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Method for producing porcine parvovirus antigen and its product

A parvovirus and antigen technology, which is applied in the preparation of porcine parvovirus antigen and its products, can solve the problems of low antigen immune activity, low expression efficiency, and high production cost, and achieve no three wastes, high expression efficiency, and low production cost. Effect

Active Publication Date: 2012-03-21
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Existing methods for preparing porcine parvovirus antigens by means of genetic engineering have defects such as low expression efficiency, high production costs, and low immune activity of the expressed antigens, which need to be improved.

Method used

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  • Method for producing porcine parvovirus antigen and its product
  • Method for producing porcine parvovirus antigen and its product
  • Method for producing porcine parvovirus antigen and its product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Preparation and purification of porcine parvovirus antigen, animal immunization experiment and virus challenge protection experiment

[0031] 1. Preparation of related solutions and medium

[0032] Solution I: 50mmol / L glucose, 25mmol / L Tris-HCl (pH8.0), 10mmol / L EDTA.

[0033] Solution II: 0.2mol / L NaOH, 1% SDS (prepared and used immediately).

[0034] Solution III: 100mL system, 5mol / L potassium acetate 80mL, glacial acetic acid 12mL, ddH 2 08mL. TAE (50×): 242g Tris base, 57.1mL glacial acetic acid, 100mL 0.5mol / L EDTA (pH8.0), sterile water to 1000mL.

[0035] TER solution: Pancreatic RNAse (RNAse A) was dissolved in 10mM Tris-HCl, 15mMNaCl, prepared as a 10mg / mL storage solution and stored at -20°C, and diluted with 1×TE buffer to a 20μg / mL working solution and stored at 4°C.

[0036] PPt Buffer: isopropanol 22mL; 5mol / mL KAc 1mL; ddH 2 O2mL.

[0037] 1×TE buffer: 10mmol / L Tris·Cl (pH8.0), 1mmol / L EDTA (pH8.0), sterilized by high temperature and hig...

Embodiment 2

[0127] Example 2 Expression of porcine parvovirus empty capsid protein gene codon-optimized sequence VP2-M in silkworm bioreactor and detection of expression products

[0128] 1. The preparation of relevant solution and culture medium: with embodiment 1.

[0129] 2. Optimization of porcine parvovirus VP2 gene and vector construction

[0130] 2.1 Codon optimization of PPV-VP2 gene

[0131] According to the silkworm codon preference, the original sequence VP2 of the porcine parvovirus capsid protein gene measured in Example 1 is optimized without changing the amino acid sequence. The optimized sequence VP2-M is shown in SEQ ID NO: 2, and the original sequence contains Rare codons in tandem, which reduce the translation sequence or even untranslate the device, the CAI value of the optimized sequence is increased from 0.86 to 0.89, the GC content and unsuitable peaks are adjusted to extend the half-life of mRNA, and the GC content is adjusted from 38.32% to 48.70% , those stem-l...

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Abstract

The invention discloses a method for producing a porcine parvovirus antigen and its product. The method comprises the following steps: porcine parvovirus capsid protein VP2 gene or optimized VP2 gene is cloned in a baculovirus carrier so as to obtain a transfer expression carrier; the constructed transfer expression carrier and baculovirus DNA are carried out cotransfection to obtain recombined baculovirus; the recombined baculovirus is used to infect insect host and cell; the infected insect host is cultured to express corresponding porcine parvovirus capsid protein; and the expressed antigen is ingathered and purified so as to obtain the porcine parvovirus antigen. The method adopts a baculovirus expression system to make safe and efficient porcine parvovirus antigen capsid particles in a domestic silkworm bioreactor; the prepared purified antigen by the method has high safety, and can be directly produced to vaccines for animal immunity. The method for producing porcine parvovirus antigen has the advantages of high expression efficiency, high immunization activity of the expressed antigen, low production cost, large scale production realization and the like.

Description

technical field [0001] The present invention relates to a method for preparing porcine parvovirus (Porcine parvovirus, PPV) antigen, in particular to a method for expressing porcine parvovirus empty capsid antigen in insects by using recombinant baculovirus, and the present invention also relates to the The antigen product and the use of the antigen to prepare oral or injectable vaccines for preventing or treating porcine parvovirus belong to the field of porcine parvovirus antigen preparation. Background technique [0002] Porcine parvovirus (Porcine parvovirus, PPV) was discovered by Mary and Mahnel in 1966 when they cultured swine fever virus tissue. It was first isolated by Cartwright and subsequently reported in many countries in Europe, America, Asia, Africa and Oceania. sex distribution. PPV is one of the important pathogens that cause reproductive disorders in sows. It can also cause fetal mummification, death of newborn piglets, dermatitis and diarrhea in piglets, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/35C12N15/866C07K14/015A61K39/23A61K48/00A61P31/20
Inventor 张志芳李轶女易咏竹王金辉王国增刘慧芬沈桂芳
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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