Method for establishing goose embryo epithelial cell line and established goose embryo epithelial cell line

A technology for epithelial cells and goose embryos, which is applied in the field of established goose embryo epithelial cell lines to achieve the effects of high purity, high success rate and less restriction

Active Publication Date: 2014-11-19
SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no commercially available goose-derived cell line

Method used

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  • Method for establishing goose embryo epithelial cell line and established goose embryo epithelial cell line
  • Method for establishing goose embryo epithelial cell line and established goose embryo epithelial cell line
  • Method for establishing goose embryo epithelial cell line and established goose embryo epithelial cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Establishment of Goose Embryo Epithelial Cell Line

[0079] Establish the goose embryo epithelial cell line of the present invention according to the following steps:

[0080] (1) Aseptically take 11-13-day-old goose embryo tissue, cut off the head, limbs and internal organs with sterilized scissors, put it into a 10mL glass beaker, add 10mL PBS, and wash for 5-10 minutes; discard; After removing the PBS, add 10-20 mL of gentamicin solution prepared with PBS with a concentration of 20% to 70% by mass for 5-10 minutes;

[0081] (2) Take out the duck embryo tissue and cut it into 0.5~1.5mm3 tissue blocks. Place the tissue blocks flat on the bottom of the 6-well culture plate, one piece for each hole, and add 2~3mL to the culture hole containing 10% fetal calf by volume. Serum, 1%-2% goose serum and 0.2%-1% goose embryo allantoic fluid in DMEM culture medium, cultured at 37℃ and 5% CO2 until the tissue block is completely attached to the wall, then add the above DMEM m...

Embodiment 2

[0092] Example 2 Analysis of biological characteristics of goose embryo epithelial cell line

[0093] 1. Morphological observation

[0094] Observed by an inverted microscope, the established goose embryo epithelial cell line F30 generation ( Figure 4 ) And F50 generation ( Figure 5 ) And primary cells ( figure 1 ) Compared to the morphology, the primary cells contain a variety of heterogeneous cells, most of the cells are long spindle-shaped, a few are polygonal and oval, and the established epithelial cell line is obtained from a single cell, its purity Above 99.9%, they are all epithelioid cells. HE staining also shows that the cells are polygonal or short spindle-shaped epithelioid cells ( Image 6 ), with a round nucleus, cell growth and division ability is very strong, population doubling time is only 17.1h.

[0095] 2. Growth curve determination

[0096] Take the 30 generations and 50 generations of the cells to be tested in a good growth state, and when they are close to con...

Embodiment 3

[0102] Example 3 Verification of the sensitivity of goose embryo epithelial cell line to Muscovy Duck Parvovirus

[0103] 1. Method

[0104] Take 50th passage cells of goose embryo epithelial cell line, aspirate and discard the culture medium when it grows to 80% monolayer, wash twice with D-Hanks medium, inoculate Muscovy parvovirus, absorb at 37°C for 1 hour, discard the virus solution And supplemented with DMEM maintenance solution (containing 1% newborn calf serum) culture, and set up a blank control (goose embryo epithelial cell line not inoculated with Muscovy Duck Parvovirus). The same method is used to inoculate gosling plague virus, type I duck hepatitis virus, and new duck hepatitis virus.

[0105] 2. Results

[0106] It was observed that the goose embryo epithelial cells were sensitive to Muscovy Duck Parvovirus, and the infected cells within 96 hours all produced unique cytopathic changes, see Picture 11 ; While the control cell morphology is normal, see Picture 10 .

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Abstract

The invention discloses a method for establishing goose embryo epithelial cell line and the established goose embryo epithelial cell line. The invention relates to the method for establishing the goose embryo epithelial cell line, which is characterized in that a primary goose tissue adherent method, a differential velocity enzymatic digestion and a monoclonal screening method are combined, so that primary culture condition can be optimized. The method has the advantages of simple operation process, and convenient popularization and application. The invention also relates to the goose embryo epithelial cell line established by the method, a preservation number of the goose embryo epithelial cell line is CCTCC NO: C2014137, The establishment of the goose embryo epithelial cell line solves the problem of no well-established goose source cell line in prior art. The invention also relates to a kit used for culturing and/or proliferating goose parvovirus, muscovy duck parvovirus and type I duck hepatitis virus and novel duck hepatitis virus, and is characterized in that a host cell to be infected is/or comprises the goose embryo epithelial cell line. The method for establishing the goose embryo epithelial cell line verifies the sensitive characteristic of the goose parvovirus, muscovy duck parvovirus and type I duck hepatitis virus and novel duck hepatitis virus.

Description

Technical field [0001] The invention belongs to the technical field of animal cell engineering, and specifically relates to a method for establishing a goose embryo epithelial cell line and the established goose embryo epithelial cell line. Background technique [0002] my country is a major producer of waterfowl, and the output of waterfowl accounts for more than 60% of the world's total, among which the output of geese accounts for more than 90% of the world's total output. Our country has a long history of raising geese. According to research, geese have been domesticated as early as the Neolithic Age about 6000 years ago. With the development of my country’s economy and the continuous improvement of people’s living standards, the increase in the proportion of animal food consumption and the improvement of consumption habits have promoted the adjustment of the agricultural industry structure and the improvement of the production status of animal husbandry. The goose industry h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12N7/00C12R1/91
Inventor 王文秀沈志强于金枝
Owner SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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