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Novel PCV (porcine circovirus) and cell strain co-culturing with novel porcine circovirus

A porcine circovirus and cell strain technology, applied in the direction of viruses, animal cells, viruses/bacteriophages, etc., can solve the problems of cell pathology and poor vaccine quality, and achieve the effects of reducing production and cultivation, increasing virus titer, and reducing operation links

Active Publication Date: 2017-05-31
WUHAN ENG SCI & TECH RESINST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In addition, because the common circovirus cannot cause pathological changes in the cells during the culture process (Yan Weidong "Swine Diseases Special Issues 2010 Academic Annual Conference Proceedings" 1184-1188), the determination of the harvest time of the virus is very blind, which is also One of the reasons for the poor quality of current vaccines

Method used

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  • Novel PCV (porcine circovirus) and cell strain co-culturing with novel porcine circovirus
  • Novel PCV (porcine circovirus) and cell strain co-culturing with novel porcine circovirus
  • Novel PCV (porcine circovirus) and cell strain co-culturing with novel porcine circovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Biological material source, preservation and culture medium

[0050] The porcine kidney cell line PK15 comes from the China Center for Type Culture Collection, with the collection number CCTCC GDC061, and researchers can purchase it from CCTCC

[0051] The PCV2 virus comes from independent isolation, and the screened porcine circovirus GY-PCV2 strain is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO:V201660. The deposit date is December 5, 2016.

[0052] GY-PCV2-PK15 was deposited in the China Center for Type Culture Collection, the preservation number is CCTCC NO: C2016201, and the preservation date is December 5, 2016.

[0053] Medium MEM+10%FBS+1% double antibody, maintenance medium MEM+2%FBS+1% double antibody

Embodiment 2

[0054] Example 2: Isolation and sequencing of porcine circovirus GY-PCV2 strain

[0055] (1) Aseptically remove the inguinal lymph nodes of pigs suffering from porcine circovirus disease with obvious clinical symptoms, and wash them more than 7 times with a mixture of 20,000 units / ml penicillin and 200ug / ml streptomycin , and smashed into a homogeneous slurry, centrifuged, filtered and sterilized to obtain the disease feed liquid;

[0056] (2) Mix the disease feed solution with DMEM maintenance medium containing 2% serum 1:1;

[0057] (3) Inoculate the mixture onto PK-15 cells in logarithmic growth phase for infection;

[0058] (4) Discard the inoculum after infection and culture for 24 hours, add a little 3mM D-glucosamine to overlay the cells, continue to culture for 2 hours, wash 5 times with PBS, add DMEM maintenance solution with 2% serum to continue culture.

[0059] (5) After culturing for 72 hours, take the supernatant and detect it with a PCV2-specific PCR detection...

Embodiment 3

[0090] Example 3. Screening and obtaining of PK15 cells producing PCV2 virus

[0091] (1) GDC061 porcine kidney cell line PK15 purchased from CCTCC to the logarithmic growth phase;

[0092] (2) Inoculate the PCV2 mutant strain into the pig kidney cell line PK15, culture for 24 hours, discard the supernatant, and treat the cells with 3 mmol of D-glucosamine in PBS solution for 2 hours, discard the D-glucosamine in PBS solution, and replace with PBS Cells were washed 5 times. Change the maintenance solution to culture the infected cells for 72 hours, take the supernatant, and use the PCV2-specific fluorescence quantitative PCR detection kit to measure PCV2. When the measured CT value is lower than or equal to the positive control CT value, it proves that at this time PK-15 cells already carry PCV2 PK-15 mixed cells;

[0093](3) Digest with trypsin, add medium to neutralize, centrifuge, use medium to adjust the cell suspension at a cell density of 1-10 cells / ml, inoculate the c...

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Abstract

The invention discloses a novel PCV (porcine circovirus) mutant strain and a porcine kidney cell strain co-culturing with the novel PCV mutant strain. Cells infected by the novel PCV mutant strain have visible minimal lesions, but cell death is not caused. The sieved porcine kidney cell strain carries a PCV2, so that stable passage can be realized, a large number of viruses are generated, and the prepared viruses can be used for preventing and treating the PCV.

Description

technical field [0001] The invention belongs to the technical field of cell engineering and virus culture. Specifically, the invention relates to a porcine circovirus, a PK15 cell strain that can be co-cultivated with the porcine circovirus, a method for preparing PCV2 virus through mass culture, and an application thereof. Background technique [0002] There are two main types of porcine circovirus vaccines currently in use in China—subunit vaccines and whole virus vaccines. Although subunit vaccines are relatively safe, they are expensive to produce and have a short immunity period. Therefore, the vast majority of pig farms in China still rely mainly on whole virus vaccines. However, the production of whole virus vaccines also has its technical bottlenecks: (existing) circovirus cultured cells in vitro have low infectivity and slow infection speed, the replication speed of the virus in different cell individuals varies greatly, and the virus culture titer is very low. ...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N5/071
CPCC12N5/0686C12N7/00C12N2750/10021C12N2750/10051C12N2750/10052
Inventor 高其双彭霞夏瑜谭珺隽杨俊红程百炼桑奥文雷长宁王利燕
Owner WUHAN ENG SCI & TECH RESINST
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