Porcine circovirus 2 Cap-cell-penetrating peptide fusion protein gene with high expression and application thereof

A technology of porcine circovirus and fusion protein is applied in the field of highly expressed porcine circovirus type 2 Cap-penetrating peptide fusion protein gene, and can solve the problem of high cost of vaccine production

Active Publication Date: 2018-03-27
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PCV2 recombinant rod-shaped subunit vaccine has been commercialized, but the vaccine production cost is high

Method used

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  • Porcine circovirus 2 Cap-cell-penetrating peptide fusion protein gene with high expression and application thereof
  • Porcine circovirus 2 Cap-cell-penetrating peptide fusion protein gene with high expression and application thereof
  • Porcine circovirus 2 Cap-cell-penetrating peptide fusion protein gene with high expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Construction and Identification of Porcine Circovirus Type 2 Recombinant Baculovirus

[0051] In this example, the porcine circovirus type 2 (PCV2) ORF2 optimized codon gene was fused with the membrane-penetrating peptide ppTG20, TAT and honeybee signal peptide (HBM) genes by PCR method, and cloned into the baculovirus vector pFast-Bac-Dual, After the correct identification by enzyme digestion and gene sequencing, the DH10Bac competent cells were respectively transformed, and the clones that were correctly transposed to Bacmid were screened by two rounds of blue and white spots, and the genome was extracted and Bacmids were transfected into Sf9 insect cells with liposomes. After collecting the cell lysates, the recombinant baculoviruses rBac-cap, rBac-ppTG20-cap, rBac-TAT-cap and rBac-HBM-cap were obtained, and were identified by Western blot and immunofluorescence techniques. The results showed that all four recombinant viruses were Cap protein can be expres...

Embodiment 2

[0108] Embodiment two, four kinds of PCV2Cap recombinant protein mouse immune characteristic comparisons expressed by baculovirus

[0109] In this study, four kinds of Cap proteins expressed by recombinant baculovirus rBac-TAT-cap, rBac-ppTG20-cap, rBac-HBM-cap and rBac-cap were used to prepare PCV2 subunit vaccines TAT-cap, ppTG20-cap, HBM-cap and cap were used for immune experiments in mice. The results showed that all four vaccines could induce the production of PCV2 antibodies, but the neutralizing antibodies and cellular immunity induced by ppTG20-cap and TAT-cap recombinant virus subunit vaccines The level is obviously higher than that of cap subunit vaccine, which has important application prospects.

[0110] 1 Materials and methods

[0111] 1.1 Main materials and experimental animals

[0112] Recombinant baculovirus rBac-TAT-cap, rBac-ppTG20-cap, rBac-HBM-cap and rBac-cap expressing Cap protein were constructed and preserved by our laboratory. High Five cells (ATCC,...

Embodiment 3

[0136] Example 3 Study on the immune protection efficacy of PCV2Cap protein recombinant baculovirus vaccine in pigs

[0137] In this study, the recombinant protein expressed by recombinant baculovirus rBac-TAT-cap and rBac-ppTG20-cap was used to prepare the vaccine, and the pig immune protection test was carried out, and compared with the imported commercial PCV2 subunit vaccine, the results were: 2 ELISA antibodies could be detected in weeks. Four weeks after immunization, the neutralizing antibody levels of TAT-Cap group, ppTG20-Cap group and commercial vaccine group were 1:90, 1:85 and 1:72, respectively. The challenge test was carried out 4 weeks after immunization. Both TAT-Cap and ppTG20-Cap vaccines could provide immune protection for challenged piglets, and the viremia, pathological changes of lymph nodes and lung tissues, and viral load of lymph node tissues in immune pigs were significantly lower than those of non-infected piglets. In the immunization control group, ...

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Abstract

The invention discloses a porcine circovirus 2 (PCV 2) Cap-cell-penetrating peptide fusion protein gene with high expression and application thereof. Coding genes of the fusion gene refer to any kindof genes in (1)-(3): (1) Cap genes of fused cell-penetrating peptide TAT gene sequence; (2) Cap genes of fused cell-penetrating peptide ppTG20 gene sequence; and (3) Cap genes of fused bee signal peptide HBM gene sequence. Study of the research group discovers a DNA vaccine prepared by mixing a cell-penetrating peptide and pVAX-Cap plasmid is capable of improving the in-vivo immune response levelof mice. According to the study, the TAT, ppTG20, HBM and PCV2 Cap protein N end are fused, the recombinant baculovirus is successfully constructed, effective expression of the Cap proteins is realized, and a foundation is laid for researching genetically engineered vaccine PCV 2.

Description

technical field [0001] The invention belongs to the technical field of biological immunity, and in particular relates to a highly expressed porcine circovirus type 2 Cap-penetrating peptide fusion protein gene and application thereof. Background technique [0002] Porcine circovirus type 2 (PCV2) is an important pathogen that endangers the swine industry in the world. The capsid cap protein encoded by the PCV2 ORF2 gene has an important immune protection function and is an important antigen gene of the currently developed PCV2 recombinant subunit vaccine. PCV2 recombinant rod-shaped subunit vaccine has been commercialized, but the production cost of the vaccine is relatively high. Many molecular adjuvants, such as some cytokines, IL-18, GMSF, etc., work together with the immunogen to stimulate the body to produce a good immune response, thereby improving the immune effect of the vaccine. [0003] Honey Bee Melittin secretion peptide (HBM) has the function of helping the se...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/866C12N7/01C07K19/00A61K39/12A61P31/20
CPCA61K39/12C07K14/005C07K2319/02C07K2319/10C12N7/00C12N15/86C12N2710/14021C12N2710/14043C12N2750/10022C12N2750/10034
Inventor 姜平白娟朱雪娇董彦鹏王先炜
Owner NANJING AGRICULTURAL UNIVERSITY
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