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Expression vectors comprising the MCMV IE2 promoter

A technology for expressing vectors and promoters, applied in vectors, using vectors to introduce foreign genetic material, viruses/bacteriophages, etc.

Active Publication Date: 2006-04-12
MERCK SERONO SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] To date, no one has expressed heterologous proteins using the IE2 region of mCMV in a vector

Method used

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  • Expression vectors comprising the MCMV IE2 promoter
  • Expression vectors comprising the MCMV IE2 promoter
  • Expression vectors comprising the MCMV IE2 promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0140] Example 1: Evaluation of expression vectors in transient transfection

[0141] Materials and methods:

[0142] Material

[0143] Cells: CHO-S from Gibco / Invitrogen (Cat no 11619).

[0144] Following the manufacturer's protocol, isolate such as figure 2 and image 3 Plasmid DNA as indicated.

[0145] Transfection:

[0146] Transfection reagent: Lipofectamine (Invitrogen, Cat No 18324-012)

[0147] Format: 24-well plate

[0148] Cells: CHO-S cells in exponential growth phase were passaged 24 hours before transfection. To avoid a stationary phase of low cell density, dilute the cells to 0.75 x 10 6 cells / ml. The total amount of cells to be transfected is 1.5 x 10 5 , resuspended in a 24-well plate with 100 μl of serum-free medium SFM II (Invitrogen, Cat No 12052-114) per well.

[0149] The transfection mix was as follows:

[0150] A) Lipofectamine: 2μl

[0151] SFM II medium: 48 μl

[0152] Total volume: 50μl

[0153]B) DNA: 1 μg (50ng expression vector...

Embodiment 2

[0165] Example 2: Evaluation of expression vectors in stable transfection

[0166] Materials and methods:

[0167] method:

[0168] Cells: CHO-S from Gibco / Invitrogen (Cat no 11619).

[0169] Plasmid DNA ( figure 2 ).

[0170] Transfection:

[0171] Transfection reagent: Lipofectamine (Invitrogen, Cat No 18324-012)

[0172] Stable transfection uses T75 flasks. CHO-S cells in exponential growth phase were passaged 24 hours before transfection. To avoid a stationary phase of low cell density, dilute the cells to 0.75 x 10 6 cells / ml. The total amount of cells to be transfected is 5 x 10 6 , resuspended in 7 ml of serum-free medium SFM II (Invitrogen, Cat No 12052-114) in a T75 flask.

[0173] The transfection mix was as follows:

[0174] A) Lipofectamine: 52.1μl

[0175] SFM II medium: 517.9 μl

[0176] Total volume: 570 μl.

[0177] B) DNA: 10 μg linearized plasmid DNA, (9 μg luciferase expression vector + 1 μg selection plasmid: SV40 promoter driving the pur...

Embodiment 3

[0189] Example 3: Co-expression of two polypeptides of interest with bidirectional expression vectors

[0190] Materials and methods:

[0191] Transfection was carried out with reference to the method of Examples 1 and 2. Briefly, 900, 500, 300 and 100 ng of vector DNA (construct C-2, see image 3 ) transiently transfected CHO-S cells (suspension, Gibco SFMII) (each condition was repeated three times). Two days after transfection, cell extracts were assayed for luciferase in RLU (relative luminance units) in triplicate. The supernatants from these wells were removed before the cells were lysed, confluent, and analyzed for IL-18BP by ELISA (see below).

[0192] ELISA test of IL-18BP

[0193] The amount of recombinant human IL-18BP (rhIL-18BP) in the supernatant was determined in a standard ELISA assay using a biotin-conjugated and protein G-purified monoclonal anti-rh-IL-18BP antibody. Extravidine-HRP (Sigma) was used as the detection reagent.

[0194] Figure 9 Shown is...

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Abstract

The invention relates to an expression vector comprising the promoter of the mCMV-IE2 gene, or a functional expression promoting fragment thereof, and / or an enhancer of the mCMV-IE2 gene, or a functional expression enhancing fragment thereof, wherein the expression vector does not contain any complete gene of the mCMV.

Description

technical field [0001] The present invention relates to an expression vector containing mCMV-IE2 gene promoter or its functional expression promoter fragment, and / or mCMV-IE2 gene enhancer or its functional expression enhancement fragment, wherein the expression vector does not contain any whole mCMV gene, the present invention The invention also relates to host cells containing these vectors, methods of using these expression vectors to produce desired polypeptides, and uses of said expression vectors. [0002] Expression vectors containing the mCMVIE2 promoter and the mCMVIE1 promoter, optionally also the novel mCMVIE2 enhancer, are preferred embodiments of the present invention, especially when the two promoters are arranged in a bidirectional manner. Background of the invention [0003] For decades, expression vectors have been used as vehicles for the expression of genes or cDNAs encoding polypeptides or proteins of interest in host cells. Traditionally, strong viral o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C07K14/045C12N5/10A61K35/12A61K35/76A61K48/00C12NC12N15/85
CPCC12N2830/205C12N2830/60C12N2710/16122C12N15/85A61P43/00C12N15/86C12N5/10C07K14/045C12N2710/16143
Inventor P·沙特拉德M·伊姆霍夫
Owner MERCK SERONO SA
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