Lamb sheep sustentacular cells as well as separation method and application thereof

A technology of testicular Sertoli cells and separation methods, applied in the separation method and application of the cells, in the field of lamb testicular Sertoli cells, can solve the problems of difficult consistency between batches, less neutralizing antibodies, and small production volumes within batches, etc. Achieve the effects of saving time for sorting, reducing production costs, and increasing the number of splits

Pending Publication Date: 2021-07-20
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

[0003] The preparation of vaccines for the prevention of oral ulcers mainly includes the following aspects: first, through continuous separation and cultivation of primary cells of cattle or sheep, the oral oral ulcer virus is isolated from them, and then continuously passaged for attenuation to prepare attenuated vaccines; second, the current There is also a trend of research and development of inactivated vaccines; third, because the surface of the sheep oral ulcer virus is covered with a capsule shaped like a small tube, which is arranged in a criss-cross pattern to cover its epitope, resulting in less neutralizing antibodies produced in the sheep, and the protection rate for sheep In view of the particularity of this immunology, some researchers used ultrasound and other means to destroy the capsule of ORFV and then prepare a mouth sore vaccine. However, the above situations are limited to laboratory research and have not been used in production large scale use
The main reasons are: the separation of primary cells is time-consuming, laborious and expensive, the number of passages of primary cells is small, the toxicity of the cultured aphthus virus is low, the production volume within a batch is small, and the repeatability between batches is difficult to be consistent due to differences in primary cells

Method used

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  • Lamb sheep sustentacular cells as well as separation method and application thereof
  • Lamb sheep sustentacular cells as well as separation method and application thereof
  • Lamb sheep sustentacular cells as well as separation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Separation and optimization of Sertoli cells of lamb testis

[0030] Routinely aseptically take out the lamb testis, carefully remove the outer membrane, wash with pH 7.0, 0.05M PBS buffer several times, cut into pieces aseptically, first digest with 0.25% trypsin at 37°C for 2 hours, and then digest with type IV collagenase 0.5-1h, add 1% serum to stop the digestion, centrifuge at 1000rpm for 15min, resuspend the pellet with digestion solution containing 0.05% trypsin and 0.02% EDTA, incubate the resuspension at 37°C for 10-30min to disperse the cells and filter, then use the filtrate in sequence Filter with one, two, or four layers of gauze to remove large pieces of undigested tissue and non-cellular components. The digestion step is very critical. If the digestion time is too short, the tissues and cells cannot be digested. If the time is too long, the cell membrane structure will be destroyed, and then 100 Mesh, 200 mesh copper mesh filter, the filtrate was centrifu...

Embodiment 2

[0034] Immortalization and screening of SCST cells

[0035] As described in Example 1, although SCST cells can increase the number of passages, the culture of SCST cells requires imported fetal bovine serum, which is expensive and limited in generation, and cannot be used for large-scale production. Therefore, the SCST cells were transfected with the telomerase gene (SCST-D for short) to make them immortalized and can be permanently passaged. Instead, use low-concentration (200ug / ml) G418 to screen first, so that untransfected fragile cells are purified first, and then use G418 (200ug / ml) medium to subseed after the cells are full, so that The fragile cells after transfection were also purified. After a long period of culture, a very small number of cells with strong vitality divided and proliferated to form several clone islands, and then were screened for resistance with high concentration (400 ug / ml) G418. This time, Most of the cells were killed again, and then after a lo...

Embodiment 3

[0037] Karyotype and Chromosome Identification of Subcloned Cells

[0038] According to the conventional karyotype experiment, 0.5-0.7ug / ml colchicine was used to make the subcloned cells in Example 2 be in metaphase, then fixed with glacial acetic acid, and Giemsa stained. The results show that the subcloned cells of the present invention are normal somatic cells, without any aberration of chromosome number and structure, showing safety such as anchorage dependence and contact inhibition, 2n=54 (see image 3 ), it can be seen that its genetic traits have not changed after immortalization.

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Abstract

The invention discloses a lamb sheep sustentacular cell as well as a separation method and application thereof, and the preservation number of the lamb sheep sustentacular cell SCST is CCTCC No.C2019203. The SCST cell disclosed by the invention can be directly used for large-scale propagation of the sheep orf virus; or transfecting a telomerase gene to immortalize the telomerase gene, then carrying out pressure coarse screening, pressure seed separation and continuous passage to obtain a subclone cell line, and the subclone cell line is applied to large-scale breeding of the orf virus. The subclone cell line can be cultured by a culture medium containing domestic serum, so that the production cost is reduced, and the subclone cell line is suitable for large-scale production and application. Compared with conventional primary cells, the subclone cell line provided by the invention has the advantages that the separation rate of the orf virus can be obviously improved, the pathogenesis time of the orf virus is obviously shortened, the copy number of the separated virus is improved by more than 100 times compared with that of the primary cells, thus providing a tool for large-scale propagation of the orf virus and research on a virus infection pathogenesis mechanism.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a lamb testicular Sertoli cell, and the invention also relates to a separation method and application of the cell. Background technique [0002] Contagious Ethyma (CE), commonly known as sheep mouth sore, is an acute and contagious infectious disease caused by sheep mouth ulcer virus (ORFvirus, ORFV) to which goats, sheep, and mainly lambs are susceptible. Zoonotic diseases that can infect humans and other animals. ORFV is mainly prevalent in Inner Mongolia, Tibet, Xinjiang, Yunnan, Gansu, Sichuan, Ningxia and other sheep-intensive areas. In recent years, it has frequently occurred in Ningxia, Inner Mongolia, Xinjiang, Tibet, Shandong, Hubei, Yunnan, Shaanxi, Heilongjiang, Qinghai and other regions . The typical symptoms of cauliflower-like hyperplasia will appear on the lips, oral mucosa, tongue and nostrils of affected sheep, which seriously affects their feeding and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/10C12N7/00C12R1/93
CPCC12N5/0683C12N7/00C12N2509/00C12N2509/10C12N2510/04C12N2710/24251
Inventor 吴锦艳田宏尚佑军杜国玉候俊玲兰喜曹小安何继军
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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