 20 μL / mouse.
[0045] 3. Take the brain of a mouse that is on the verge of death, weigh it, prepare a 10% (W / V) homogenate, centrifuge to harvest the supernatant, name it CV-A4-M3, and store it at -20°C.
[0046] 4. Continue to infect 7-day-old suckling mice with the harvested virus according to the same infection method, 30 μL / mouse, and the harvested 7-day-old mouse brain homogenate (named CV-A4-M7).
[0047] 5. Infect 10-day-old suckling mice with the 7-day-old rat brain homogenate (named after CV-A4-M7) harvested at 30 μL / , and take the ...
Embodiment 2
[0052] Embodiment 2: CV-A4-M14-RP3 strain purification
[0053] 1. Plaque purification of CV-A4-M14-RP3 virus clone
[0054] (1) Carry out 10-fold serial dilution of CV-A4-M14-RP3 venom, the dilution range is 10 -1 ~10 -6 .
[0055] (2) Take a 6-well plate with RD cell confluence of 90%-95%, add 200 μL / well of diluted virus suspension, and incubate for 2 hours.
[0056] (3) After incubation, discard the virus solution. After the pre-prepared 2 × low melting point agarose is melted, mix it with 2 × MEM virus maintenance solution 1:1 and spread it in a 6-well plate, 2 mL / well.
[0057] (4) Microscopically check the CPE condition of each well, pick a CPE-positive clone from the well corresponding to the maximum dilution, suspend the virus in 300 μL virus maintenance solution, and blow and mix well.
[0058] (5) Take 100 μL of the harvest solution for ten-fold dilution, and the dilution range is 10 -1 ~10 -4 , repeat the above steps for three consecutive plaque purification...
Embodiment 3
[0064] Example 3: CV-A4-M14-613131 and CV-A4-M14-623131 Whole Genome Sequence Determination
[0065] 1. Viral RNA extraction
[0066] Use the Sangon Bioengineering (Shanghai) Co., Ltd. Column Viral RNA Extraction and Purification Kit (Product No.: B518667) to extract and purify viral RNA.
[0067] 2. cDNA synthesis by reverse transcription
[0068] Reverse transcription synthesis cDNA reaction system is shown in Table 6 and Table 7
[0069] (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.
[0070] (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.
[0071] Table 6
[0072]
[0073]
[0074] 3. PCR amplification
[0075] (1) Amplification primers: See Table 8 for CV-A4 full sequence determination and extension primers.
[0076] Table 8: CV-A4 whole genome seque...
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