Mass preparation of SARS virus capable of meeting the production of inactivated vaccine and inactivation method
A SARS virus and inactivation technology, applied in the field of SARS virus liquid, can solve problems such as danger
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Embodiment 1
[0032]Example 1 Preparation of Stromal Cell Scale Working Seed Batch
[0033] 1) Take one main seed of the human embryonic lung diploid cell 2BS strain that meets the vaccine production requirements, resuscitate routinely, passage to 24-30 passages, inoculate with CF10, digest in a single layer, make a cell suspension, and centrifuge (800- 1000rpm), discard the supernatant, and adjust the cell concentration to 10 after suspending the cells with the cell freezing medium 6 ~10 7 After / ml, add DMSO and mix evenly, and distribute into cryopreservation tubes, 1.0-2.0ml / tube.
[0034] 2) Take one branch of the main seed of human embryonic lung diploid cell MRC-5 strain that meets the requirements for vaccine production, routinely resuscitate, subculture to 24-30 generations, inoculate with CF10, digest in a single layer, make a cell suspension, and centrifuge ( 800~1000rpm), discard the supernatant, suspend the cells with cell freezing medium, adjust the cell concentration to 10 ...
Embodiment 2
[0037] Example 2 Preparation of Working Cell Batches of Stromal Cells
[0038] 1) Preparation of working cell batches of human embryonic lung diploid cells 2BS strain
[0039] Take one working seed of the human embryonic lung diploid cell 2BS strain described in Example 1, resuscitate through the conventional cell resuscitation process as shown in Figure 2, and conventionally (1:2~1:4) for 4 to 5 consecutive passages , spread to 8-16 10-layer (or 2-4 40-layer) cell factories (cultivation area up to 50560-101120cm 2 / batch, equivalent to 40~80 3000ml cell culture spinner bottles), used for inoculating SARS virus.
[0040] 2) Preparation of working cell batches of human embryonic lung diploid cells MRC-5 strain
[0041] Take 1 working seed of the human embryonic lung diploid cell MRC-5 strain described in Example 1, resuscitate through the conventional cell resuscitation process as shown in Figure 2, and conventionally (1:2~1:4) for 4 to 4 consecutive passages. 5 times, sprea...
Embodiment 3
[0044] Example 3 Conventional isolation method of SARS virus in clinical SARS patient specimen
[0045] Specific steps are as follows:
[0046] (1) Stool suspension (5%); and / or
[0047] (2) Throat swab 1-2ml (original times);
[0048] (3) Sterilization: After 0.22um filtration, add 10-20% PS and mix well, then overnight at 4°C;
[0049] (4) Inoculation: 25cm 2 After washing the monolayer cells, inoculate the sterilized sample (1-2 bottles, 1-2ml / bottle);
[0050] (5) Adsorption: Adsorb at 37°C for 1-4 hours, add maintenance solution to 10ml / bottle;
[0051] (6) Cultivation: constant temperature cultivation at 33-37°C;
[0052] (7) Observation: Directly observe cell lesions under a microscope, and record the earliest time, type and degree of lesions in detail;
[0053] (8) Harvest: Harvest the obtained virus samples in different ways according to the presence or absence of lesions:
[0054] a) No disease: If there is no disease after 3-5 consecutive generations of blind...
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