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Nucleotide sequence tWPRE and gene expression vector and application thereof

A technology of nucleotide sequence and gene expression, which is applied in the field of nucleotide sequence tWPRE and its gene expression carrier, can solve problems such as ignoring virus titer detection, achieve the effects of expanding the scope of action, efficient expression tools, and expanding the capacity of the carrier

Pending Publication Date: 2019-06-25
OBIO TECH SHANGHAI CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that the WPRE element that retains the gamma and alpha parts can be fused with SV40poly(A) to form a fragment of only 399bp in length, which can maintain the packaging of AAV virus and the expression of transgenes, but the detection of virus titer is ignored

Method used

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  • Nucleotide sequence tWPRE and gene expression vector and application thereof
  • Nucleotide sequence tWPRE and gene expression vector and application thereof
  • Nucleotide sequence tWPRE and gene expression vector and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0051] This embodiment discloses a method for constructing a pAAV-CAG-MCS-EGFP-3FLAG-tWPRE vector containing a tWPRE nucleotide sequence, comprising the following steps:

[0052] 1) Synthesizing the tWPRE nucleotide sequence shown in SEQ ID NO: 1 with restriction sites and upstream and downstream homology arms;

[0053] 2) The plasmid pAAV-CAG-MCS-EGFP-3FLA-WPRE-hGH polyA was double digested with AgeI and PmlI. 50μL enzyme digestion reaction system contains pLenti-CMV-SaCas9-P2A-Puro-linker-EGFP-S11 10ug, 10×Cutsmart (NEB) 5μL, Age I and BstE II each 2μL, make up to 50μL with water, digest at 37℃ for 4h Perform 1% agarose gel electrophoresis, cut off large fragments with a blade under ultraviolet light and perform recovery and purification.

[0054] 3) Perform seamless cloning of the purified enzyme-cleaved product obtained in step 2) and the synthetic sequence of SEQ ID NO: 1 in step 1). The 50 μL reaction system contained 2) 102.2 ng of digested product, 14.3 ng of synthet...

Embodiment 2

[0058] This embodiment discloses a method for cell transfection with the pAAV-CAG-MCS-EGFP-3FLAG-tWPRE vector containing the tWPRE nucleotide sequence, and the specific steps are as follows:

[0059] (1) Spread 293T cells (purchased from the ATCC cell bank in the United States) in 24-well plates;

[0060] (2) Discard the cell supernatant when the confluence of the cells reaches 70% to 80%, add Opti-MEM medium, and add the backbone plasmid (as a control group) or the pAAV-CAG- 0.5 μg of each MCS-EGFP-3FLAG-tWPRE plasmid (as the experimental group) was transfected into 293T cells by Lipo2000 reagent, and fluorescent photos were taken after 48 hours of transfection to compare the fluorescent signal to detect the transfection efficiency. The test results are as follows figure 2 As shown, it can be seen from the figure that the fluorescent signal of pAAV-CAG-MCS-EGFP-3FLAG-tWPRE plasmid transfection is stronger than that of the control group, indicating that the transfection effic...

Embodiment 3

[0062] This embodiment discloses a method for cell infection by AVV virus, comprising the following steps:

[0063] (1) packaging the backbone plasmid (as a control group) or the pAAV-CAG-MCS-EGFP-3FLAG-tWPRE (as an experimental group) obtained in Example 1 into an AAV virus;

[0064] (2) Cultivate 293T cells (purchased from the ATCC cell bank in the United States), spread 3 wells of a 24-well plate, and arrange infection when the cell confluence reaches about 50-70% the next day; when infecting, according to (number of cells × MOI value / virus stock solution Titer (MOI=5 and virus stock solution titer=4.76E+12v.g. / ml))×10 3 = virus addition (μl), add the corresponding virus volume in each hole; discard the medium after 12-20h of infection, add fresh cell culture medium; take pictures after 48h of infection, compare the fluorescent signal to detect the infection efficiency, from image 3 It can be seen that there is no significant difference in the infection efficiency of th...

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Abstract

The invention belongs to the field of genetic engineering and particularly relates to a nucleotide sequence tWPRE including a WPRE-polyA element and gene expression vector and application thereof. Thenucleotide sequence tWPRE including the WPRE-polyA element has the nucleotide sequence shown in SEQ ID NO:1. The gene expression vector comprises the nucleotide sequence tWPRE. The invention furtherimproves the WPRE-polyA element. The optimized tWPRE is obtained, the effectiveness of the tWPRE element is verified on the aspects of plasmid transfection and virus infection, the high-efficiency transgene expression efficiency and virus infection efficiency are guaranteed while the capacity of the vector is expanded, more specific promoters and genes can be carried by an AAV vector, the action range of an AAV virus is expanded, and a new more specific and efficient expression tool is provided for basic scientific research and clinical research.

Description

technical field [0001] The invention belongs to the field of genetic engineering, more specifically the invention relates to a nucleotide sequence tWPRE and its gene expression vector and application. Background technique [0002] The adeno-associated virus vector system (AAV), with its low immunogenicity, ability to infect both dividing and non-dividing cells, and the maintenance of long-range expression in host cells, has shown absolute promise in achieving gene therapy for human diseases compared with lentiviral vector systems Advantage. At present, the adeno-associated virus vector system (AAV) has been used in a variety of gene therapy abroad and entered clinical trials, and achieved good results, such as blindness, heart disease, muscular dystrophy, hearing loss, lung cancer, and Parkinson's brain damage. and other diseases. At the same time, there has not been any case of side effects or serious deaths in clinical trials using the adeno-associated virus vector as th...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/864C12N7/01C07K14/02
Inventor 杨蕊菊杨兴林刘晓芬夏清梅杨佳丽
Owner OBIO TECH SHANGHAI CORP LTD
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