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Cell culture method for duck flavivirus

A technology of cell culture and duck yellow virus, applied in the direction of virus/bacteriophage, biochemical equipment and methods, microorganisms, etc., can solve the problems that cannot meet the needs of virus molecular biology research, fail to find cell culture methods, limit molecular pathogenic mechanisms and Genetic engineering vaccine research and other issues, to achieve the effect of an intuitive virus operation platform

Inactive Publication Date: 2011-10-19
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the flavivirus that caused a sudden drop in egg production in breeding (egg) ducks was reported for the first time in my country, only a part of the research on the biological characteristics and detection methods of the pathogen has been carried out so far. This technology cannot meet the needs of virus molecular biology research. At present, no suitable cell culture method has been found, which greatly limits the research on its molecular pathogenic mechanism and genetic engineering vaccine.

Method used

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  • Cell culture method for duck flavivirus
  • Cell culture method for duck flavivirus
  • Cell culture method for duck flavivirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, the adaptive passage of virus

[0026] 1. Cultivate BHK21 cells in a plastic culture dish with a diameter of 6 cm to form a monolayer.

[0027] 2. Inoculate the seed virus on BHK21 cells (ATCC Number: CCL-10), and place in 5% CO at 37°C 2 Incubate in the incubator for 45-60 minutes, and shake well every 10 minutes during this period.

[0028] 3. Wash the unadsorbed virus with sterile PBS buffer, discard the washing solution, and repeat 1-2 times.

[0029] 4. Add 12-15mL cell maintenance solution (DMEM (Gibco) containing 2-3% fetal bovine serum (Gibco) and 1% double antibody (Fermentas)) and place at 37°C, 5% CO 2 to cultivate.

[0030] 5. Observe twice a day, record the cell state and pathological changes, and observe continuously for 5 days.

[0031] 6. Freeze and thaw the cell culture 3 times on the 5th day or after serious cell lesions appear, take an appropriate amount of culture as seed poison for the next round of subculture, and repeat steps 1-6...

Embodiment 2

[0033] The RT-PCR identification of embodiment 2 virus

[0034] During the passaging process, the cell cultures of the 1st, 3rd, 5th, 7th, 9th, 11th, and 13th passages were selected, and duck flavivirus-specific detection primers (P1: 5'-ctt gca aaa cgt ggt aaa tat ggc-3 ';P2: 5'-tgt acc aaa tag ctc tac ta-3') was identified by RT-PCR. This primer can specifically amplify some gene fragments of duck yellow virus. The fragment size is 400bp. The operation steps refer to the literature (2) method.

[0035] 1. Extraction of viral RNA

[0036] Extraction of total RNA from cell culture was carried out according to the instructions of Trizol LS Reagent (Invtrogen). Specifically: Take 0.25ml of culture supernatant, add 0.75ml TRIZOL, mix well, place at 15-30°C for 5min, add 0.2ml of chloroform, shake the centrifuge tube for 15sec, place at room temperature for 2-5min, centrifuge at 2-8°C at no more than 12000g for 15min , transfer the upper liquid phase to another tube (don't suck...

Embodiment 3

[0051] Example 3 Growth Characteristics of Cell Adaptation Toxins

[0052] In order to understand the growth characteristics of the cell-adapted virus on BHK21 cells, the 8th generation cell culture was frozen and thawed three times and then used as seed virus to inoculate BHK21 cells, and the virus titers at different time points were measured (the results were expressed as TCID 50 Indicated), the determination method refers to literature (3), specifically:

[0053] After virus seeds were inoculated into BHK21 cells, 100ul of cell supernatants were taken at 0, 12, 24, 36, 48, 60, 72, 84, 96, 108 and 120 hours after inoculation respectively, and the virus content in cell supernatants at different time points was measured (with TCID 50 express).

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Abstract

The invention provides a cell culture method for duck flavivirus. The method comprises the following steps: inoculating to attach an anchorage-dependent kidney cell BHK21 of a baby hamster to the surface of a cell culture container to form a monolayer; inoculating the monolayer with virus; culturing the monolayer inoculated with the virus in an incubator with 5% CO2 at 37 DEG C; and continuously passing by taking a cell culture product as an inoculum for the next passage. According to the cell culture method provided by the invention, after the virus is continuously passed for 5-7 generations on the monolayer, stable multiplication can be obtained; and after the cell is inoculated for 3-4 days, an obvious cytopathic effect can be formed. The method provides a convenient and visual virus operating platform for studying the biological characteristics of the virus and the molecular basis of virus pathopoiesia and for the vaccine research developed for effectively preventing and controlling the disease.

Description

technical field [0001] The invention relates to a cell culture method of duck yellow virus, which is suitable for the isolation, culture and propagation of the virus, and belongs to the field of biotechnology. Background technique [0002] Since April 2010, there has been an outbreak of a disease of breeding ducks and laying ducks bred in Jiangsu, Zhejiang, Fujian, Shandong, Shanghai, Henan, Jiangxi, Hebei, Beijing and other regions in my country. The disease that has been reduced to the main feature has caused economic losses of up to 3 billion yuan to the duck industry in my country. Epidemiological surveys show that the virus mainly harms Cherry Valley breed ducks, muscovy duck females, various laying ducks, wild ducks, etc., especially for laying ducks. The incidence rate in the flock is almost 100%, and the fatality rate is 0-12%; the egg production rate of laying ducks drops rapidly from high egg production rate (such as 80%-95%) to 10%-30%, serious cases even stop pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00
Inventor 傅光华黄瑜施少华万春和程龙飞陈红梅林建生林芳
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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