Pig parvovirus immune composition as well as preparation method and application of pig parvovirus immune composition

A technology of parvovirus and vaccine composition, which is applied in the field of porcine parvovirus vaccine composition and its preparation, can solve the problems of inability to induce cellular immune response, poor safety, large dosage, etc., and achieve reduction and prevention of related diseases, good Immunogenic effects

Pending Publication Date: 2018-02-06
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, inactivated vaccines have the advantages of good safety, long antibody production time, and no need for low-temperature storage. However, inactivated vaccines produce antibodies slowly and cannot induce cellular immune responses. Compared with live vaccines, the antibody level is lower. Repeated vaccination is required, and the dosage is large. ; The attenuated vaccine has strong immune efficacy, rapid antibody production, less dosage, and low cost, but its safety is poor; the genetic engineering subunit vaccine has better immunogenicity, but its cost and technical requirements are relatively high; the genetic engineering live virus vector Vaccines can prevent two or more diseases at the same time and can induce a strong immune response, but the production technology is complicated and the cost is high

Method used

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  • Pig parvovirus immune composition as well as preparation method and application of pig parvovirus immune composition
  • Pig parvovirus immune composition as well as preparation method and application of pig parvovirus immune composition
  • Pig parvovirus immune composition as well as preparation method and application of pig parvovirus immune composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Construction of Porcine Parvovirus VP2 Protein Kluyveromyces marx Recombinant Expression Vector

[0050] The amino acid sequence encoding porcine parvovirus capsid protein VP2 is shown in SEQ ID No.1. According to the codon preference of Kluyveromyces marxis, the codon optimization of the gene sequence of VP2 was carried out without changing the amino acid sequence. The optimized sequence is shown in SEQ ID No.2. Insert the optimized VP2 gene sequence into Kluyveromyces marx expression vector pUKD-N125 to construct the recombinant expression vector-PUKD-N125 / VP2, the specific process is as follows:

[0051] with specific primers

[0052] VP2-N125-F (SEQ ID No. 3)

[0053] 5'-TTTTTTTGTTAGATCCGCGGATGAGCGAAAACGTGGAGC-3'

[0054] VP2-N125-R (SEQ ID No. 4)

[0055] 5'-AGCTTGCGGCCTTAACTAGTCTAGTACAACTTTCTTGGG-3'

[0056] The VP2 gene was amplified by PCR, and the amplified product was subjected to 1% agarose gel electrophoresis to recover a fragment of about 174...

Embodiment 2

[0057] Example 2 Construction of Kluyveromyces marx genetically engineered bacteria Fim-1-pUKD-N125 / VP2

[0058] The yeast expression host strain used in the present invention is Kluyveromyces marxense Fim-1ura3Δ, and the preparation method of this strain can be constructed by the method disclosed in Example 1 of the Chinese patent publication CN105112313A for the auxotrophic strain Fim-1ura3Δ. Porcine parvovirus VP2 protein Kluyveromyces marx genetic engineering strain Fim-1-pUKD-N125 / VP2 is obtained by transforming the recombinant expression vector pUKD-N125 / VP2 to construct the Fim-1ura3Δ strain, the implementation process is as follows: The yeast 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 pH 7.5, 1 mM EDTA). Add carrier DNA, recombinant expression vector pUKD-N125 / VP2, PEG solution ...

Embodiment 3

[0060] Example 3 Expression of porcine parvovirus VP2 protein in Kluyveromyces marx

[0061] Recombinant engineered bacteria Fim-1-pUKD-N125 / VP2 and control bacteria Fim-1ura3Δ were respectively inoculated in 50ml of YD medium (1% yeast extract, 2% glucose), cultured at 30°C, 220rpm for 66h and then centrifuged The cells were collected, resuspended with 10 mM Tris-HCl (pH 7.5), and then homogeneously disrupted by high pressure. Take 100 μl of cell lysate and add protein electrophoresis loading buffer to mix, boil water for 5 minutes, then conduct protein electrophoresis SDS-PAGE (polyacrylamide gelelectrophoresis, PAGE) and Western Blot with PPV-VP2 antibody to detect the expression level of VP2 protein. The PPV-VP2 antibody used for Western Blot detection is a polyantibody serum isolated from mice immunized with PPV-VP2 protein recombinantly expressed in Escherichia coli, and the secondary antibody is a goat anti-mouse polyantibody labeled with horseradish peroxidase.

[006...

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Abstract

The invention discloses a pig parvovirus immune composition which contains the following a) and b), or is basically prepared from the following a) and b), or is prepared from the following a) and b):a) viroid particles formed by self-assembly of pig parvovirus VP2 protein expressed by Kluyveromyces marxianus recombinant bacterium; b) pharmaceutically or veterinarily acceptable accessories used for injection preparations. The invention also discloses a preparation method and application of the pig parvovirus immune composition. Pig parvovirus viroid particle vaccine injection immunization canobtain high-level serum IgG antibodies, has good immunogenicity and can reduce and prevent related diseases caused by pig parvovirus infection.

Description

technical field [0001] The invention relates to the fields of biotechnology and veterinary medicine, in particular to a porcine parvovirus vaccine composition and a preparation method and application thereof. Background technique [0002] Porcine parvovirus (porcin parvovirus, PPV) is one of the important pathogens that cause reproductive disorders in pigs. Aberdeen can also cause skin diseases and diarrhea. In 1966, British Mary et al. discovered the virus when they used pig kidney primary cells for tissue culture of swine fever virus. In 1967, Cartwright et al. isolated porcine parvovirus from aborted pig fetuses, and verified its pathogenicity for the first time. Subsequently, the virus was also reported in Japan, the Netherlands and other parts of the world. Since the 1980s, it has been reported from all over the country. Different genotypes of PPV were also isolated successively. Through the prevalence and reports of various countries in the world in recent years, it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/23A61P31/20A61P1/12A61P9/00A61P11/00A61P17/00C07K14/015C12N15/81
Inventor 吕红陈蕾余垚周峻岗
Owner FUDAN UNIV
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