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Recombinant feline herpesvirus type 1 gB-gD protein and application thereof

A feline herpes virus, gb-gd technology, applied in the direction of recombinant DNA technology, application, virus, etc., can solve the problem that the infection or disease development of cats with virus can not be prevented, and achieve the improvement of stability and immunogenicity, expression High level, the effect of reducing production costs

Active Publication Date: 2021-12-28
SUZHOU MIDI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing FHV-1 vaccines confer reasonable protection against disease in naïve cats but do not prevent infection or disease development in infected cats

Method used

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  • Recombinant feline herpesvirus type 1 gB-gD protein and application thereof
  • Recombinant feline herpesvirus type 1 gB-gD protein and application thereof
  • Recombinant feline herpesvirus type 1 gB-gD protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1 Construction of recombinant eukaryotic expression vector pCI-gB-GS

[0062] 1. FHV-1 gB gene amplification and purification

[0063] The codon-optimized FHV-1 gB gene (SEQ ID NO: 1) was synthesized in Shanghai Sunny Biotechnology Co., Ltd. and cloned into the pUC-57 vector to obtain the pUC-gB plasmid vector. Using pUC-gB as a template and gB-F and gB-R as primers for PCR amplification (the gene sequences of gB-F and gB-R are shown in SEQ ID NO: 3 and 4), 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 cycles; extension at 72°C for 10 minutes, and storage at 4°C.

[0064] Table 1 FHV-1 gB gene amplification system

[0065]

[0066] Perform gel electrophoresis on the PCR product to identify the size of the target gene, such as figure 1 As shown, a band appeared near the positio...

Embodiment 2

[0081] Embodiment 2 Construction of recombinant eukaryotic expression vector pCI-gB-gD-GS

[0082] 1. Amplification and purification of FHV-1 gD gene expression cassette

[0083]The FHV-1 gD gene expression cassette was synthesized in Shanghai Sunny Biotechnology Co., Ltd. and cloned into the pUC-57 vector to obtain the pUC-gD plasmid vector. The expression cassette includes CMV promoter, codon-optimized gD gene (SEQ ID NO: 5) and SV40 polyA transcription termination signal. Using pUC-gD as a template and gD-F and gD-R as primers for PCR amplification (the gene sequences of gD-F and gD-R are shown in SEQ ID NO: 7 and 8), the amplification system is shown in Table 5. 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 cycles; extension at 72°C for 10 minutes, and storage at 4°C.

[0084] Table 5 FHV-1 gD gene expression cassette amplification system ...

Embodiment 3

[0103] Example 3 Construction and Screening of Recombinant CHO Cells

[0104] 1. Cell Transfection

[0105] 1.1 Cell preparation

[0106] Take CHO cells in the logarithmic growth phase, sample and count, and use 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.

[0107] 1.2 Plasmid and cell mixing

[0108] Take 5 μg of the pCI-gB-gD-GS plasmid vector in Example 2, add it to the EP tube, add 0.7ml of the CHO cell suspension obtained in step 1.1, mix well, and let stand for 15 minutes.

[0109] 1.3 Electric transfer

[0110] 280V 20ms electric shock for 2 pulses. Immediately after the electric shock ...

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Abstract

The invention discloses a recombinant feline herpesvirus type 1 gB-gD protein and application thereof. The recombinant feline herpesvirus type 1 gB-gD protein comprises a first protein having a sequence as shown in SEQ ID NO:2 and / or a second protein having a sequence as shown in SEQ ID NO:6. A vaccine comprises the recombinant feline herpesvirus type 1 gB-gD protein and a pharmaceutically acceptable carrier. The recombinant feline herpesvirus type 1 gB-gD protein provided by the invention is long in half-life period, high in molecular stability, capable of forming a dimer, especially a heterodimer, strong in immunogenicity, capable of being prepared in a bioreactor large-scale serum-free suspension culture mode through a mammalian cell expression system, high in expression level and good in protein immunogenicity; and the prepared vaccine also has the advantages of easy quality control, batch-to-batch stability, low production cost and the like.

Description

technical field [0001] The invention relates to a genetic engineering vaccine, in particular to a recombinant feline herpesvirus type 1 gB-gD protein and its application, such as the application in the preparation of feline herpesvirus type 1 recombinant subunit vaccine, which belongs to the technical field of animal immune medicines. Background technique [0002] Feline Herpesvirus type 1 (FHV-1) is the causative agent of feline infectious rhinotracheitis (FIR) and one of the main pathogens causing acute upper respiratory disease in felines. FHV-1 only infects cats, among which young cats are most susceptible, and the incidence rate can reach 100%. After FHV-1 infection, it mainly affects the upper respiratory tract and eyes of cats. The sick cats will have symptoms of upper respiratory tract such as elevated body temperature, depression, coughing and sneezing, as well as ocular symptoms such as purulent secretions in the eyes and keratoconjunctivitis. Infection can cause ...

Claims

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Application Information

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IPC IPC(8): C07K14/03C12N15/38C07K19/00C12N15/62C12N15/85C12N5/10G01N33/569A61K39/245A61P31/22
CPCC07K14/005C12N15/85G01N33/56994A61K39/12A61P31/22C07K2319/00C12N2710/16722A61K2039/552A61K2039/54C12N2710/16734G01N2333/03
Inventor 孔迪方鹏飞滕小锘曹文龙张大鹤
Owner SUZHOU MIDI BIOTECH CO LTD
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