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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
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  • 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 orally in the current PCV2 vaccine, the present invention provides an orally available PCV2 vaccine

Method used

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  • 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

Embodiment 1

[0064] Embodiment 1, the construction of PCV capsid protein recombinant expression vector pUKD-N125 / Cap

[0065] In this embodiment, the gene encoding the amino acid sequence of porcine circovirus capsid protein (hereinafter also referred to as "porcine circovirus capsid Cap protein", or "Cap protein") has the sequence of SEQ ID No.3. According to the codon preference of Kluyveromyces marx, the codons of the Cap gene sequence were 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 (such as SEQ ID No.2).

[0067] Using the method of PCR amplification, the Cap gene was amplified with primers PCVN125-F (5'-TTTTTTTTGTTAGATCCGCGGATGACATATCCAAGGAGGCGTTTC-3') and PCVN125-R (5'-AGCTTGCGGCCTTAACTAGTTCA CTTAGGGTTAAGTGGAGGGTCCTTAAG-3'). After 1% agarose gel electrophoresis, a fragment of about 700...

Embodiment 2

[0074] Example 2, Construction of PCV Capsid Protein Cap Protein in Kluyveromyces marx Genetic Engineering Bacteria Fim-1ura3Δ-pUKD-N125 / Cap and Cap Expression Analysis

[0075] A uracil auxotrophic strain Fim-1ura3Δ of Kluyveromyces marxense 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 overnight on a shaker at 30°C until the OD600 was 12-15. The cells were collected and washed with LiAc-TE solution (100 mM LiAc, 10 mM Tris-HCl, 1 mM EDTA).

[0077] Add carrier DNA, recombinant carrier pUKD-N125 / Cap, PEG solution (40% PEG4000, 100mM LiAc, 10mM Tris-HCl pH 7.5, 1mM EDTA) and a final concentration of 10mM DTT to the cells in sequence, and mix thoroughly. Water bath at 30°C for 15 minutes, water bath at 47°C for 15 minutes, spin off at 8000 ...

Embodiment 3

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

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

[0081]Compared with the control bacteria Fim-1ura3Δ (lane 1), there is an extra protein of 35-40kD in the whole cell lysate and supernatant (lanes 2 and 3) of the genetically engineered bacteria Fim-1ura3Δ-pUKD-N125 / Cap strips (such as image 3 ), this protein band is the Cap protein of porcin...

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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

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Application Information

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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
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