Equine herpesvirus 1 gB-gD fusion protein, recombinant vector, eukaryotic cell strain, preparation method of recombinant vector, and vaccine

A equine herpes virus and fusion protein technology, applied in the directions of virus/bacteriophage, chemical instruments and methods, biochemical equipment and methods, etc., can solve problems such as no equine rhinopneumonia can be prevented, and achieve good recombination effect, good stability, Effects with high accuracy and success rate

Active Publication Date: 2019-01-04
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 1 gB-gD fusion protein, recombinant vector, eukaryotic cell strain, preparation method of recombinant vector, and vaccine
  • Equine herpesvirus 1 gB-gD fusion protein, recombinant vector, eukaryotic cell strain, preparation method of recombinant vector, and vaccine
  • Equine herpesvirus 1 gB-gD fusion protein, recombinant vector, eukaryotic cell strain, preparation method of recombinant vector, and vaccine

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preparation example Construction

[0051] The present invention provides a method for preparing the above 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 the eukaryotic expression vector fragment, and recombining 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 high accuracy and success rate.

[0053] In a preferred embodiment of the present invention, the preparation method includes the following steps: separately processing the equine herpesvirus type 1 gB-gD gene fragment and the eukaryotic expression vector fragment to obtain cohesive ends, and then performing enzyme 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 equine herpesvirus type 1 gB-gD gen...

Embodiment 1

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

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

[0088]1.1 After optimization, the gB gene (GeneID: 1487545) is designed to be 720bp in length, and the gD gene (GeneID: 2948561) is optimized to be 747bp in length. The gB and gD gene fragments 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. Restriction sites were designed upstream and downstream: HindⅢ, XbaI. The target fragment sequence is shown in Figure SEQ ID NO.1.

[0089] The synthesis of target fragments with enzyme cleavage sites was entrusted to Shanghai Sangon Biotechnology Co., Ltd.

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

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

Embodiment 2

[0145] Example 2 Transfection of CHO cells and screening of monoclonal eukaryotic cell lines expressing equine herpesvirus 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 8mL PBS, and discard the PBS;

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

[0149] 3) Add 4mL DMEM / F12 (containing 10% serum, 1% double antibody) to terminate the digestion reaction, and blow the cells away 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 cells to 2×10 5 cells / mL, take 2mL of mixed cells and add them to a six-well culture dish, place at 37°C, 5% CO 2 Incubate overnight ...

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Abstract

The invention relates to the technical field of biology and particularly provides equine herpesvirus 1 gB-gD fusion protein, a recombinant vector, a eukaryotic cell strain, a preparation method of therecombinant vector, and a vaccine. The equine herpesvirus 1 gB-gD fusion protein recombinant vector provided herein comprises a eukaryotic expression vector fragment and an equine herpesvirus 1 gB-gDgene fragment; the recombinant vector herein allows both equine herpesvirus 1 glycoproteins gB and gD to be expressed in an exogenous system at any time. A construction method provided herein includes: screening CHO (Chinese hamster ovary ) cells containing the recombinant vector are screened to obtain a CHO cell strain that may stably express the equine herpesvirus 1 gB-gD fusion protein; the construction method is simple, easy to perform, and suitable for successful screening of eukaryotic cell strains to stably and highly express eukaryotic equine herpesvirus 1 gB-gD fusion protein; a feasible technical scheme is provided for subsequent various researches, such as vaccine preparation and other applications.

Description

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

Claims

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Application Information

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Patent Type & Authority Applications(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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