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Novel double promoter structural unit

A dual-promoter and structural unit technology is applied to cells modified by introducing foreign genetic materials, DNA/RNA fragments, and vectors to introduce foreign genetic materials. It can solve the problems of high cost and expensive equipment, and achieve strong The effect of transcription efficiency

Inactive Publication Date: 2015-01-14
SHANGHAI MBR BIOMEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the entire process of biopharmaceuticals includes raw materials, expensive equipment, and high costs

Method used

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Examples

Experimental program
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Effect test

preparation example Construction

[0053] Preparation of the expression vector of the present invention

[0054] The expression vectors of the present invention can theoretically be prepared by conventional methods known in the art, such as those described by Sambrook et al. (1989). Sambrook also describes the functional components of the vector, such as appropriate promoters (except the hamster ubiquitin / S27a promoter), enhancers, termination and polyadenylation signals, antibiotic resistance genes, selectable markers, replication origin and splicing signals. The expression vectors can be prepared using conventional cloning vectors such as plasmids, phages, phagemids, cosmids, or viral vectors (such as baculoviruses, retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses), as well as synthetic or Artificial chromosomes / minichromosomes. Eukaryotic expression vectors typically also contain prokaryotic sequences, such as origins of replication and antibiotic resistance genes that allow...

Embodiment 1

[0092] Embodiment 1, the impact of different promoters on the expression of GFP

[0093] The promoter unit of the present invention and the traditional CMV promoter are used to construct reporter gene plasmids respectively, so as to evaluate the efficiency of the promoter unit to promote protein expression. The reporter gene plasmids are named respectively: Pcmv, Pcmv-Enhl, Pcmv-HBV333, Pcmv-HBV333-Pcmv plasmid map see figure 2 , image 3 , the reporter gene is Firefly luciferase gene. The plasmids were transfected into CHO cells, and the luciferase activity was detected. Specific steps are as follows:

[0094] (1) Add 4 μl of liposome Lipofactamine2000 into 150 μl of Opti-MEM medium.

[0095] (2) Add 2.5 μg of plasmid to 150 μl of Opti-MEM medium.

[0096] (3) Gently mix the above solutions and let stand at room temperature for 10 minutes.

[0097] (4) Add the above mixture into a 6-well cell culture plate (50% coverage of CHO cells).

[0098] (5) Continue culturing f...

Embodiment 2

[0100] Example 2, the influence of different promoters on the expression of Avastin antibody

[0101] The promoter structural unit of the invention is constructed as an expression vector, and the commercialized monoclonal antibody drug Avastin gene sequence is inserted downstream of it, see Figure 6 . At the same time, the traditional single CMV promoter was used to construct an expression vector, and the Avastin gene sequence was inserted downstream of it, see Figure 7 . Two plasmids containing Avastin light chain and heavy chain were electrotransfected into CHO cells, the specific process is as follows:

[0102] (1) Prepare 2×10 7 CHO cells in the exponential growth phase were added to two electric shock cups, each electrode cup contained 10 7 cells, with a volume of 0.7ml.

[0103] (2) Add 100 μl containing 40 μg plasmid DNA (20 μg each for the light and heavy chain plasmids) to the electric shock cup, set the voltage to 300 V, the capacitance to 900 μF, and set the ...

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Abstract

The invention belongs to the technical field of bioengineering and particularly relates to a novel double promoter structural unit. The novel double promoter structural unit disclosed by the invention comprises two CMV promoters shown in a nucleotide sequence SEQ ID NO. 2 and a sequence shown in a nucleotide sequence SEQ ID NO. 1. The double promoter structural unit is integrated by chromosomes, so that transcription or expression of a target gene in an expression system is increased. The promoter structural unit provided by the invention can be used for constructing expression vectors of cells of mammals and the constructed plasmid can be used for transient transfection for cells of mammals and construction of a stable expression cell strain. Compared with the conventional CMV promoter, the promoter unit provided by the invention is stronger in transcription efficiency.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a novel dual-promoter structural unit. Background technique [0002] In the entire biopharmaceutical industry, about 70% of protein drugs are expressed by Chinese hamster ovary (CHO) cells. At present, the CHO cell expression systems widely used in the market mainly include the DG44 cell expression system of the American Life Technologies Company and the CHO K1 expression system of the British Lonza Company. [0003] A common feature of the DG44 cell expression system / CHO K1 cell expression system is the use of a single-copy CMV promoter, which is a strong promoter that can efficiently drive the transcription of foreign genes in CHO cells. However, the entire process of biopharmaceuticals includes raw materials, expensive equipment, and high costs. Major pharmaceutical companies are paying more and more attention to how to improve protein expression levels, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/79C12N5/10C12P21/00
Inventor 陈亮王加龙
Owner SHANGHAI MBR BIOMEDICAL TECH
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