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Recombinant vaccinia virus carrying EB virus latent membrane antigen 2 gene and application of recombinant vaccinia virus

A technology of vaccinia virus and Epstein-Barr virus, applied in application, antiviral agent, genetic engineering, etc., to achieve rapid detection effect

Inactive Publication Date: 2017-12-19
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the above-mentioned vaccines have undergone phase I clinical trials, there is currently no basic experimental data and relevant literature support. This experiment discusses the construction of the MVA-LMP2A vaccine strain and the study of its immunological effects

Method used

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  • Recombinant vaccinia virus carrying EB virus latent membrane antigen 2 gene and application of recombinant vaccinia virus
  • Recombinant vaccinia virus carrying EB virus latent membrane antigen 2 gene and application of recombinant vaccinia virus
  • Recombinant vaccinia virus carrying EB virus latent membrane antigen 2 gene and application of recombinant vaccinia virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] 1. Example 1: Construction and Identification of PZL-GFP-LMP2 Recombinant Plasmid

[0045] 1.1 Acquisition of EBV LMP2 gene fragment

[0046] Pst1 and Not1 double digestion T-LMP2 plasmid (Zhang Lixia. EBV-LMP2 recombinant vaccinia virus immune effect research. Beijing University of Technology), the enzyme digestion system is 20ul (DNA plasmid 2μL; NEBuffer3.1 1μL; BSA: 2μL; Pst1 0.5μL ; Not 10.5 μL; ddH 2 O 14 μL), digestion at 37°C for 2 hours. Load 10 μL of the digested product, electrophoresis on 1% agarose gel at 110V, observe whether the target band appears under ultraviolet light, and recover the target fragment using Qiagen’s gel cutting recovery kit. The specific operation is as follows: according to 0.1 Add 300 μL of QG to g gel, mix 2-3 times intermittently at 50°C for 10 min; transfer supernatant to QIA quick spin column, centrifuge at 12000 rpm for 1 min; add 700 μL of PE Buffer, centrifuge at 12000 rpm for 1 min; centrifuge again at 12000 rpm for 1 min; ...

Embodiment 2

[0053] Embodiment 2: MVA-GFP-LMP2A recombinant virus construction

[0054] 2.1 Optimum conditions for the construction of MVA-GFP-LMP2A

[0055] Table 1 MVA-GFP-LMP2A Construction Conditions

[0056]

[0057] Note: "+" in the table represents the expression intensity of GFP under the fluorescence microscope.

[0058] rMVA (Sutter, G. and C. Staib, Vaccinia vectors as candidate vaccines: the development of modified vaccinia virus Ankara for antigen delivery. CurrDrug Targets Infect Disord, 2003.3 (3): p.263-71.) according to 10 -1 , 10 -2 , 10 -3 Infect the BHK-21 cells in the 6-well plate, infect two wells for each dilution, wash the infected cells twice with PBS after 2 hours, and use 4ug and 6ug of PZL-GFP-LMP2 plasmid content for each dilution For transfection (see Table 1), refer to the manual of Roche reagent (X-tremeGENE HP DNA Transfection Reagent manual) for specific operation steps. Place the infected cells in a cell incubator at 37°C, observe GFP expression u...

Embodiment 3

[0070] Embodiment 3: Obtaining and identification of MVA-LMP2A recombinant virus

[0071] 3.1 MVA-LMP2A recombinant virus harvest and morphology observation

[0072] Homologous recombination to obtain MVA-GFP-LMP2A, because the probability of homologous recombination is low, the virus is screened by the same extreme dilution method as above to obtain the MVA-LMP2A recombinant virus, that is, the GFP gene is removed, and only the white color carrying the EBV LMP2 foreign gene is obtained. For the recombinant virus, the virus was stored at -80°C. Observed under a fluorescent microscope, although the infected cells were diseased, there was no green fluorescent expression ( Figure 6 ). Freezing and thawing at -80°C and 37°C were repeated three times (to break the cells and release the virus), centrifuge at 3000rpm / min for 10min, absorb the supernatant, that is, MVA-LMP2A recombinant virus, and store at -80°C.

[0073] 3.2 PCR identification of EBV LMP2 gene expression

[0074...

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Abstract

The invention provides a recombinant vaccinia virus carrying an EB (epstein-barr) virus latent membrane antigen 2 gene and an application of the recombinant vaccinia virus. A preparation method of the recombinant virus comprises the steps of constructing a joint recombinant plasmid and MVA (modified vaccinia virus ankara) infection BHK-21 cell carrying an EBV (epstein-barr virus) LMP2 exogenous gene and a green fluorescin exogenous gene, and obtaining an MVA recombinant virus (MVA-LMP2A) only carrying the EBV LMP2 exogenous gene. Mouse spleen lymphocytes immunized by MVA-LMP2A are detected by an IFN (interferon)-gamma immunodotting method, and a result shows that MVA-LMP2A can well induce EBV LMP2 specific immune response generated in a body of a mouse.

Description

technical field [0001] The invention relates to the field of animal vaccines, in particular to an MVA-LMP2A recombinant virus carrying an EBV LMP2 exogenous gene and its application. Background of the invention [0002] Epstein-Barr virus (EBV) belongs to the gamma subfamily of herpesviruses, most of which are first infected in childhood, have no obvious clinical symptoms, and are carried for life. EBV is widespread in humans and is associated with many human neoplasms, including Burkitt lymphoma, nasopharyngeal carcinoma NPC, Hodgkin's disease HD, and lymphomas caused by lymphoproliferative disorders (PTLD) in a variety of immunosuppressed and transplanted patients and other diseases [1] . Therefore, the existence of this virus provides an important potential target for CTL-based tumor therapy. The latent membrane proteins LMP1 and LMP2 expressed by EBV play multiple tumorigenic roles by activating multiple signal transduction and regulating the expression of various tum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/863C12N15/38A61K39/245A61P35/00A61P31/22C12N5/0783
Inventor 曾毅王湛孙立莹
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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