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Porcine circovirus-like particle, and vaccine and preparation method thereof

A porcine circovirus, virus-like technology, applied in antiviral agents, virus antigen components, pharmaceutical formulations, etc., to achieve the effect of preventing infection

Inactive Publication Date: 2013-12-11
CHONGQING AULEON BIOLOGICALS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the PCV-2 VLP vaccine, so there is an urgent need for an antigen that can assemble VLP, which can induce a good immune response in experimental animals after being mixed with an adjuvant

Method used

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  • Porcine circovirus-like particle, and vaccine and preparation method thereof
  • Porcine circovirus-like particle, and vaccine and preparation method thereof
  • Porcine circovirus-like particle, and vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Preparation of the expression vector of porcine circovirus-like particle vaccine

[0024] Take the nucleotides from the CAP protein of BJ-HB, GD, GD-TS, BJ0401, HB0402, TJ04, ZJ0401, TJ05, HB0601, CQ08, HuB08, LN08, HN08, SC07 and JX0601 subtypes of PCV-2b, wherein The nucleotide sequence of the CAP protein of the BJ-HB subtype is shown in SEQ ID NO.1; the nucleotide sequence of the CAP protein of the GD subtype is shown in SEQ ID NO.2; the CAP protein of the GD-TS subtype The nucleotide sequence of the CAP protein is shown in SEQ ID NO.3; the nucleotide sequence of the CAP protein of the BJ0401 subtype is shown in SEQ ID NO.4; the nucleotide sequence of the CAP protein of the HB0402 subtype is shown in SEQ ID NO. 5; the nucleotide sequence of the CAP protein of the TJ04 subtype is shown in SEQ ID NO.6; the nucleotide sequence of the CAP protein of the ZJ0401 subtype is shown in SEQ ID NO.7; the CAP protein of the TJ05 subtype The nucleotide sequence of the C...

Embodiment 2

[0026] Example 2 Cell Culture and Cell Batch Bank Establishment

[0027] Taking UMNSAH / DF-1 cell culture and building a bank as an example, UMNSAH / DF-1 cells come from the American Type Culture Collection (ATCC). The medium adopts DMEM medium (Dulbecco's modified Eagle medium), and 100 U / mL penicillin and 100 μg / mL streptomycin are added to the medium, and then fetal calf serum with a volume fraction of 2-10% is added, and then in the cell factory or The cell fermenter is used for culturing, and the batch bank of cell working seeds is established, and the exogenous factors, tumorigenicity and stability after passage are comprehensively tested. The number of cell generations used is controlled within 60 generations, which all meet the requirements of vaccine production media.

[0028] Other animal cells can also be used in this embodiment, such as: PK-15 cells; ② VERO cells; ③ COS cells; ④ human diploid cells; ⑤ BHK cells; ⑥ CHO cells; ⑦ MDCK cells; Other cells commonly used i...

Embodiment 3

[0029] Example 3 Plasmid transfection and stable high-expression cell line screening and library construction

[0030] The UMNSAH / DF-1 cells established in Example 2 were selected as target cells, and the recombinant pHWD2000-CAP expression vector constructed in Example 1 was used to transfect UMNSAH / DF-1 cells by liposome method, and then the stable and high expression PC VLP- 2 cell lines. The specific steps are as follows: ① Add 1.5 μg of pHWD2000-CAP expression vector to a 35 mm well; ② Add 5 μL of liposomes prepared in OptiMEM, and incubate at room temperature for 45 minutes. Then add it to the host cells rinsed with OptiMEM and incubate for 5h; ③Centrifuge to remove the carrier-liposome mixture, and add DMEM to the cells for culture; ④The pHWD2000-CAP recombinant vector is expressed in the cell line and can self-assemble PC VLP-2; ⑤The UMNSAH / DF-1 cell line expressing PCVLP-2 was screened by pressure. The results showed that within 10 generations of UMNSAH / DF-1 cells, ...

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Abstract

The invention discloses a porcine circovirus-like particle, a vaccine and a preparation method thereof. The virus-like particle is composed of a main structural protein-nucleocapsid protein of 2-type porcine circovirus, can excite cell and humoral immune response, can be used as a virus-like particle vaccine (VLP vaccine) to immune different fauna and can safely and effectively prevent PCV-2 infections after being used. The VLP can be made into injections, nose drops and drinking preparations by adding adjuvants or not. An ideal vaccine is provided for security of different populations of sows, piglets, fattening pigs and for effective immune prevention and control of PCV-2 infections.

Description

field of invention [0001] The invention belongs to the field of biopharmaceuticals, and relates to a porcine circovirus-like particle, in particular to a virus-like particle vaccine and a preparation method thereof. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) is a virus that causes a decrease in herd immunity, and is associated with postweaning multisystemic wasting syndrome (Postweaning Multisystemic Wasting Syndrome, PMWS), porcine dermatitis and nephrotic syndrome (Porcine Dermatitis) and Nephropathy Syndrome, PDNS), porcine reproductive failure (Porcine reproductive failure), porcine respiratory disease syndrome (Porcine Respiratory Disease Syndrome, PRDC), porcine proliferative necrotizing pneumonia (Porcine Necrotizing Pneumonia, PNP), porcine congenital tremor ( Porcine Congenital Tremor, CT) and other diseases are linked. PCV was first discovered in 1974 in an in vitro cultured pig kidney cell line PK-15. At that time, it was considere...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04A61K39/12A61K39/39A61P31/20
Inventor 李阳春曹政
Owner CHONGQING AULEON BIOLOGICALS
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