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A natural passage cell line of lamb testis Sertoli cells and its application in the isolation, culture and propagation of sheep pox virus

A technology of testicular Sertoli cells and passaged cells, which is applied in the field of veterinary vaccine preparation, can solve the problems of complex manufacturing process, high production cost, and unreachable virus titer, and achieve simplified production process, reduced production cost, and stable quality Effect

Active Publication Date: 2019-07-26
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 in the primary cells of sheep testis, kidney cells, BHK 21 and Vero cells, and produce cytopathic (CPE), but the virus titer is low, especially passage cells (such as BHK 21 , 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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  • A natural passage cell line of lamb testis Sertoli cells and its application in the isolation, culture and propagation of sheep pox virus
  • A natural passage cell line of lamb testis Sertoli cells and its application in the isolation, culture and propagation of sheep pox virus
  • A natural passage cell line of lamb testis Sertoli cells and its application in the isolation, culture and propagation of sheep pox virus

Examples

Experimental program
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Effect test

Embodiment 1

[0028] [Example 1] Isolation, cultivation and identification of Sertoli cells of sheep testis

[0029] 1.1 Preparation of primary sheep testis cells

[0030]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. Use ophthalmic tweezers and scissors to cut the testicular parenchyma into small pieces of about 1mm×3mm, gently pipette several times in D-hanks solution, remove the supernatant, put the small tissue pieces into a 50mL centrifuge tube and add 10 times the volume of The digestion solution containing 0.1wt% type IV collagenase (GIBCO) and 0.25wt% trypsin (GIBCO) was digested at 4°C for 12h or overnight, and cultured with an equal volume of DMEM containing 10% (v / v) newborn bovine ser...

Embodiment 2

[0046] [Example 2] Proliferation of sheep pox virus

[0047] Goatpox virus can grow on tissue cells derived from cattle, sheep, and goats, among which primary or secondary lamb testicular cells (LT) and lamb kidney cells (LK) are the most sensitive; the disease material can appear as early as 3 days after infection after the cells are inoculated A small amount of cytopathic effect (CPE), after 4-6 days CPE rapidly spread to the entire cell layer, observe whether CPE appears and last up to 14 days. Typical CPE is cell rounding, cell aggregation in a fascicular structure, cell shrinkage or swelling, nuclei vacuolation, chromatin fragmentation, loss of coherence in the cell monolayer, and inclusion bodies in the cytoplasm (Adla et al., 1971; Joshi et al., 1994). In addition, sheeppox virus can also propagate on BHK-21 cells and Vero cells. Some strains also grow in fetal bovine muscle cells, calf kidney cells, and bovine kidney epithelial (MDBK) cell lines.

[0048] Therefore,...

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Abstract

The invention discloses a natural passage cell line for lamb testis sertoli cells and application of the natural passage cell line to separate culture and proliferation of goatpox viruses. The cell line disclosed by the invention is a lamb testis sertoli natural passage cell line which is obtained by separating and purifying primary cells of a lamb testis to obtain the lamb testis sertoli cells and performing natural passage on the lamb testis sertoli cells. A research shows that the cell line is relatively sensitive to inoculation of the goatpox viruses; the virus titer proliferated by the cell line is greater than that of the primary cells, Vero cells and BHK21 cells of the testis of a newborn lamb by about 1.5, 2.5 and 2.3 respectively. The cell line for separating the goatpox viruses can guarantee the uniformity and the stability of the viruses and prevent the risk that the separated viruses carry other pathogenies due to the fact that the primary cells are used for multiple times. Furthermore, by the use of the passage cell line for preparing vaccines, the production process is simplified, the production period is shortened, and the production cost is reduced; meanwhile, the quality of the vaccines are kept stable; the cell line has certain practical significance for large-scale production of the vaccines.

Description

technical field [0001] The present invention relates to a subcultured cell line used for sheeppox virus propagation and vaccine preparation and its preparation method and application, in particular to a lamb testicular Sertoli cell line obtained through natural subculture and the use of the cell line to proliferate and cultivate sheep Pox virus method. The invention belongs to the technical field of animal vaccine preparation. Background technique [0002] Capripox virus (CPV) is the most important animal poxvirus, belonging to the genus Capripoxvirus (Capripoxvirus) in the vertebrate poxvirus subfamily (Chordopoxrinae) of the family Poxviridae (Poxviridae) in taxonomic status, including goat Pox virus (Goatpox virus, GTPV), sheep pox virus (Sheep poxvirus, SPPV) and bovine nodular herpes virus (Lumpy skin disease virus, LSDV). Goatpox is an acute, contagious infectious disease caused by goatpox virus. It is listed as one of the 93 animal diseases that must be reported by ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N7/00A61K39/275A61P31/20C12R1/93
CPCA61K39/12C12N5/061C12N7/00C12N2509/00C12N2710/24021C12N2710/24034
Inventor 王光祥张志东尚佑军刘湘涛王艳华吴锦艳陈妍张克山田宏尹双辉
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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