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A purification method and application of porcine parvovirus virus-like particles

A technology of parvovirus and purification method, which is applied in peptide preparation methods, chemical instruments and methods, biochemical equipment and methods, etc., and can solve problems such as low added value of products and increased production costs

Active Publication Date: 2021-05-04
扬州优邦生物药品有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Especially for veterinary vaccines, the added value of the product itself is low. If the purification process with cumbersome steps is adopted, the production cost will inevitably increase greatly
[0004] At present, there is no technology for the industrial purification of porcine parvovirus virus-like particles

Method used

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  • A purification method and application of porcine parvovirus virus-like particles
  • A purification method and application of porcine parvovirus virus-like particles
  • A purification method and application of porcine parvovirus virus-like particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The preparation process of embodiment 1 porcine parvovirus semi-finished product

[0023] The nucleotide sequence encoding the porcine parvovirus virus-like particle gene is connected to the baculovirus to construct a recombinant baculovirus seed, and the virus seed infects insect cells (Sf9 cells), and the culture conditions of Sf9 cells are 27°C, 140rpm, 96h~ 120h. When the cells were lysed to release the virus, the venom was collected and the porcine parvovirus virus-like particles were inactivated by binary ethylenimine (BEI).

Embodiment 2

[0024] Example 2 Purification of porcine parvovirus virus-like particles

[0025] The inactivated venom is clarified to remove the precipitate, the pH of the semi-finished product is adjusted to 5.5-6.0 with 2% acetic acid, and the semi-finished product before purification of porcine parvovirus virus-like particles is obtained.

[0026] 1) weak anion exchange chromatography

[0027] The pretreated semi-finished product was added to a weak anion exchange column (DEAE Focusose 6XL, Wuhan Huiyan Biotechnology Co., Ltd.) equilibrated with sodium acetate buffer A (20mM sodium acetate, 0.1M NaCl, pH=5.5), Use sodium acetate buffer B (20mM sodium acetate, 1.0M NaCl, pH=5.5) for elution, the elution volume is 3 column volumes, the flow rate is 2.0mL / min, and the peak components are collected, and the weak anion exchange Chromatograms such as figure 1 shown.

[0028] 2) Determination of active protein components by polyacrylamide gel electrophoresis (SDS-PAGE) Take 80 μL of samples ...

Embodiment 3

[0050] Example 3 Safety Test of Vaccines Containing Porcine Parvovirus Virus-like Particles

[0051] 1. The same amount of adjuvant was added to the samples before and after the purification of the experimental vaccine porcine parvovirus virus-like particles to prepare the vaccine. The vaccine batch before purification was defined as VP2-01, and the vaccine batch after purification was defined as VP2-02.

[0052] 2. Test piglets were purchased from Yunli Animal Husbandry Co., Ltd., Dantu District, Zhenjiang City, and all of them were negative for porcine parvovirus antigen antibodies.

[0053] 3. Experimental method

[0054] 3.1 Single-dose vaccination safety experiment

[0055] 3.1.1 Safety test of piglets

[0056] 20 weaned piglets aged 3 to 5 weeks, 5 piglets in each batch of vaccine, intramuscular injection in the neck, 2mL / head, and the remaining 5 piglets as a control, inoculated with normal saline, carefully observed the reaction of the pigs after injection, and obser...

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Abstract

The invention discloses a method for purifying porcine parvovirus virus-like particles and an application thereof, belonging to the technical field of separation and purification of biological products. The method comprises the following steps: clarifying the culture of the expressed porcine parvovirus virus-like particles, collecting the supernatant and inactivating it with diethyleneimine to obtain a semi-finished product; adjusting the pH value of the semi-finished product to be acidic; The semi-finished product is added to the weak anion exchange column equilibrated with the first buffer solution, and the weak anion exchange column is eluted with the second buffer solution, and each peak component is collected; the active protein of each peak determined by polyacrylamide gel electrophoresis components. The purity of porcine parvovirus virus-like particles obtained according to the purification method of the present invention is as high as 90%, and the recovery rate is as high as 99%, and the purification method is convenient for production operation, and provides safer preparation for porcine parvovirus baculovirus vector inactivated vaccines. reliable way.

Description

technical field [0001] The invention relates to a purification method and application of porcine parvovirus virus-like particles, belonging to the technical field of separation and purification of biological products. Background technique [0002] Porcine parvovirus (PPV) belongs to the genus Parvovirus of the family Parvoviridae, and is an autonomously replicating virus. Clinically, it is characterized by a series of symptoms such as abortion, stillbirth, deformed fetus, fetal mummification and weak offspring in pregnant sows. . Porcine parvovirus is often co-infected with other pathogens that cause reproductive disorders in sows to cause disease, which has brought serious losses to the pig industry. [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 Ther...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/015C07K1/18C12N15/866A61K39/125A61P31/20
CPCA61K39/12A61K2039/5252A61K2039/552A61P31/20C07K14/005C12N15/86C12N2710/14043C12N2750/14023C12N2750/14034C12N2750/14051
Inventor 范娟丁国伟李群潘杰徐萍叶正琴李甜甜魏荣荣孙舒李琛
Owner 扬州优邦生物药品有限公司
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