A purification method and application of porcine circovirus type 2 virus-like particles

A technology of porcine circovirus and type 2 virus, applied in the direction of microorganism-based methods, biochemical equipment and methods, antiviral agents, etc.

Active Publication Date: 2019-10-11
扬州优邦生物药品有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] So far, the vaccines commonly used in China are mainly inactivated whole virus vaccines and recombinant subunit vaccines expressed by baculovirus. Compared with inactivated whole virus vaccines, subunit vaccines have the advantage of high antigen expression, but There are some allergic reactions (such as vomiting, tremors, etc.) in the clinical application of the recombinant subunit vaccine expressed by the virus. These side effects are mainly caused by the unpurified subunit vaccine

Method used

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  • A purification method and application of porcine circovirus type 2 virus-like particles
  • A purification method and application of porcine circovirus type 2 virus-like particles
  • A purification method and application of porcine circovirus type 2 virus-like particles

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Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1 is the preparation process of the porcine circovirus type 2 semi-finished product in the purification method according to the present invention.

[0021] The nucleotide sequence encoding the porcine circovirus type 2 virus-like particle gene is connected to the baculovirus to construct a recombinant baculovirus seed, the virus seed infects insect cells (Sf9 cells), and the Sf9 cell culture conditions are 27 ° C, 140 rpm , 96h~120h. When the cells were lysed to release the virus, the venom was collected and inactivated by binary ethyleneimine (BEI) to obtain porcine circovirus type 2 virus-like particles.

[0022] The inactivated venom was clarified to remove the precipitate with 0.2M Na 2 HPO 4 Adjust the pH of the semi-finished product to about 6.5-6.8 to obtain the semi-finished product before purification of porcine circovirus type 2 virus-like particles.

Embodiment 2

[0023] Example 2 Purification of porcine circovirus type 2 virus-like particles

[0024] 1) Strong cation exchange chromatography

[0025] Add the pretreated semi-finished product to a strong cation exchange column equilibrated with PBS buffer A (20mM PBS, 0.1M NaCl, pH=6.8), and use PBS buffer B (20mM PBS, 1.0M NaCl, pH=8.0 ) for elution, the elution volume is 3 column volumes, the flow rate is 2.0ml / min, and each peak component is collected, the strong ion exchange chromatogram is as follows figure 1 shown.

[0026] 2) Polyacrylamide gel electrophoresis (SDS-PAGE) to determine the active protein components

[0027] Take 80 μl of samples from each stage in a 1.5ml EP tube, then add 20 μl of 5×SDS-PAGE loading buffer, and mix well. Each EP tube was placed in a boiling water bath for 5 minutes, and centrifuged at 4000 rpm for 3 minutes to obtain SDS-PAGE samples. The SDS-PAGE of each component was as follows: figure 2 as shown, figure 2 Middle, lane M: protein marker; la...

Embodiment 3

[0033] Example 3 is a safety test of a vaccine containing porcine circovirus type 2 virus-like particles obtained by the above-mentioned purification method of porcine circovirus type 2 virus-like particles.

[0034] 1. Experimental Vaccine Vaccines were prepared by adding the same appropriate amount of adjuvant to samples before and after purification of porcine circovirus type 2 virus-like particles.

[0035] 2. Test piglets were purchased from Yunli Animal Husbandry Co., Ltd., Dantu District, Zhenjiang City, and all of them were negative for porcine circovirus type 2 antigen antibodies.

[0036] 3. Experimental method

[0037] 3.1 Single-dose vaccination safety experiment

[0038] Piglets aged 2 to 3 weeks were selected, and 200 piglets were injected intramuscularly with vaccines prepared from viruses before and after purification, 1ml / head. After the injection, observe the mental state, drinking water, and food intake of pigs in each group every day; whether there is an ...

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Abstract

The invention relates to a method for purifying porcine circovirus type 2 virus-like particles and a use thereof. The method comprises clarifying the culture of porcine circovirus type 2 virus-like particles, collecting the supernatant, inactivating the viruses through pyrrole to obtain a semi-finished product, adjusting pH of the semi-finished product to less than 7, adding the semi-finished product into a strong cation exchange column balanced by a first buffer solution, eluting the strong cation exchange column through a second buffer solution, collecting components producing peaks and determining active protein components producing the peaks through polyacrylamide gel electrophoresis. The porcine circovirus type 2 virus-like particles purified by the method have purity of 87.40% and arecovery rate is 95%. The method is conducive to production and provides safe and reliable approaches for preparation of a porcine circovirus type 2 baculovirus vector inactivated vaccine.

Description

technical field [0001] The invention relates to the technical field of separation and purification of biological products, in particular to a purification method and application of porcine circovirus type 2 virus-like particles. Background technique [0002] Porcine circovirus type 2 (PCV2) is an essential pathogen of multisystemic wasting syndrome (PMWS) in weaned piglets, and destroys the immune system of pigs, leading to porcine reproductive and respiratory syndrome virus, pseudorabies virus and Secondary infections such as Haemophilus parasuis have brought huge economic losses to pig raising. Vaccination can effectively control PCV2 infection. [0003] So far, the vaccines commonly used in China are mainly inactivated whole virus vaccines and recombinant subunit vaccines expressed by baculovirus. Compared with inactivated whole virus vaccines, subunit vaccines have the advantage of high antigen expression, but There are some allergic reactions (such as vomiting, tremor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/02A61K39/12A61P31/20C12R1/93
Inventor 宋庆庆丁国伟吴李君许兆君李群徐晓婷李琛潘杰范娟
Owner 扬州优邦生物药品有限公司
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