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Porcine circovirus type II virus-like particle vaccine and preparation method thereof

A porcine circovirus and virus-like technology, applied in biochemical equipment and methods, viruses, viral peptides, etc., can solve the problems of high process requirements, long production cycle, poor protein immunogenicity, etc., and achieve simple cultivation operation and low production cost Low, good immune 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

However, prokaryotic expression is prone to inactive inclusion bodies, and protein renaturation is required to improve the vaccine effect. The cost is high, the production cycle is long (about 2-3 months per batch), and the efficiency is low. The obtained protein immunogen In particular, although the protein expressed by E. coli has been purified, endotoxin may still remain, and immunized animals will have side effects
[0008] B) Another class of virus-like particles (eukaryotic expression) expressed by the baculovirus / insect cell expression system to produce PCV2 virus-like particle vaccines by the baculovirus / insect cell expression system, using the ORF2 gene (Cap Protein) inserted into insect baculovirus, cultured with insect cells, to obtain recombinant PCV-2Cap protein, obtained after purification and mixing with immune adjuvant, clinically immunized by injection, eukaryotic expression requires high technology, Baculovirus particles may be mixed in the product, causing side effects and posing certain biosafety risks

Method used

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  • Porcine circovirus type II virus-like particle vaccine and preparation method thereof
  • Porcine circovirus type II virus-like particle vaccine and preparation method thereof
  • Porcine circovirus type II virus-like particle vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1, Construction of PCV Capsid Protein Recombinant Expression Vector pUKD-N115 / Cap

[0058] In this embodiment, the gene encoding the amino acid sequence of porcine circovirus capsid protein (also referred to as "porcine circovirus Cap protein" or "Cap protein" in the context of the present invention) has the sequence of SEQ ID No.2. 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.

[0059] The amino acid sequence of the encoded Cap protein can be as shown in SEQ ID No.1.

[0060] Using the PCR amplification method, the Cap gene was amplified with primers PCVN125-F (5'-GGGGCCCGGGACATATCCAAGGAGGCGTTTCAGAAG-3') and PCVN125-R (5'-GGGGGCGGCCGCTCACTTAGGGTTAAGTGGAGGGTCCTTAAG-3'). After 1% agarose gel electrophoresis, a fragment of about 700kb was recovered with a DNA gel kit (such as figure 1 ).

[0061] Provide pUKD-N115 vector, pUKD-N115 ve...

Embodiment 2

[0065] Example 2, Construction of PCV Capsid Protein Protein Recombinant Kluyveromyces marx Genetic Engineering Bacteria Fim-1ura3Δ-pUKD-N115 / Cap

[0066] A uracil auxotrophic strain Fim-1ura3Δ of Kluyveromyces martensis is provided, and the preparation method of the strain can adopt the construction method of the auxotrophic strain Fim-1 (ura3Δ) disclosed in Example 1 of Chinese patent publication CN105112313A.

[0067] 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).

[0068] Add carrier DNA, recombinant carrier pUKD-N115 / 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 rpm, discard ...

Embodiment 3

[0070] Example 3, PCV Cap protein expressed in Kluyveromyces marx genetically engineered bacteria Fim-1ura3Δ-pUKD-N115 / Cap

[0071] The constructed engineering bacteria Fim-1ura3Δ-pUKD-N115 / Cap and the control bacteria Fim-1ura3Δ were respectively inoculated in 50 ml of YP medium (1% Yeast Extract, 2% glucose), at 30°C, After culturing at 220rpm for 96 hours, the cells were collected by centrifugation, and the cells were broken by high-pressure homogenization. The cell lysate was subjected to protein electrophoresis SDS-PAG (polyacrylamide gel electrophoresis, PAGE) and PCV antibody immunoblotting Western Blot detection to analyze porcine circovirus (PCV) The expression level of Cap protein in Kluyveromyces. Anti-circovirus PCV pig serum polyantibody and goat anti-pig HRP enzyme-labeled secondary antibody were used for Western Blot.

[0072] From the protein electrophoresis detection and Western Blot detection results of cell lysates (such as image 3 and Figure 4 ), it ca...

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Abstract

The invention provides a kluyveromyces marxianus engineering bacterium obtained from recombinant expression of porcine circovirus type II capsid protein; and porcine circovirus type II virus-like particles are obtained by cloning porcine circovirus type II capsid protein genes into an expression vector and conducting recombinant expression in kluyveromyces marxianus. The invention also provides a preparation method of a porcine circovirus type II virus-like particle vaccine, wherein the preparation method comprises the following steps: preparing the porcine circovirus type II virus-like particles through high-density fermentation of the recombinant kluyveromyces marxianus, and then conducting separation and purification so as to prepare the vaccine. The porcine circovirus type II virus-like particle vaccine provided by the invention, which can generate a high-level serum IgG antibody just by implementing injection immunization once, has the advantages of being good in immunizing effect, high in safety, simple in culture operation, high in yield, low in production cost and the like, and the porcine circovirus type II virus-like particle vaccine can achieve large-scale enlarged production; and the porcine circovirus type II virus-like particle vaccine can be used for relieving and preventing related clinical symptoms caused by the infection of porcine circovirus type II (PCV2).

Description

technical field [0001] The invention relates to a recombinant porcine circovirus type II virus-like particle vaccine and a preparation method thereof, in particular to a method for preparing porcine circovirus II virus-like particle vaccine with high yield and low cost by using Kluyveromyces marxe, and A porcine circovirus type II virus-like particle vaccine with good immune effect and high safety. 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] PCV is very harmful to the pig industry and is widely prevalent in the world. After infection, pigs manifest as emaciation, swollen lymph nodes, panting, diarrhea, and reproduc...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12N7/04A61K39/125A61P31/14
CPCA61K39/12A61K2039/5258C07K14/005C12N7/00C12N15/815C12N2750/10023C12N2750/10034
Inventor 吕红周峻岗段进坤余垚
Owner FUDAN UNIV
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