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A bovine Sertoli cell cancer cell and its application in the isolation and culture of poxvirus

A technology of Sertoli cells and Sertoli cells, which is used in the separation and culture of newborn bovine Sertoli cells and cancer cells, cancer cells and their separation, and application fields in poxviruses, which can solve the problem of high production costs, difficulty for enterprises to obtain profits, Carrying other pathogens and other problems to achieve the effect of simplifying the production process, shortening the production cycle and reducing the production cost

Active Publication Date: 2021-11-30
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Abstract
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  • Application Information

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Problems solved by technology

ORFV can grow on skin cells, kidney cells of cattle and sheep, and testicular cells of calves and lambs, and produce cytopathic changes, but the virus titer is low. Ovine infectious impetigo virus has the advantages of convenient cell culture, high virus isolation efficiency, and high virus proliferation titer, but there are problems in obtaining the cells (sheep embryos are required), and a large number of animals are required to provide tissues in vaccine production.
[0007] In China, although the Veterinary Research Institute of Qinghai Academy of Animal Husbandry and Veterinary Sciences and the Gansu Provincial Institute of Animal Husbandry and Veterinary Medicine have developed an attenuated vaccine for impetigo contagiousis, but the preparation process is complicated and backward, and newborn cattle must be used to breed the attenuated vaccine for impetigo contagiosum. primary testis cells
However, the procedures for preparing this kind of cells are complicated, the production cost is high, and the production efficiency is low, which brings inconvenience to the large-scale production of sheep infectious impetigo attenuated vaccine by vaccine factories, and it is difficult for enterprises to obtain huge profits.
At the same time, the variety of newborn bovine testicles used for each breeding of sheep aphthous antigen is different, which makes it difficult to ensure the quality and stability of the attenuated vaccine produced. In addition, newborn bovine testicles have the potential risk of carrying other pathogens
In addition, the current veterinary vaccines have been developed in the direction of highly efficient antigen concentration, purification, and minimizing animal immune side effects (standards for the production of human medical vaccines). Therefore, it is possible to use cancer cells to produce vaccine antigens

Method used

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  • A bovine Sertoli cell cancer cell and its application in the isolation and culture of poxvirus
  • A bovine Sertoli cell cancer cell and its application in the isolation and culture of poxvirus
  • A bovine Sertoli cell cancer cell and its application in the isolation and culture of poxvirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] [Example 1] Discovery and isolation of newborn bovine testicular Sertoli cell carcinoma cells (BTSCC)

[0039] 1.1 Transfection of NBSC cells with pCI-neo-hTERT plasmid

[0040] The limited passage cell line (NBSC) of bovine testis Sertoli cells (NBSC), which can be limited to 20 passages, was isolated and purified from the cultured bovine testis primary cells, and the eukaryotic expression plasmids encoding hTERT and neo genes were transferred by lipofection method pCI-neo-hTERT was introduced into the established limited-passage cell line NBSC, and the NBSC limited-passage cell line was immortalized. The pCI-neo-hTERT expression vector map and enzyme digestion identification results are as follows figure 1 shown. Gradient concentration of G418 was used to screen, and anti-G418 positive clones were obtained: hTERT-NBSC ( figure 2 ). RNA was extracted from the screened hTERT-NBSC cells, and the expression of exogenous hTERT in transfected cells was detected by RT-PC...

Embodiment 2

[0058] [Example 2] Newborn bovine testicular Sertoli cell carcinoma cell BTSCC serum dependence test

[0059] The serum dependence of bovine testicular Sertoli cell carcinoma BTSCC cells was detected by MTT method, as follows:

[0060] (1) The 30th generation, 40th generation, 50th generation, 60th generation BTSCC and the 12th generation BTSCC cells or NBSC cells were divided into 1×10 4 cells / well were seeded in a 96-well culture plate.

[0061] (2) 24 hours after inoculation, the cell solution was replaced with DMEM / F12 culture medium of 50mL / L FBS, 100mL / L FBS, and 200mL / L FBS respectively. Make 3 replicate wells for each gradient, and set 3 blank wells at the same time.

[0062] (3) Incubate for 14 hours in an incubator at 37° C. with a condition ratio of 5% CO2 and saturated humidity.

[0063] (4) Add 10 μL of MTT (10 mg / mL) reaction solution, and continue to incubate at 37° C. for 2 to 4 hours until obvious needle-like purple crystals can be seen under the microscope...

Embodiment 3

[0067] [Example 3] Passage of BTSCC cell and its proliferation test to ORFV and GTPV

[0068] The BTSCC cells were continuously subcultured, and the time for each generation of cells to grow into a monolayer was recorded. For each passage of 5 generations, the ORFV cell virulence (lgTCID) of the same generation was inoculated. 50 ≥6.0) and ORFV cytotoxicity (lgTCID 50 ≥4.0), observe the CPE of ORFV and GTPV on the BTSCC cell line, and measure its TCID 50 .

[0069] Experiments have confirmed that when BTSCC cells are subcultured to 65 generations, the cell lines still maintain vigorous vitality. And it maintains a high proliferative ability for ORFV and GTPV viruses, the cells produce CPE more regularly, and the titer of ORFV virus (lgTCID 50 ) are above 6.20; GTPV virus titer (lgTCID 50 ) are above 4.20, the results are shown in Table 3.

[0070] Table 3 Passaging of BTSCC and its proliferation to ORFV

[0071]

[0072] In summary, the present invention successfully ...

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Abstract

The invention discloses a bovine testicular Sertoli cell carcinoma cell and its application in poxvirus isolation and cultivation. The bovine testicular Sertoli cell carcinoma cell is named bovine testicular Sertoli cell carcinoma cell BTSCC, and its preservation number is CCTCC NO: C201931. Furthermore, the present invention also proposes the application of the bovine testicular Sertoli cell carcinoma cells in the isolation and cultivation of poxviruses such as ORFV and GTPV. The cells are sensitive to ORFV and GTPV cytotoxicity, can efficiently multiply ORFV and GTPV viruses, and can ensure the uniformity and stability of the viruses. The virus titer propagated by the cells is equivalent to that of newborn bovine testicular Sertoli cells (NBSC), and the growth speed is very fast, the separation rate is high, and it can grow under low serum culture conditions. Using the cells to isolate and cultivate ORFV and GTPV viruses can improve test efficiency, reduce test costs, and at the same time ensure the stability of the quality of vaccines prepared by using the cells. The proposal of the present invention provides a new technical means for the cultivation of poxviruses and the large-scale production of vaccines thereof.

Description

technical field [0001] The present invention relates to a kind of cancer cell and its separation method and application, in particular to a kind of separation of newborn bovine testicular Sertoli cell cancer cell and its culture method, and its use in the propagation and cultivation of poxviruses such as sheep infectious impetigo virus and sheep pox virus in the application. The invention belongs to the technical field of cell engineering. Background technique [0002] Sertoli cell (SC) is the epithelial cell on the basement membrane of the seminiferous tubule and the only somatic cell in the seminiferous tubule. Structurally, it provides a scaffold for the differentiation and maturation of germ cells, provides support, nutrition and protection for germ cells, and is called the "nanny cell" 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/09C12N7/00
CPCC12N5/0693C12N7/00C12N2710/24021C12N2710/24221
Inventor 王光祥王艳华张志东李彦敏贾宁杨能能
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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