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Recombinant Swine pox virus (SPV) vector vaccine for the expression of Streptococcus equi subsp zooepidemicus (SEZ) M-like protein (SzP)

A technology of recombining swine pox virus and Streptococcus equi is applied in the direction of antibacterial drugs, bacterial antigen components, biochemical equipment and methods, etc., to achieve broad application prospects, consistent antibody levels, and lasting protective effects.

Inactive Publication Date: 2011-09-28
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there has been no successful report on constructing a recombinant vaccine using pox virus as a vector, let alone a recombinant vaccine using a recombinant pox virus vector to express the M protein gene of Streptococcus equi subspecies zooepidemicus

Method used

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  • Recombinant Swine pox virus (SPV) vector vaccine for the expression of Streptococcus equi subsp zooepidemicus (SEZ) M-like protein (SzP)
  • Recombinant Swine pox virus (SPV) vector vaccine for the expression of Streptococcus equi subsp zooepidemicus (SEZ) M-like protein (SzP)
  • Recombinant Swine pox virus (SPV) vector vaccine for the expression of Streptococcus equi subsp zooepidemicus (SEZ) M-like protein (SzP)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Amplification, cloning, identification and sequencing analysis of embodiment 1SzP gene

[0038] 1.1 PCR primer design and synthesis

[0039] According to the gene sequence of the SEZ standard strain ATCC 35246, a pair of specific primers for the SzP gene (GenBank EU624402) were designed, and the 5' ends of the primers contained restriction endonucleases BamHI and SalI respectively. Primers were synthesized by Shanghai Invitrogen Biological Company.

[0040] SzP1: 5'-gtc gac gat tct gtt gag tca gct aag-3' (SEQ ID NO: 1)

[0041] SzP2: 5'-gga tcc tta tta gtt ttc ttt gcg tct tg-3' (SEQ ID NO: 2).

[0042] 1.2PCR amplification

[0043] Take 25.0 μL of 2*Taq PCR Mix, 11.0 μL of SzP, 1.0 μL of SzP2, 3.0 μL of SEZ bacteria (ATCC35246) solution, and make up to a total volume of 50.0 μL with sterile ultrapure water. Perform the reaction on a PCR machine. The cycle parameters were pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, ...

Embodiment 2

[0058] Preparation and determination of embodiment 2 recombinant pox virus

[0059] 2.1 Recovery, culture and cryopreservation of PK15 cells

[0060] Gently thaw a tube of frozen PK15 cells in a water bath at 37°C; carefully wash the outer wall with 70% alcohol and put it in an ultra-clean workbench; transfer the cells into a sterile 15mL centrifuge tube, add 10% Serum in MEM, centrifuge at 600g for 5min to pellet the cells; discard the supernatant, resuspend the cells with 10.0mL of 10% newborn bovine serum in MEM, then add to a 100mL cell flask; place in a 5% carbon dioxide incubator at 37°C to cultivate. Digest the cells with trypsin at room temperature for 1-3 minutes to separate the cells until a single layer is formed; tap the cell bottle wall to move the cells, and use an inverted microscope to observe whether the cells are rounded and separated; use new nutrient solution to separate the cells as needed bottle. In order to preserve the specific phenotype of a cell su...

Embodiment 3

[0074] The immune protection experiment of embodiment 3 recombinant swine pox virus vaccine

[0075] 3.1 Mice immunization test of recombinant swine pox virus

[0076] Take 32 4-week-old healthy and clean ICR mice and divide them into four groups randomly, 8 in each group; one group uses 2*10 7 The recombinant pox virus rSPV-SzP of PFU was immunized by intramuscular injection, and the other three groups were the negative control immunized with wild-type pox virus, the positive control immunized with inactivated ATCC 35246 bacteria and the blank control immunized with PBS; On the 14th day and 35th day after the initial immunization, the second and third immunizations were carried out in the same way; the experimental mice were observed every day to record their immune responses; On days 34, 41 and 48, blood was collected from the orbits of the mice, the serum was separated, and the antibody titer was measured by indirect ELISA.

[0077] see results figure 2 . The results s...

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Abstract

The invention belongs to the field of biological pharmacy. The invention provides a recombinant vaccine, comprising a SPV and one pharmaceutically acceptable vector and / or adjuvant or a plurality of such vectors and / or adjuvants. The SPV comprises an SPV vector and the encoding genes of SzP. The recombinant SPV vaccine provided in the invention can proliferate in large quantities in immune animalbodies, express target protein SzP and induce the generation of high titer antibodies in animal bodies, and exerts a good protective effect on immune animals.

Description

1. Technical field [0001] The invention belongs to the field of biological products. The present invention relates to a recombinant swine pox virus vector vaccine, in particular to a recombinant swine pox virus (rSPV-SzP) vector vaccine expressing Streptococcus equi zooepidemic subspecies (SEZ) class M protein (SzP). It can provide immune protection against SEZ and reduce the economic loss of pig industry. 2. Background technology [0002] Streptococcus equi subsp zooepidemicus SEZ is one of the important pathogens of swine streptococcosis in my country, and it is also an important pathogen of zoonosis. The Streptococcus suis disease caused by it was once widespread in South China, Southwest my country and East China, causing great harm to the pig industry in my country. Streptococcus suis is also an important bacterial infection that seriously endangers the pig industry in the world. disease and can cause infection in humans. [0003] M-like protein (M-Like Protein) is a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/09A61K48/00A61P31/04C12N15/863
Inventor 范红结蔺辉星陆承平
Owner NANJING AGRICULTURAL UNIVERSITY
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