Lamb testis support cell immortalized cell line and establishment method and application thereof

A technology for Sertoli cells and immortalized cell lines, applied in the field of cell engineering, can solve the problems of complex manufacturing process, difficulty in obtaining profits for enterprises, and high production costs, and achieve the effects of reducing production costs, shortening production cycles, and simplifying production processes

Active Publication Date: 2019-06-04
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Goat pox virus (GTPV, SPPV) can grow on sheep testicular primary cells, kidney cells, BHK21 and Vero cells, and produce cytopathic changes (CPE), but the virus titer is low, especially in passage cells (such as BHK21, Vero cells) ) The virus liquid prepared cannot reach the virus titer required for vaccine preparation
In China, the attenuated sheeppox vaccine is produced by primary cells of newborn sheep testis, and its manufacturing process is complex and backward. The attenuated sheeppox antigen must be produced by primary cells of newborn sheep testis, but the preparation of such cells is complicated and the production cost is too high , the production efficiency is low, which brings inconvenience to the large-scale production of sheep pox vaccine for vaccine factories, and it is difficult for enterprises to obtain huge profits
At the same time, the variety of newborn sheep testes used to breed sheep pox attenuated antigen each time is different, which makes it difficult to maintain the quality of the attenuated vaccine produced. In addition, newborn sheep testes have the potential risk of carrying other pathogens

Method used

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  • Lamb testis support cell immortalized cell line and establishment method and application thereof
  • Lamb testis support cell immortalized cell line and establishment method and application thereof
  • Lamb testis support cell immortalized cell line and establishment method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] [Example 1] Isolation, purification and cultivation of lamb Sertoli cells (LSC)

[0035] 1.1 Isolation, purification and culture of lamb testis Sertoli cells (LSC)

[0036] Select healthy male lambs born to healthy ewes (whose physique is healthy and negative for foot-and-mouth disease, brucellosis, and tuberculosis), and collect the scrotum after slaughter (the root of the scrotum is ligated, and the outside of the scrotum is sterilized with 75% alcohol), in an incubator, for 2 hours sent back to the laboratory. The testicular parenchyma was aseptically collected, and the lamb testicular primary cells were prepared by digestion with collagenase and trypsin mixed enzymes (0.1wt% type IV collagenase (GIBCO), 0.25wt% trypsin (GIBCO)).

[0037] Using 6h differential speed attachment, 50mmol / L Tris_HCl, the cultured lamb testis primary cells were subjected to hypotonic treatment, 38.5°C high temperature culture, separation and purification to obtain lamb testis Sertoli cel...

Embodiment 2

[0045] [Example 2] Establishment of Lamb Sertoli Cell Immortalized Cell Line (hTERT-LSC)

[0046] 2.1 Transfection of LSC cells with pCI-neo-hTERT plasmid

[0047] Example 1 was isolated and purified from lamb testis primary cells to obtain lamb testis Sertoli cells. According to the requirements of the Lipofectamine 2000Regent transfection instructions of Invitrogen Company, the eukaryotic expression plasmid pCI-neo-hTERT (vector map Such as Figure 4 (shown) was introduced into LSC (17th passage) to immortalize the LSC passage-limited cell line.

[0048] 2.2 Determination of optimal screening concentration of neomycin (G418)

[0049] Prepare LSC (17th passage) cells into a cell suspension at 1×10 5 cells / mL were inoculated into 24-well plates. Add G418 at a gradient of 50 μg / mL in the range of 100 μg / mL to 1 000 μg / mL, and add different concentrations of screening medium to each well. Four replicate wells were made for each gradient. According to the growth of the cell...

Embodiment 3

[0056] [Example 3] hTERT-LSC Biological Characteristic Measurement

[0057] 3.1 Growth characteristics of hTERT-LSC monoclonal cells and virus proliferation

[0058] The hTERT-LSC monoclonal cell strains 1#~16# screened in Example 2 were respectively inoculated into 6-well plates, and the seed separation rate (referring to how many cells of the same volume can be inoculated from the primary cells in 1 bottle of cell bottles) Subculture in the flask), the time for the cells to grow into a monolayer; then inoculate the hTERT-LSC monoclonal cell lines 1#~16# that are covered with a monolayer with GTPV cell virulence (lgTCID 50 ≥6.0), observe the CPE produced by the virus on the cells, the results are shown in Table 2.

[0059] Table 2 Growth characteristics of hTERT-LSC monoclonal cells and virus proliferation

[0060]

[0061]It can be seen from Table 2 that the growth characteristics of each monoclonal cell line of hTERT-LSC are stable, the growth rate and cell division ra...

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Abstract

The invention discloses a lamb testis support cell immortalized cell line and an establishment method and application thereof. The lamb testis support cell immortalized cell line is named a lamb testis support cell immortalized cell line hTERT-LSC with a collection number CCTCC NO: C2018202. Tests find that the hTERT-LSC cell line can realize continuous passage for more than 60 generations, and the growth and proliferation characteristics are not changed. The cell line is relatively sensitive to inoculation of the sheep pox virus, can efficiently proliferate the sheep pox virus, and can ensurethe uniformity and stability of the virus. The titer of the sheep pox virus proliferated by the cell line is equivalent to that of the lamb testis support cell LSC, and the cell division rate is higher than that of the LSC and can reach 1:3. Furthermore, by utilizing the immortalized cell line for preparing a vaccine, the production process is simplified, the production cycle is shortened, the production cost is reduced, and simultaneously, the quality stability of a prepared sheep pox virus vaccine is also ensured. Therefore, the invention provides a new technical means for large-scale production of the sheep pox virus vaccine.

Description

technical field [0001] The invention relates to an immortalized cell line and its establishment method and application, in particular to an immortalized cell line of lamb testicular Sertoli cells, its establishment method and its application in the propagation and cultivation of sheep pox virus. The invention belongs to the technical field of cell engineering. Background technique [0002] Sertoli cells (Sertoli cells, SC) are epithelial cells on the basement membrane of the seminiferous tubules, which provide support, energy and nutrition for the differentiation and maturation of germ cells, and are called "nanny cells" of germ cells. In the process of sperm differentiation and maturation, Sertoli cells not only deliver gonadotropins to the seminiferous epithelium, control a series of dynamic processes of mitosis, meiosis and subsequent differentiation of spermatogenic cells, but also support, nourish, phagocytize, and release sperm cells. , secretion and other functions. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N7/00C12R1/91
Inventor 张志东王光祥王艳华秦晓东李彦敏杨能能贾宁苏雅杰
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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