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Porcine circovirus (pcv2) subunit vaccine using recombinant yeast whole cells and method for manufacturing same

A porcine circovirus, whole cell technology, applied in veterinary vaccines, vaccines, viruses, etc., can solve the problem of difficulty in maintaining antigens stably, and achieve the effect of simplifying the vaccine production process

Inactive Publication Date: 2016-01-13
KOREA RES INST OF BIOSCI & BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] It is known that if yeast cells are lysed to extract antigens expressed in the cells, antigens such as virus-like particles are difficult to stably remain in solution after preparation of cell lysates

Method used

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  • Porcine circovirus (pcv2) subunit vaccine using recombinant yeast whole cells and method for manufacturing same
  • Porcine circovirus (pcv2) subunit vaccine using recombinant yeast whole cells and method for manufacturing same
  • Porcine circovirus (pcv2) subunit vaccine using recombinant yeast whole cells and method for manufacturing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Preparation of virus by expressing PCV2 ORF2 in Saccharomyces cerevisiae strain Poison-like particles

[0040] 1-1: Construction of a vector for expressing PCV2 ORF2 in Saccharomyces cerevisiae strain, and analysis of expression

[0041] A yeast codon-optimized ORF2 sequence (SEQ ID NO: 1 ) was synthesized by Bioneer for efficient expression in yeast Saccharomycescerevisiae strains. The synthesized gene was cloned into the yeast expression vector YEGα-HIR525. Expression of ORF2 was determined using two promoters (GAL10 and ADH1) and adding a 6-histidine tag to the C-terminus. For the GAL10 promoter, primers (Table 1) having EcoRI and SalI restriction enzyme recognition sites in the 5' and 3' end regions were constructed, and inserted into expression vector YEGα-HIR525 digested with the same restriction enzymes. The resulting vector was digested with SmaI and EcoRI to remove the GAL10 promoter and insert the ADH1 promoter in its place. The primers u...

Embodiment 1-2

[0049] Example 1-2: Fusion expression and analysis of cellulose binding domain and ORF2

[0050] It is known that insoluble particles can be formed in cells when the cellulose binding domain (CBD) is expressed as a fusion protein with a different protein. Therefore, fusion expression of CBD and ORF2 was attempted in anticipation of having functions similar to those of virus-like particles. Fusion expression with CBD was attempted in two cases: the case where the α-amylase signal sequence was used for extracellular secretion (SS-CBD); and the case where the α-amylase signal sequence was not used. Here, the ORF2 gene does not contain a nuclear localization signal (NLS). Each of CBD and ORF2 was amplified, and then the amplified products were ligated by overlap PCR and inserted into the EcoRI and SalI sites of YEGa-HIR525. The primers used are shown in Table 3 below, and the constructed plasmid ( Figure 4 ) into Y2805.

[0051] table 3

[0052]

[0053] The plasmid c...

Embodiment 1-3

[0054] Examples 1-3: Observation of disease formation by ORF2 expressed in Saccharomycescerevisiae strains Poison-like particles

[0055] Among the ORF2 expression cassettes prepared in various ways, three yeast strains showing effective expression of ORF2 were selected based on Western blotting. In addition, the pGAL10-ORF2 plasmid without His tag expressing only ORF2 was prepared according to the method of Example 1-1, thereby preparing the following four plasmids: pGAL10-ORF2, pGAL10-ORF2-His, pGAL10-Ty1-ORF2(-NLS ) and pGAL-CBD-ORF2(-NLS)-His. Y2805 with each plasmid was cultured in YPDG for 48 hours at 30°C and 180 rpm. Each culture was centrifuged at 13,000 rpm for 10 minutes to harvest the cells. TEN buffer (10 mM Tris-HCl pH 7.4, 2 mM EDTA, 140 mM NaCl) and beads were added to the cells, followed by vortexing for about 10 minutes to lyse the cells. The cell lysate was centrifuged at 13,000 and 4°C for 20 minutes to collect the supernatant, which was then subject...

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Abstract

The present invention relates to a recombinant porcine circovirus (PCV2) subunit vaccine using yeast whole cells or lysates thereof and a method for manufacturing the same. The yeast whole cells or lysates thereof according to the present invention have an excellent effect as a vaccine composition, and can also significantly simplify processes, such as cell disruption, antigen extraction, purification, stabilization, and the like, which cannot be avoided in a procedure for manufacturing a porcine circovirus vaccine using various advantages of yeast and recombinant microorganisms.

Description

technical field [0001] The present invention relates to a recombinant porcine circovirus (PCV2) subunit vaccine for vaccinating sows and piglets, comprising recombinant yeast whole cells, and a method for its production And it can prevent porcine circovirus (PCV) related diseases. Background technique [0002] Porcine circovirus 2 (PCV-2) is known to be the main causative virus of postweaning multisystemic wasting syndrome (PMWS). The reason why it is difficult to develop a vaccine capable of preventing damage caused by porcine circovirus is that relatively expensive media and animal cell culture systems are required for the production of vaccines using animal cells, and various heterologous proteins contained in serum media may be in Side effects during vaccination. [0003] Four PCV2 vaccines based on the ORF2 antigen from the PCV2a virus are on the global market. In foreign countries, the company at the highest level of technology is Merial (Merial, France), which has ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/34A61K39/12
CPCA61K39/12A61K2039/552C12N2750/10023C12N2750/10034C12N1/18C12N15/11
Inventor 崔毅星朴景民徐成和安廷梧李殷教金千锡尹寅重俞成植沈宁贞
Owner KOREA RES INST OF BIOSCI & BIOTECH
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