Dairy cattle mammary epithelial cell immortalizing method

Abstract

The invention provides a dairy cattle mammary epithelial cell immortalizing method. The dairy cattle mammary epithelial cell immortalizing method is characterized in that when primary generation dairy cattle mammary epithelial cells are infected through HPV E6 and E7 viruses to be immortalized, pantropic and facultative virus particles are added, and thus the virus infection efficiency is improved. The invention further provides a method for establishing an immortalized dairy cattle mammary epithelial cell line. The method for establishing the immortalized dairy cattle mammary epithelial cell line comprises the following steps that firstly, dairy cattle mammary tissue is subjected to tissue block primary generation culture in a culture bottle; secondly, the primary generation dairy cattle mammary epithelial cells are infected through the virus method, and the primary generation dairy cattle mammary epithelial cells are immortalized; thirdly, positive clone is selected, and the cell line is cultured. By means of the methods, the dairy cattle mammary epithelial cells can be efficiently immortalized, the dairy cattle mammary epithelial cell line high in growth speed and capable of achieving stable continuous passage is established, the dairy cattle mammary epithelial cells with the physiological function are obtained, and experimental cell materials are provided for researching control over lactation nutrition of the dairy cattle mammary epithelial cells and the pathogenesis of diary cattle mastitis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for primary cell culture, monoclonal cell acquisition, western blot detection of the expression of E6 and E7 viruses in cow mammary gland epithelial cells and establishment of dairy cow mammary gland epithelial cell lines. Background technique [0002] Bovine mammary epithelial cells (BMEC, Bovine mammary epithelial cell) have the function of synthesizing and secreting milk protein, and have a defense effect against microorganisms invading mammary tissue. At the same time, it is an important response cell of breast tissue. Therefore, primary culture and cell immortalization of dairy cow mammary gland epithelial cells are regarded as important cell models for the study of nutritional regulation of dairy cow lactation and the pathogenesis of dairy cow mastitis. [0003] At home and abroad, the primary cultured dairy cow mammary gland epithelial cells are basically isolated and ...

AI Technical Summary

Problems solved by technology

However, in the process of subculture using dairy cow mammary epithelial cell lines, as the number of cell passages increases, dairy cow mammary gland epithelial cell lines will lose the physiological functions of mammary gland tissue, such as: chromosome karyotype changes, presenting haploid or polyploid body

Changes in the number of chromosomes will cause errors in experimental data, and even make some dairy cow mammary epithelial cell lines unable to express milk protein at all, thus losing the significance of using dairy cow mammary epithelial cells to study lactation nutrition regulation and lactation mechanism

However, there are no reports that HPVE6 and E7 can immortalize dairy cow mammary epithelial cells

Therefore, applying the above-mentioned immortalization method of human epithelial cells to dairy cow mammary gland epithelial cells has the following difficulties and doubts: 1) when the target gene is transduced into dairy cow mammary gland epithelial cells by virus method, the envelope on the viral skeleton Whether the glycoprotein Vsvg can bind the lipids of dairy cow mammary gland epithelial cells, although in the current research, Vsvg protein can bind most of the lipids on human or mouse epithelial cell membranes, but it has not been found that it can bind lipids on dairy cow mammary gland epithelial cell membranes Report; 2) Due to the low titer of the virus, coupled with the characteristics of the primary dairy cow mammary epithelial cells that "cannot be quickly separated, resulting in slow cell reproduction", the efficiency of infecting cells will be low

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