PK-15 clone cell strain and application thereof

Abstract

The invention relates to a PK-15 clone cell strain and application thereof. The cell strain provided by the invention is preserved in the China Center for Type Culture Collection, the preservation number is CGMCC NO: 45845, the cloned cell strain is sensitive to the porcine circovirus type 3, stable and efficient passage can be performed on the porcine circovirus type 3, the replication level of the virus on the cell is remarkably improved, compared with a female parent cell, the cell strain can be used for obtaining a high-titer virus solution, and the cell strain has the advantages that the cell strain is simple in structure and convenient to use. The cell strain provides an important culture medium for culturing, separating and efficiently breeding PCV3, can be used for production of inactivated vaccine of porcine circovirus type 3, and has high application value in research and production of vaccines and diagnostic reagents.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a PK-15 clone cell strain and application thereof. Background Art

[0002] Porcine circovirus (PCV) is the smallest known DNA virus among mammalian viruses and belongs to the genus Circovirus of the family Circoviridae. At least four PCVs have been found in pigs, named PCV1 to PCV4. Among them, PCV3 is an emerging pathogen first reported in North America in 2016. Studies have shown that PCV3 has been circulated in pigs in Asia, Europe, South America and North America for a long time, attracting widespread attention in the pig industry. Since the first report of PCV3, it has been widely found in the lungs, heart, kidneys and other organs or tissues. Several studies have further confirmed that PCV3 infection is associated with respiratory, digestive, reproductive disorders and multi-system diseases. There are also studies reporting that PCV3 is co-infected with other pathogens (such as PEDV, PCV2, PRRSV or PRV, etc.) in some pig farms. The specific biology and pathogenic mechanism of PCV3 are still under further study. All evidence suggests that PCV3 may be another important pathogen in the pig industry. However, the difficulty of isolating PCV3 and the limited culture cells limit its in vitro reproduction and research. How to effectively obtain cell lines that can efficiently increase the titer of porcine circovirus type 3 is a technical problem that needs to be solved urgently. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a PK-15 clone cell line and application thereof.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] A PK-15 cloned cell line was submitted to the General Microbiology Center of the China Microbiological Culture Collection Administration for preservation on April 17, 2024. The classification name is PK-15 cells, and its preservation number is CGMCC NO: 45845. It can be used to effectively proliferate porcine circovirus type 3.

[0006] The PK-15 clone cell line described above was used for virus culture.

[0007] Furthermore, the virus is porcine circovirus, more specifically, the porcine circovirus is porcine circovirus type 3.

[0008] Use of the PK-15 clone cell line as described above in the preparation of porcine circovirus type 3.

[0009] The use of the PK-15 cloned cell line as described above in the preparation of a vaccine.

[0010] Furthermore, the use of the PK-15 cloned cell line as described above in the preparation of porcine circovirus type 3 vaccine.

[0011] The beneficial effects of the present invention are:

[0012] The PK-15 cloned cell strain provided by the present invention is sensitive to porcine circovirus type 3, can significantly improve the replication level of porcine circovirus type 3 on cells, and compared with the mother cell, the cell strain can obtain a high-titer virus liquid, providing an important culture matrix for culturing PCV3, and has high application value in the research and production of vaccines and diagnostic reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The cell morphology of different PK-15 cell clones.

[0014] Figure 2 Shows the growth curves of different clones of cells. DETAILED DESCRIPTION

[0015] The present invention adopts the limiting dilution method to clone PK-15 single cells, screens out a PK-15 clone cell line 3C5 cell, studies and analyzes the cell morphology, growth characteristics and adaptability to PCV3 strains, and the results show that compared with the mother cell and other cloned cells, the cell growth rate and sensitivity to 0.1% trypsin are different. After inoculation with PCV3, the virus replication level in the cell can be significantly increased, and the virus titer TCID50 increases from 10 3.5 / ml stable to 10 6.2 / ml. The present invention obtains a cultured cell strain for stable and efficient propagation of PCV3 virus through cell cloning and screening, which provides an important culture matrix for efficient proliferation of PCV3 and also provides a certain basis for the development of PCV3 vaccine and diagnostic reagent.

[0016] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the present invention all belong to the scope of the present invention.

[0017] If not otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents used in the present invention are all analytically pure or above, and commercially available products can be used.

[0018] Example 1

[0019] 1. Cell cloning

[0020] 1.1 Cloning of PK-15 cell line

[0021] 1.1.1 Cell recovery and passaging: PK-15 cells (porcine kidney epithelial cells, from ATCC American Type Culture Collection) were revived and placed in a T25 cell culture flask. After culturing in complete culture medium until the cells reached a monolayer, they were passaged at a ratio of 1:3 and were passaged several times before cloning.

[0022] 1.1.2 Cell cloning by limiting dilution: PK-15 single cell cloning was performed by limiting dilution in a 96-well plate. After digesting the cells to obtain single cells, they were diluted in a culture medium containing 20% ​​serum. The diluted cells were spread in a 96-well plate and cultured in a 37°C, 5% CO2 incubator until they became monolayer cells. The cells of a single clone were further cultured and selected for further subcloning. After three subclones, the selected single clones were expanded and observed for identification.

[0023] The morphological identification results of PK-15 mother cells and cloned cell lines marked as 3C1, 3C5, and 1A7 are shown in Figure 1 As shown, it has a typical epithelial-like, apposed growth pattern.

[0024] 1.2. Analysis of proliferation characteristics of PK-15 clone cell line

[0025] Different clones of cells were inoculated into 96-well plates, with a volume of 100 μl (5×10 4 ), set 4-6 duplicate wells. Set zero wells and control wells at the same time. After a certain period of culture, remove the old culture solution, add 10μl of MTT solution to each well, incubate for 4 hours, stop the culture, add 100μl of formazan solution to each well, shake to fully dissolve, and continue incubation for 4 hours. After the end, select 570nm wavelength to measure the light absorption value of each well to determine the growth curve.

[0026] The results are as follows Figure 2 The results showed that PK-15 clone cell line 3C1 (PK-15-3C1) grew rapidly after 48 hours, while the growth rate of PK-15 clone cell line 3C5 (PK-15-3C5) and PK-15 clone cell line 1A7 (PK-15-1A7) was lower than that of the parent cell. Therefore, PK-15 clone cell line 3C1 has better growth performance.

[0027] 1.3 Analysis of the sensitivity of PK-15 clone cell line to trypsin

[0028] The obtained cloned cell lines were inoculated in equal amounts in 6-well cell culture plates, and the culture medium was discarded after culturing to a monolayer, and an equal amount of trypsin was added for digestion. The digestion speed of different cell lines was compared, and the time required for each cell line to be digested to a single cell was analyzed. The results of the trypsin sensitivity test of different cell lines are shown in Table 1.

[0029] Table 1 Results of clone cell trypsin sensitivity test

[0030]

[0031] The experimental results showed that the sensitivity of 3C5 to pancreatic enzyme digestion efficiency was significantly lower than that of other cells.

[0032] The PK-15 clone cell line 3C5 was submitted to the General Microbiology Center of China Microbiological Culture Collection Administration for preservation on April 17, 2024, and the classification name is: PK-15 cells; its microbial preservation number is: CGMCC NO: 45845.

[0033] Study on the adaptability of 1.4PK-15 clone cell line to porcine circovirus type 3

[0034] The PK-15 cloned cell lines were cultured to monolayers, inoculated with equal amounts of porcine circovirus type 3, and after adsorption for 2 hours, MEM maintenance solution was added and cultured at 37°C, 5% CO2. The virus solution was collected and observed daily, and frozen and thawed three times. The virus solution was diluted 10 times and inoculated in 96-well plates, with 6 parallels in each well. A control group was also set up. After culturing for 3-5 days, the cell lesions were observed and the TCID50 was calculated. The method for calculating the TCID50 concentration was the Reed-Muench method for calculating the virus TCID50. The results are shown in Table 2.

[0035] Table 2 Virus titers after PCV3 virus inoculation in different cells

[0036]

[0037] Note: The “*” in the table indicates significant difference, p<0.05.

[0038] The results showed that the virus titer inoculated with PK-15 clone cell line 3C5 was the highest, and the TCID50 of the harvested virus titer reached 10 6.2 / ml, which is much higher than that of the mother cells and other cloned cells, providing an important cell culture matrix that can be used for the cultivation of PCV3 virus to prepare inactivated vaccines, and has high application value in vaccine production.

Claims

1. A PK-15 clone cell line, characterized in that: Its deposit number is CGMCC NO:45845.

2. Use of the PK-15 clone cell line according to claim 1 in virus culture.

3. The use according to claim 2, characterized in that The virus is porcine circovirus.

4. The use according to claim 3, characterized in that: The porcine circovirus is porcine circovirus type 3.

5. Use of the PK-15 clone cell line as described above in the preparation of porcine circovirus type 3.

6. Use of the PK-15 clone cell line according to claim 1 in preparing a vaccine.

7. The use according to claim 6, characterized in that: The vaccine is porcine circovirus type 3 vaccine.

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