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A large-scale full suspension culture method of porcine circovirus type 2

A porcine circovirus and culture method technology, applied in the directions of microorganism-based methods, viruses, antiviral agents, etc., can solve the problems of uncertainty in the culture of semi-finished products, unfavorable PK15 cells, low reproductive capacity, etc., to reduce stress, improve Immunity levels, the effect of reducing human resources

Active Publication Date: 2020-10-16
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Porcine circovirus type 2 (PCV2) whole virus particles have good immunogenicity and are suitable for whole virus inactivated vaccines, but the low reproductive ability of PCV2 on PK15 cells is one of the biggest bottlenecks affecting the quality of PCV2 whole virus vaccines
[0003] During the cultivation of PCV2, it was found that bovine serum has a great influence on the reproduction of PCV2, which is not conducive to the infection of PK15 cells by PCV2 virus, and the uncertainty of the cultivation of semi-finished products of PCV2 vaccine is caused by the source of bovine serum and the difference between batches. Foreign studies have found that D-glucosamine, ammonium chloride and amphotericin also have certain limitations in the proliferation effects of PCV2.

Method used

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  • A large-scale full suspension culture method of porcine circovirus type 2
  • A large-scale full suspension culture method of porcine circovirus type 2
  • A large-scale full suspension culture method of porcine circovirus type 2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Adaptation of pig kidney cells sPK15-YP adapted to large-scale serum-free full suspension culture

[0024] 1. Materials and Methods

[0025] 1.1 Materials

[0026] Adherent PK-15 cells were preserved by Shanghai Yuanpei Biotechnology Co., Ltd. VirusPro PK-15-S cell serum-free medium, D-PBS, Trpzyme recombinant trypsin digestion solution were products of Shanghai Yuanpei Biotechnology Co., Ltd.

[0027] 1.2 Method

[0028] The PK-15 cells cultured with serum attached to the wall were adapted as a suspension cell line adapted to serum-free culture according to the following method:

[0029] (1) When the confluency of T-75 adherent cultured PK-15 cells reaches 50%-80%, discard the supernatant, add 5 ml of D-PBS without calcium and magnesium ions, and wash twice.

[0030] (2) Discard the PBS, add 5ml Trpzyme, let it react for a while at room temperature, discard the trypsin digestion solution, leave a little trypsin enough to cover the cells, incubate at 37°C, ...

Embodiment 2

[0040]The establishment of the large-scale full suspension culture method of embodiment 2 porcine circovirus type 2

[0041] 1. Materials and methods

[0042] 1.1 Viruses and Cells

[0043] Porcine circovirus type 2 (PCV2 / LG strain) was preserved by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences; sPK15-YP cells (CGMCC NO.13846) were domesticated by Example 1.

[0044] 1.2 Method

[0045] 1.2.1 Whole suspension culture of sPK15-YP cells

[0046] 1.2.1.1 Optimization of initial seeding density of cells

[0047] The sPK15-YP cell concentration was 10×10 3 / mL, 10×10 4 / mL, 30×10 4 / mL, 50×10 4 / mL and 80×10 4 / mL were cultured in a bioreactor at a temperature of 37°C, and the culture medium was MEM medium. Samples were taken every 24 hours for cell counting and viability testing.

[0048] 1.2.1.2 Optimization of the way of rehydration

[0049] Culture in the bioreactor by half-changing liquid and gradually adding liquid respectively, samp...

Embodiment 3

[0088] Preparation and immune efficacy test of embodiment 3PCV2 / LG strain vaccine

[0089] 1. Materials and methods

[0090] 1.1 Plasmids and strains

[0091] The plasmid pMD-18-PCV2 containing PCV2 virus ORF2 was constructed by our laboratory and kept for future use.

[0092] 1.2 Other reagents

[0093] The DNA extraction kit was purchased from Tiangen Biotechnology Co., Ltd., rTaq DNA polymerase and dNTP were purchased from Dalian Bao Biotechnology Co., Ltd., and the plasmid extraction kit was purchased from Beijing Biotech Co., Ltd.

[0094] 1.3 Method

[0095] 1.3.1 Preparation of PCV2 / LG strain vaccine

[0096] The PCV2 / LG virus purified in Example 2 was inactivated with 0.2% formaldehyde solution for 24 hours, and then emulsified with the French Sepic company adjuvant ISA15A VG. The amount of virus contained in each ml of vaccine was 10 6.5 TCID 50 / mL. The prepared PCV2 / LG inactivated vaccine was tested for immune efficacy.

[0097] 1.3.2 Primer design and amplifi...

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Abstract

The invention discloses a large-scale full-suspension production method of a porcine circovirus type 2. The inventors of the invention domesticate a porcine kidney cell adaptable to large-scale serum-free full-suspension culture, which is named as sPK15-YP, is collected in the China General Microbiological Culture Collection Center and has the culture collection number of CGMCC NO.13846. The sPK15-YP cell adaptable to full-suspension culture is used for achieving serum-free large-scale culture of the porcine circovirus type 2 (PCV2), so that the conventional spinner bottle culture technology is replaced, the human resource is reduced, the product quality is improved, and the bottleneck that the virus content is low during PCV2 full-virus culture is solved; by a full-suspension sPK15-YP cell technology, a high-potency PCV2 semifinished product is prepared; by a hollow fiber method, a PCV2 virus culture solution is concentrated and purified to obtain a more pure PCV2 virus concentrated antigen. By the large-scale full-suspension production method, a solid foundation is laid for studying a PCV2 multivalent vaccine, the dosage of the vaccine is reduced, the stress of a swine herd is reduced and the immune level of the swine herd is enhanced.

Description

technical field [0001] The present invention relates to a method for cultivating porcine circovirus type 2, in particular to a full-suspension culture method for cultivating porcine circovirus type 2 on a large scale, and also relates to a sPK15-YP cell suitable for large-scale serum-free full-suspension culture , The invention belongs to the technical field of medicine. Background technique [0002] Porcine circovirus type 2 (PCV2) whole virus particles have good immunogenicity and are suitable for whole virus inactivated vaccines, but the low reproductive ability of PCV2 on PK15 cells is one of the biggest bottlenecks affecting the quality of PCV2 whole virus vaccines . [0003] During the cultivation of PCV2, it was found that bovine serum has a great influence on the reproduction of PCV2, which is not conducive to the infection of PK15 cells by PCV2 virus, and the uncertainty of the cultivation of semi-finished products of PCV2 vaccine is caused by the source of bovine ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N7/00A61K39/12A61P31/20C12R1/93
CPCA61K39/12A61K2039/5252A61K2039/552C12N5/0686C12N7/00C12N2750/10034C12N2750/10051
Inventor 危艳武孙奇威刘长明李智力黄立平吴洪丽冯力
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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