Oral porcine circovirus II-like particle vaccine, and preparation method and application thereof

A porcine circovirus and defect-type technology, applied in biochemical equipment and methods, vaccines, viruses, etc., can solve problems such as low production efficiency, inability to take oral administration, and large side effects, and achieve low production costs, good immune effects, and safety sex high effect

Active Publication Date: 2017-02-15
FUDAN UNIV
View PDF4 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] Aiming at the defects of low production efficiency, large side effects and inability to take

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Oral porcine circovirus II-like particle vaccine, and preparation method and application thereof
  • Oral porcine circovirus II-like particle vaccine, and preparation method and application thereof
  • Oral porcine circovirus II-like particle vaccine, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0064] Example 1. Construction of PCV capsid protein recombinant expression vector pUKD-N125 / Cap

[0065] In this example, the gene encoding the amino acid sequence of the porcine circovirus capsid protein (also referred to as "porcine circovirus capsid Cap protein" or "Cap protein" in the context) has the sequence of SEQ ID No.3. According to the codon preference of Kluyveromyces marxianus, the Cap gene sequence was codon optimized, and the optimized Cap gene sequence was artificially synthesized.

[0066] The amino acid sequence of the encoded Cap protein can be as shown in SEQ ID No. 1, or the phenylalanine at position 226 in SEQ ID No. 1 is replaced by leucine (for example, SEQ ID No. 2).

[0067] Using PCR amplification method, PCVN125-F (5'-TTTTTTTGTTAGATCCGCGGATGACATATCCAAGGAGGCGTTTC-3') and PCVN125-R (5'-AGCTTGCGGCCTTAACTAGTTCA CTTAGGGTTAAGTGGAGGGTCCTTAAG-3') were used as primers to amplify the Cap gene. After 1% agarose gel electrophoresis, use DNA gel kit to recover fragme...

Example Embodiment

[0074] Example 2. Construction of PCV capsid protein Cap protein in Kluyveromyces marxianus genetically engineered strain Fim-1ura3Δ-pUKD-N125 / Cap and Cap expression analysis

[0075] The Kluyveromyces marxianus uracil auxotrophic strain Fim-1ura3Δ is provided, and the preparation method of the strain can refer to the construction method of the Fim-1(ura3Δ) strain disclosed in Example 1 of Chinese Patent Publication CN105112313A.

[0076] Fim-1ura3Δ was inoculated in a glass test tube containing 3 mL of YEPD medium and cultured in a shaker at 30°C overnight until the OD600 was 12-15. The cells were collected and washed with LiAc-TE solution (100mM LiAc, 10mM Tris-HCl, 1mM EDTA).

[0077] Add carrier DNA, recombinant vector pUKD-N125 / Cap, PEG solution (40% PEG4000, 100mM LiAc, 10mM Tris-HCl pH 7.5, 1mM EDTA) and final concentration of 10mM DTT to the bacteria in sequence. After mixing well, 30°C water bath for 15 minutes, 47°C water bath for 15 minutes, 8000rpm instant separation, di...

Example Embodiment

[0079] Example 3, Kluyveromyces marxianus genetically engineered strain Fim-1ura3Δ-pUKD-N125 / Cap expressing Cap protein and assembly of virus-like particles

[0080] The clones that were verified by PCR were inoculated into a flask containing 50ml YP medium (1% Yeast Extract, 2% glucose), cultured at 30°C and 220 rpm for 96 hours, then centrifuged to collect the cells, and homogenized by high pressure The cells were broken by the method, and the cell lysate was subjected to polyacrylamide gel electrophoresis (SDS-PAG) (polyacrylamide gel electrophoresis, PAGE) and PCV antibody western blotting and Western Blot to detect the expression of porcine circovirus (PCV) Cap protein in Kluyveromyces.

[0081] Compared with the control strain Fim-1ura3Δ (lane 1), the whole cell lysate and supernatant (lanes 2 and 3) of the genetically engineered strain Fim-1ura3Δ-pUKD-N125 / Cap has an extra protein in the size of 35-40kD Strips (e.g. image 3 ), the protein band is the Cap protein of porcine ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides kluyveromyces marxianus recombinant engineering bacteria and an application of the kluyveromyces marxianus recombinant engineering bacteria to the production of an oral porcine circovirus II-like particle vaccine. The recombinant engineering bacteria express porcine circovirus II capsid proteins through kluyveromyces marxianus expression vectors to prepare the oral porcine circovirus II-like particle vaccine. Regulation control elements of the kluyveromyces marxianus expression vectors provided by the invention are all from food-safety-level kluyveromyces, and have the characteristics of no antibiotic resistance genes and escherichia coli sequences. The invention further provides a method for preparing the oral porcine circovirus II-like particle vaccine. By utilizing the oral porcine circovirus II-like particle vaccine provided by the invention, related clinical symptoms caused by PCV2 infection can be reduced and prevented; and the oral porcine circovirus II-like particle vaccine has the advantages of good immune effect, high safety, simple culture operation, high yield, low production cost, capability of performing large-scale enlarged production and the like.

Description

technical field [0001] The present invention relates to an oral vaccine and its preparation method and application, in particular to an oral porcine circovirus vaccine and its preparation method and application Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) is one of the smallest animal viruses discovered so far. It belongs to the family Circoviridae and belongs to the genus Circovirus. -20nm, PCV is known to have two serotypes, namely porcine circovirus type I (PCV1) and porcine circovirus type II (PCV2), wherein PCV 1 is a non-pathogenic virus and PCV2 is pathogenic Virus. [0003] Although the genome structure of porcine circovirus is simple, it does great harm to animal husbandry. Porcine circovirus disease is one of the most important viral infectious diseases affecting the world's pig industry in recent years. [0004] Porcine circovirus type II (PCV2) infection in China has the following three characteristics: [0005] 1) The infection r...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N1/19C12N7/04A61K39/12A61P31/20A61P13/12A61P15/08A61P17/00C12R1/645
CPCA61K39/12A61K2039/5258A61K2039/53A61K2039/542C07K14/005C12N7/00C12N2750/10022C12N2750/10023C12N2750/10034
Inventor 吕红段进坤周峻岗余垚
Owner FUDAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap