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Preparation method and application of porcine pseudorabies virus subunit vaccine

A technique for porcine pseudorabies virus and immune response is applied in the field of preparation of porcine pseudorabies virus subunit vaccine, and can solve the problems of strong virulence of the strain, weak immune protection, and high cost of vaccine production

Active Publication Date: 2019-10-15
苏州世诺生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, inactivated vaccines have weak autoimmune protection and incomplete inactivation, resulting in the risk of spreading the virus; attenuated live vaccines have the risk of the strain returning to stronger virulence; subunit vaccines have high vaccine production costs and immunogenicity. The problem of being inferior to attenuated vaccines and inactivated vaccines

Method used

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  • Preparation method and application of porcine pseudorabies virus subunit vaccine
  • Preparation method and application of porcine pseudorabies virus subunit vaccine
  • Preparation method and application of porcine pseudorabies virus subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: Construction of recombinant eukaryotic expression vector pCI-gB-GS

[0049] 1.1. Construction of pUC-S1 plasmid vector

[0050] The codon-optimized gB gene is from Nanjing GenScript Biotechnology Co., Ltd., and the optimized gB gene sequence is shown in SEQ ID NO:1. In this example, the Escherichia coli cloning plasmid vector used in all the cloning steps is preferably pUC-57, and the codon-optimized gB gene was cloned into the pUC-57 vector to construct the pUC-gB plasmid vector.

[0051] 1.2. gB gene amplification

[0052] With pUC-gB as template, gB-F, gB-R are used as primers to carry out PCR amplification (wherein the gene sequence of gB-F is shown in SEQID NO.11, and the gene sequence of gB-R is shown in SEQ ID NO.12 ), the amplification system is shown in Table 1. The reaction conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 95°C for 45 seconds, renaturation at 60°C for 45 seconds, extension at 72°C for 2 minutes, 30 cycle...

Embodiment 2

[0072] Example 2: Construction and screening of recombinant CHO cells

[0073] 1. Cell Transfection

[0074] 1.1. Prepare the cells and take CHO cells in the logarithmic growth phase. The CHO cells can be CHO-DG44 cells or CHO-DXB11 cells or CHO-K1 cells or CHO-S cells. In this embodiment, CHO-S cells are selected for sampling and counting. Take 1×10 6 The cell density of cells / ml continues to be subcultured, maintain the seeds, centrifuge the remaining cells, centrifuge at 1000rpm for 4 minutes, discard the supernatant, resuspend with about 20ml of fresh CHO-WM medium, centrifuge again, centrifuge at 1000rpm for 4 minutes, discard the supernatant After resuspending with a small amount of medium for counting, the final cell density was adjusted to 1.43×10 7 cells / ml.

[0075] 1.2. Plasmid and cell mixing Take 5 μg of the pCI-gB-GS plasmid vector obtained in step 1.6 of Example 1, add it to an EP tube, add 0.7ml of cells, mix well, and let stand for 15 minutes.

[0076] 1.3...

Embodiment 3

[0087] Embodiment 3: SDS-PAGE detection

[0088] The harvested cell culture supernatants were subjected to SDS-PAGE respectively, and empty CHO cells were used as negative controls. The specific operation is as follows: take 40 μl of harvested cell culture, add 10 μl of 5×loading buffer, bathe in boiling water for 5 minutes, centrifuge at 12000 r / min for 1 minute, take the supernatant and carry out SDS-PAGE gel (12% concentration gel) electrophoresis, After electrophoresis, the gel was stained and decolorized to observe the target band.

[0089] The cell culture detection result of gained expression gB albumen is as follows Figure 16 As shown, the target band appears around the molecular weight of about 90kDa, and the negative control has no band at the corresponding position.

[0090] The cell culture detection result of gained expression gC protein is as follows Figure 17 As shown, the target band appears around the molecular weight of about 50kDa, and the negative cont...

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Abstract

The invention discloses a preparation method and application of a porcine pseudorabies virus subunit vaccine. The porcine pseudorabies virus subunit vaccine is prepared from porcine pseudorabies virusrelated proteins encoded by nucleic acid molecules of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:9 or encoded by nucleic acid molecules with more than 95% of nucleotide sequences the same as SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:9. The vaccine is expressed by eukaryotes, the antigenicity and immunogenicity of the porcine pseudorabies virus subunit vaccine are similar to that of natural proteins, the expression level is high, the immunogenicity is high, the protective effect is good, no pathogenicity is caused to animals, the vaccine can be prepared by large-scale serum-free suspension culture in a bioreactor, and meanwhile, the cost of vaccine production is greatly reduced.

Description

technical field [0001] The invention belongs to the technical field of animal vaccines, in particular to a preparation method and application of a porcine pseudorabies virus subunit vaccine. Background technique [0002] Pseudorabies is an acute infectious disease caused by a variety of domestic and wild animals caused by Pseudorabies virus (PrV). Pigs are the storage hosts of pseudorabies virus, and sick pigs, infected pigs and infected rodents are the main sources of infection for this disease. The disease is fulminant in pigs, and piglets and other susceptible animals are mostly acutely fatal, with obvious neurological symptoms, and the mortality rate is as high as 100%. Adult pigs are mostly recessively infected, showing reproductive disorders and respiratory symptoms. Abortion and stillbirth in pregnant sows, infertility in boars, dyspnea and growth stagnation in fattening pigs are one of the major infectious diseases that endanger the global pig industry. [0003] Ps...

Claims

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Application Information

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IPC IPC(8): A61K39/245A61P31/22C12N15/85C12N15/38G01N33/569G01N33/561
CPCA61K39/12A61P31/22C12N15/85C07K14/005G01N33/56983G01N33/561A61K2039/552A61K2039/575C12N2800/107C12N2710/16722C12N2710/16734C12N2710/16751
Inventor 曹文龙孔迪滕小锘易小萍张大鹤
Owner 苏州世诺生物技术有限公司
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