A kind of bovine testis cell line and its establishment method and application

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 testicular cell line and its establishment method and application. Background technique [0002] Vaccine is the main preventive measure to prevent swine fever. The production of vaccines with heterologous animal cells can prevent the virulence of the species from returning to strong and avoid the use of homologous animals to cause other diseases to spread, such as the primary cell proliferation CSFVC strain vaccine such as cattle testis, goat testis, goat kidney, etc. [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 Chinese swine fever lapinized attenuat...

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

In order to meet the production needs, constant temperature storage is usually used in production, and the short-term low temperature (not constant temperature) often caused by the refrigeration mechanism of the refrigerator affects the production of testicular cell culture and vaccines

[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

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