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Equine herpesvirus type 1 gb-gd fusion protein, recombinant vector and eukaryotic cell strain and preparation method and vaccine thereof

A technology of equine herpes virus and fusion protein, applied in virus/bacteriophage, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of equine rhinopneumonia can not be prevented, achieve good recombination effect, good stability, The effect of high accuracy and success rate

Active Publication Date: 2022-07-29
天康制药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no subunit vaccine that can prevent equine rhinopneumonia

Method used

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  • Equine herpesvirus type 1 gb-gd fusion protein, recombinant vector and eukaryotic cell strain and preparation method and vaccine thereof
  • Equine herpesvirus type 1 gb-gd fusion protein, recombinant vector and eukaryotic cell strain and preparation method and vaccine thereof
  • Equine herpesvirus type 1 gb-gd fusion protein, recombinant vector and eukaryotic cell strain and preparation method and vaccine thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0051] The present invention provides a method for preparing the above-mentioned equine herpesvirus type 1 gB-gD fusion protein recombinant vector, comprising the following steps: inserting the equine herpesvirus type 1 gB-gD gene fragment into a eukaryotic expression vector fragment, and recombining and constructing to obtain Equine herpesvirus type 1 gB-gD fusion protein recombinant vector.

[0052] The method is simple and easy to operate, has good recombination effect, and has high accuracy and success rate.

[0053] In a preferred embodiment of the present invention, the preparation method comprises the following steps: treating the equine herpesvirus type 1 gB-gD gene fragment and the eukaryotic expression vector fragment respectively to obtain sticky ends, and then performing enzymatic ligation to obtain equine herpesvirus type 1 gB -gD fusion protein recombinant vector. The double restriction sites at both ends of the eukaryotic expression vector fragment and the equi...

Embodiment 1

[0086] Example 1 Construction of pcDNA3.1-gB-gD recombinant plasmid

[0087] The schematic diagram of the pcDNA3.1-gB-gD recombinant plasmid is as follows figure 1 shown.

[0088]1.1 After optimization of gB gene (GeneID: 1487545), the design length is 720 bp, and the gD gene (GeneID: 2948561) is designed to be 747 bp in length after optimization. The fragments of gB and gD genes are connected with the sequence 5'-ggaggaggaggatctggaggaggaggatct-3' to obtain the target fragment gB-gD gene, the full length of the target fragment is 1497bp. The upstream and downstream sites are designed respectively: HindIII, XbaI. The target fragment sequence is shown in Figure SEQ ID NO.1.

[0089] The synthesis of the target fragment with the restriction site was entrusted to Shanghai Sangon Biological Company.

[0090] 1.2 gB-gD gene and vector were subjected to double digestion reaction

[0091] 1.2.1 Construct a 50 μL reaction system: 10×buffer 5 μL, DNA sample 2 μg, HindIII 2.5 μL, Xb...

Embodiment 2

[0145] Example 2 Transfection of CHO cells and screening of monoclonal eukaryotic cell lines expressing equine herpes virus type 1 gB-gD fusion protein

[0146] 2.1 Transfection of CHO-K1 cells

[0147] 1) Take out the cells, discard the supernatant medium, wash once with pre-warmed 8 mL PBS, and discard the PBS;

[0148] 2) Add 2 mL of 0.25% trypsin-EDTA to each petri dish, digest at room temperature for 2 minutes, and observe the cells become round and single cells under the microscope;

[0149] 3) Add 4 mL of DMEM / F12 (containing 10% serum, 1% double antibody) to stop the digestion reaction, and blow off the cells with a pipette;

[0150] 4) Transfer to a 15mL centrifuge tube and centrifuge at 200rpm for 5min;

[0151]5) DMEM / F12 (containing 10% serum, 1% double antibody) resuspended cells and counted;

[0152] 6) Dilute the cells to 2×10 5 cells / mL, add 2 mL of mixed cells to a six-well culture dish, set at 37°C, 5% CO 2 Incubate overnight in a cell incubator;

[015...

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Abstract

The invention relates to the field of biotechnology, and in particular, provides a equine herpesvirus type 1 gB-gD fusion protein, a recombinant vector and a eukaryotic cell strain, a preparation method and a vaccine thereof. The present invention provides a equine herpesvirus type 1 gB-gD fusion protein recombinant vector, comprising a eukaryotic expression vector fragment and a equine herpesvirus type 1 gB-gD gene fragment. The recombinant vector can realize the simultaneous expression of equine herpesvirus type 1 glycoproteins gB and gD in an exogenous system at any time. The construction method provided by the present invention includes screening the CHO cells containing the above-mentioned recombinant vector to obtain a CHO cell strain stably expressing the equine herpes virus type 1 gB-gD fusion protein. The construction method is simple and easy to operate, and can successfully screen out a stable and highly expressing eukaryotic cell line of the equine herpes virus type 1 gB-gD fusion protein, which provides a feasible technical solution for subsequent studies including vaccine preparation and other applications.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a equine herpesvirus type 1 gB-gD fusion protein, a recombinant vector and a eukaryotic cell strain, a preparation method and a vaccine thereof. Background technique [0002] Equine rhinopneumonia is an equine animal infectious disease caused by two closely related equine herpesviruses, namely equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4). Equine herpes virus belongs to the genus Varicella virus and is highly contagious. Glycoproteins include gB, gC, gD, gE, gG, gI, gK, gM, gH and gL. Among them, gB and gD have been confirmed to induce neutralizing antibodies in infected hosts. [0003] EHV-1 has a broader host than EHV-4. EHV-1 can bind to various cellular receptors through gD, thereby entering host cells, and the resulting host range is very wide. On the other hand, the gD of EHV-4 can only bind to immobilized receptors, thus limiting the host range of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10C07K19/00A61K39/295A61K39/25A61P31/22
CPCA61K39/12A61P31/22C12N15/85C07K14/005A61K2039/552A61K2039/70C07K2319/00C12N2710/16722C12N2710/16734C12N2510/00
Inventor 贺笋任立松李延涛陈泽良任郭子君
Owner 天康制药股份有限公司
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