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Bovine testicle cell line as well as establishment method and application thereof

A bovine testicular cell line and a technology for establishing a method, which are applied in the field of animal cell engineering, can solve problems such as difficulty in meeting production needs, inconvenient production arrangements, and affecting production, and achieve the effects of cost reduction, fast growth, and easy cultivation

Active Publication Date: 2014-06-11
SHANDONG LVDU BIO SICIENCE & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for a long time, there have been many problems in the production of my country's swine fever rabbit testicular attenuated testicular cell vaccine: in the production process of swine fever calf testicular cell vaccine, because the newborn calf testicles eliminated by dairy farms are used as raw materials for production, The production of each batch is affected by the limitation of the number of donors. If only calf testes collected on the same day are used, it is often inconvenient for production arrangements due to the small number
[0004] With the rapid development of the pig industry, the demand for swine fever bovine testicular cell vaccines is increasing, and it is difficult to meet the needs of production by simply relying on the primary culture of calf testicular cells, so the establishment of bovine testicular cell lines is imminent
At the same time, primary bovine testicular cells need to be collected from calf testicular tissue for isolation, and its source is very limited

Method used

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  • Bovine testicle cell line as well as establishment method and application thereof
  • Bovine testicle cell line as well as establishment method and application thereof
  • Bovine testicle cell line as well as establishment method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Embodiment 1. The preparation of liquid DMEM medium

[0094] The liquid DMEM medium is self-prepared, and the preparation method is as follows:

[0095] (1) Prepare fresh triple-distilled water or millipore ultrapure water. The preparation steps of the millipore ultrapure water are as follows: Tap water is filtered through the first stage filter, then filtered through the second stage CTO activated carbon filter, and enters the third stage After being filtered by the security filter, it is processed by an ultra-clean high-pressure water pump, then filtered by RO reverse osmosis membrane, and finally ultra-pure water is obtained after multi-stage pure water column treatment;

[0096] (2) Add a pack of DMEM medium dry powder (purchased from Dulbeccos Modified Eagle Medium, Gibco Company) to about half of the three-distilled water; wash the inner surface of the packaging bag twice with water, and introduce it into the medium to ensure that all the dry powder is dissolved i...

Embodiment 2

[0100] Embodiment 2. Preparation and cultivation of bovine testis cell line

[0101] (1) Primary culture: Aseptically collect the testes of newborn calves, rinse with PBS 3 to 5 times to remove the adherent tissue, and cut into 0.5 to 2.0mm 3 The tissue blocks were digested and separated by trypsinization method to obtain high-viability primary bovine testicular cells; the isolated high-viability primary bovine testicular cells were added with complete DMEM medium, and counted according to 5000 cells / mL Inoculate in a 24-well plate (liquid DMEM medium containing 10% fetal bovine serum) at 37°C, 5% CO 2 cultured under conditions to obtain primary bovine testicular cells;

[0102] The complete DMEM medium is the DMEM medium in which 10% fetal bovine serum is added by volume to the liquid DMEM medium.

[0103](2) Transfection and screening: Take out the DH5a bacteria containing the pCI-neo-hTERT plasmid stored at -80°C, inoculate them in LB medium containing ampicillin, culture...

Embodiment 3 Embodiment 2

[0126] Embodiment 3. The effect verification of bovine testis cell line obtained in embodiment 2

[0127] The bovine testis cell line cultivated in Example 2 was checked for biological characteristics, and the methods and conclusions were as follows.

[0128] 1. Observation of cell morphology

[0129] Methods: During the in vitro culture of the cells, the cells were routinely inspected to observe the growth status of the cells, the color change of the medium and whether the cells were polluted.

[0130] Conclusion: if Figure 1 to Figure 4 As shown, when the cells were growing well, their morphology was fusiform or irregular triangle. Observing the overall view of the cell population, the cultured cells all showed radial, flame-like or swirl-like trends, which proved that the cells grew healthy and in good shape.

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Abstract

The invention provides a bovine testicle cell line and an establishment method thereof. The establishment method comprises the following main steps: (1) primary culture, namely collecting a bovine testicle of a newborn calf in a sterile manner, cleaning adhesive tissues, separating by virtue of a trypsinization method so as to obtain bovine testicle cells with high vitality, culturing the separated bovine testicle cells with high vitality in a DMEM (Dulbecco modified eagle medium), and culturing at 37 DEG C under the condition of 5% CO2 to obtain primary bovine testicle cells; (2) transfection and screening, namely extracting and purifying eukaryotic expression plasmids pCI-neo-hTERT containing hTERT genes, leading the extracted and purified eukaryotic expression plasmids pCI-neo-hTERT to the primarily cultured bovine testicle cells in the step (1) to perform transfection by adopting a lipidosome transfection method; (3) screening and enlarged culture. The bovine testicle cells provided by the invention are easy in culture and relatively fast in growth speed, can be stably inherited, are high in motility rate and purity, and can be well preserved; the method provided by the invention is simple and convenient in operation and convenient in popularization and application. The cell line can be used for producing hog cholera vaccines, sheep poxes, ecthyma vaccines and the like.

Description

technical field [0001] The invention belongs to the technical field of animal cell engineering and relates to a new cell line, in particular to a bovine testis cell line and its establishment method and application. Background technique [0002] Vaccination is the main preventive measure against swine fever. The use of heterologous animal cells to produce vaccines can prevent the virulence of the seed virus from returning to strength and avoid the use of homologous animals to cause other diseases to spread, such as bovine testis, sheep testis, sheep kidney and other primary cell proliferation CSFV C strain vaccines. [0003] Classical swine fever virus (CSFV) can proliferate in bovine testicular cells without causing cell pathology (CPE), and the proliferation of the virus can increase the number of passages and maintenance time of primary bovine testicular cells. However, for a long time, there have been many problems in the production of my country's swine fever rabbit te...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12R1/91
Inventor 王文秀沈志强管宇魏凤
Owner SHANDONG LVDU BIO SICIENCE & TECH
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