A kind of attenuated strain of respiratory syncytial virus and its application
A technology of syncytial virus and respiratory tract, applied in the field of attenuated strains of respiratory syncytial virus, can solve the problems of over-attenuated virulence, attenuation, and insufficient attenuation, and achieve good immunogenicity, effectiveness assurance, and reduced The effect of production costs
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Embodiment 1
[0027] Example 1: Vero cell culture
[0028] Take out Vero cells (purchased from ATCC, USA, No. CCL-81) from liquid nitrogen and quickly put them into hot water at 37°C to melt the cell suspension quickly; then add MEM or DMEM containing 10% newborn bovine serum (purchased at GIBCO Invitrogen Corporation) culture solution, adjust the pH to 7.2 and incubate at 37°C for about 4 h, discard all the culture solution, add fresh culture solution to continue the culture, and after growing into a dense monolayer, wash the cells with PBS (purchased from GIBCO Invitrogen Corporation). Digest with trypsin-EDTA (purchased from GIBCO Invitrogen Corporation), add the above culture medium, and subculture the cells at a split ratio of 1:2 to 1:4 until enough cells are obtained for seeding, such as figure 1 Shown are normal Vero cells.
Embodiment 2
[0029] Example 2: Isolation and Identification of Respiratory Syncytial Virus RSV
[0030] Take 1ml of sputum samples from 20 children with pneumonia obtained from the First People’s Hospital of Yunnan Province, suspend them in PBS to 20ml, mix well, filter and sterilize with a 0.22μm filter membrane, inoculate Vero cells with 1ml of the obtained filtrate, and incubate at 37°C , use maintenance solution (maintenance solution: MEM or DMEM culture medium with 2% newborn bovine serum, 3% NaHCO 3 ) culture, screen out a strain of RSV that has syncytia, proliferates, and has the fastest pathological changes, such as figure 2 shown.
[0031]Take 200 μL of the obtained virus strain, put it into a 1.5mLEP tube treated with DEPC (purchased from Shanghai Haoran Biotechnology Co., Ltd.), and then add 800 μL of Tripure reagent (purchased from Tiangen Biochemical Technology Co., Ltd. (Beijing)), vigorously After oscillating and standing still, 100 μL of chloroform (purchased from Chongq...
Embodiment 3
[0033] Embodiment 3: the adaptive attenuation culture process of RSV
[0034] The RSV strain KM516-WT harvested above was passed to the cells (Vero cells) in Example 1 for 5 generations at 37°C, 10 generations at 32°C, 15 generations at 28°C, and 10 generations at 25°C, and finally obtained KM516–AS strain; then passage Vero cells to 35cm 2 In the culture flask, when the cells grow to 70-80% of the monolayer, discard the supernatant medium, add 0.5ml respiratory syncytial virus attenuated KM516-AS strain and 0.5ml diluent (diluent: to the non-bovine serum Add 1% NaHCO to the MEM or DMEM culture medium 3 ), incubated at a constant temperature of 25°C, shaking once every 30min, and added 10ml of maintenance solution after 2h and incubated at a constant temperature of 25°C. Such as figure 2 As shown, when the cell lesion area is above 80%, discard the supernatant, add 2ml of PBS, freeze and thaw repeatedly to make the cells fall off completely, harvest the virus, take 0.5ml t...
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