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Preparation method and application of a kind of immortalized duck embryo liver cell line

A technology for immortalization of duck embryo hepatocytes is applied in the field of preparation of duck embryo hepatocytes, which can solve the problems of inconspicuous cytopathic changes, high cost, large differences between batches, etc., and achieves simple culture conditions and high accuracy. , easy-to-control effects

Active Publication Date: 2017-12-29
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the isolation and culture of primary duck embryo liver cells must use duck embryos every time. The production procedure is cumbersome, the cost is high, it is easy to pollute, and the cell state is not easy to control.
In addition, it contains miscellaneous cells, the difference between batches is large, and the cytopathy is not obvious, so this method is difficult to promote

Method used

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  • Preparation method and application of a kind of immortalized duck embryo liver cell line
  • Preparation method and application of a kind of immortalized duck embryo liver cell line
  • Preparation method and application of a kind of immortalized duck embryo liver cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The construction of embodiment 1 duck embryonic liver cell line

[0033] 1. pCI-neo-hTERT eukaryotic expression system

[0034] pCI-neo-hTERT can be constructed by vector ligation or commercially purchased, and the pCI-neo-hTERT vector specifically used in the present invention is purchased from Addgene.

[0035] 2. Isolation and culture of duck embryonic liver cells

[0036] Aseptically take 15-day-old duck embryo liver, cut into about 1mm 3 The tissue pieces were washed three times with 37°C preheated PBS solution to remove blood stains. Add an appropriate amount of EDTA-trypsin solution (containing 0.05% trypsin and 0.02% EDTA, 10ml / embryo), digest at 37°C for 5min, and discard the supernatant. Add EDTA-trypsin solution again, add a magnetic stirring bar, put it on a magnetic stirrer, 120 rpm, stir and digest for 10 minutes. Collect the supernatant, centrifuge at 600g for 10min at 2-8°C, resuspend with M199 medium containing an appropriate amount of 15% serum, fi...

Embodiment 2

[0053] Example 2 Determination of Duck Hepatitis Virus Egg Yolk Antibody Titer with Duck Embryo Hepatocytes

[0054] 1. Preparation of refined yolk antibody against duck viral hepatitis

[0055] According to the conventional method, the refined egg yolk antibody of duck viral hepatitis is prepared, and the basic steps are as follows:

[0056] (1) adopt duck viral hepatitis DHAV-1 strain to inoculate 10 days old susceptible chicken embryo, then harvest allantoic fluid, formaldehyde inactivation and prepare vaccine;

[0057] (2) Use the prepared vaccine to immunize laying hens, and after immunization, take a sample to determine the neutralization titer of anti-DHAV-1 antigen antibody in the egg yolk of the chicken hyperimmune egg ≥ 1: 8192, and then collect the hyperimmune eggs of the chicken;

[0058] (3) Disinfect the eggshell of the high-free egg, collect the egg yolk, add an equal volume of distilled water to the egg yolk, stir and mix, and pasteurize at low temperature; pu...

Embodiment 3

[0071] The preparation of embodiment 3 duck viral hepatitis vaccines

[0072] After the transfected duck embryonic hepatocytes grow into a monolayer, discard the culture medium, inoculate the DHV-1-LY strain virus solution diluted 1:100 with sterilized PBS solution, absorb at 37°C for 1 hour, and discard the virus solution , add 10% M199 medium, put into CO 2 In the incubator, culture was continued at 37°C, and a control group was set at the same time. The cell changes were observed every day after inoculation. Compared with the control group, CPE appeared in the cells 24 hours after inoculation, and the lesion was obvious at 72 hours. The lesion cells showed vacuoles, netting phenomenon, and some cells were lysed. To collect poison and measure the price of poison.

[0073] DHV-1-LY strain virus seeds of duck hepatitis virus were diluted 1:100 with sterilized PBS solution, and 10-12 days old susceptible duck embryos were inoculated through the allantoic cavity, 0.2ml per emb...

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Abstract

The invention provides a method for preparing an immortalized duck embryo liver cell line, the method comprising: (1) digesting the duck embryo liver with a trypsin digestion method, cultivating the digestive juice, and obtaining primary cultured duck embryo liver cells; (2) Using liposome transfection method, the eukaryotic expression plasmids encoding hTERT and Marker genes were introduced into the primary cultured duck embryo liver cells; (3) The transfected duck embryo liver cells were cultured, and the G418 was used to screen the cells hTERT-positive clones; (4) culturing the screened hTERT-positive clones for more than 50 passages to obtain immortalized cell lines. The cell line still has good vigor after more than 50 passages in vitro, maintains division and proliferation, does not undergo senescence and apoptosis, and is an immortalized cell line. The immortalized duck embryo hepatocyte has regular shape, simple culture conditions, easy control and convenient use, and provides a good carrier for studying duck diseases, especially duck hepatitis virus.

Description

technical field [0001] The invention relates to a preparation method and application of an immortalized duck embryo liver cell line. Background technique [0002] Duck liver is the main digestive organ of ducks. Duck liver cells are the main functional cells of duck liver. Many duck pathogens can cause disease in duck liver cells. Therefore, the establishment of duck embryonic liver cell lines can provide a carrier for the study of duck diseases. Research on duck diseases at cellular level. [0003] Duck viral hepatitis is a rapidly spreading and highly fatal infectious disease of ducklings caused by duck hepatitis virus. Duck viral hepatitis yolk antibody can effectively prevent and treat this disease. At present, duck embryos are used to breed duck viral hepatitis virus in China. , and then make antigens, immunize laying ducks, and use duck embryos to breed duck viral hepatitis virus. The cost is high and the operation process is cumbersome. [0004] At present, the meth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10A61K39/29A61P31/14G01N33/569C12R1/91
Inventor 张许科孙进忠白朝勇
Owner PU LIKE BIO ENG
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