Method for improving Japanese encephalitis virus titer
A type of Japanese encephalitis virus, Japanese encephalitis technology, applied in the direction of antiviral agents, virus/phage, virus antigen components, etc., can solve the problems of long production cycle, clinical immune failure, large differences, etc.
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[0026] According to the method for preparing Japanese encephalitis vaccine of the present invention, since the freezing and thawing operation is omitted in the process of multiplying Japanese encephalitis virus, the titer of the harvested virus liquid is significantly improved, so it can be used to pass The Japanese encephalitis virus vaccine with high potency, stability and good immunogenicity was prepared by mixing vaccines. As an embodiment, the Japanese encephalitis vaccine is an inactivated vaccine and / or a live attenuated vaccine.
[0027] To sum up, the method of the present invention omits the freezing and thawing operation in the conventional method, and directly harvests the virus liquid without freezing and thawing after the cells reach the required lesion level, and mixes the virus liquid to prepare the Japanese encephalitis vaccine. In this way, on the one hand, higher quality and more stable Japanese encephalitis virus can be produced by significantly increasing ...
preparation Embodiment 1
[0031]Treatment of hamster kidneys: The collected 10-14-day-old hamster kidneys (commercially purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.) were treated with 500 ml of Chinese medicine containing 400 units / ml double antibody (commercially purchased from GIBCO brand). Wash twice with Kirschner's solution (commercially purchased from Gibco brand). One 10,000-mL cell bottle is suitable for the kidney tissue of 20 hamsters. Mince the tissue with scissors and cut evenly. Then rinse the tissue in the beaker repeatedly with Hank's solution containing 400 units / ml double antibody until the supernatant in the beaker is clear, and finally fully precipitate, and pour off the supernatant.
[0032] Trypsin digestion: use 7.5 mass% NaHCO3 to adjust the pH of trypsin to 7.2-7.4, and then pour the above precipitated tissue into the digestion bottle (aseptic operation). Add 0.25 mass% trypsin (commercially purchased from GIBCO brand) according to the volume ratio of try...
preparation Embodiment 2
[0037] Prepare according to the method identical with Preparation Example 1, difference is:
[0038] In the trypsin digestion step, the volume ratio of trypsin:hamster kidney tissue was 2.5:1, and the digestion was performed at 7°C for 12 hours.
[0039] In the step of cell dispersion culture, the volume ratio of Ruhan liquid and RPMI1640 is 1:0.9, and the cell concentration for culture is 2×10 8 number of cells / L.
[0040] During virus inoculation and harvesting, after inoculating JE seed virus at 0.8% by volume of the maintenance liquid, culture it at 37°C, and harvest the virus liquid when the cytopathic pathology reaches 100%.
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