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shRNA expression vector expressed by specificity inhibitor set protein, construction method and application thereof

An expression vector and protein expression technology, applied in the field of genetic engineering, can solve the problems of low transfection efficiency, large amount of vector, unstable interference effect, etc., so as to overcome the problems of low transfection efficiency, stable interference effect and high transfection efficiency. Effect

Active Publication Date: 2011-01-19
上海埃秀马生物科技有限公司
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  • Application Information

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Problems solved by technology

[0006] In the prior art, no shRNA expression vectors with long-term stable expression and specific inhibition of SET protein expression have been found, and other existing shRNA expression vectors often have low transfection efficiency, unstable interference effects, large amount of vectors, and Defects such as multiple transient transfections are often required

Method used

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  • shRNA expression vector expressed by specificity inhibitor set protein, construction method and application thereof
  • shRNA expression vector expressed by specificity inhibitor set protein, construction method and application thereof
  • shRNA expression vector expressed by specificity inhibitor set protein, construction method and application thereof

Examples

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Effect test

Embodiment 1

[0046] Design and Synthesis of Short Hairpin Circular RNA (shRNA) Oligonucleotide Strands Targeting Human SET

[0047] In this example, a lentiviral interference recombinant vector targeting human SET gene was constructed using lentivirus-mediated RNA interference technology, and the virus was packaged and transfected into L-02 hepatocytes. SET protein and mRNA levels were used to verify the expression of SET, and the best interference fragment that efficiently inhibited SET expression was screened out, and an shRNA lentiviral expression vector that efficiently inhibited SET expression was constructed. The specific methods and steps are as follows:

[0048] Materials The lentiviral expression vector pLVX-shRNA1 vector and lentiviral transfection packaging kit (Lenti-X Packaging system) were purchased from Clontech Company, with human U6 promoter, puromycin (puromycin) resistance resistance gene, BamH I and EcoR I restriction site (see figure 1 ); Restriction enzymes BamH I an...

Embodiment 2

[0055] Construction of shRNA recombinant plasmids

[0056] Dissolve and dilute the top strand and bottom strand of oligonucleotide chains synthesized by Shanghai Sangon Company to 100 μmol with TE buffer, take equimolar top strand and bottom strand, heat at 95°C for 30s, 72°C for 2min, 37°C for 2min, 25°C After 2 minutes, put it on ice to form double-stranded shRNA oligonucleotide chains with sticky ends of BamHI and EcoR I at the 5' and 3' ends respectively. The lentiviral vector pLVX-shRNA1vector was digested with BamH I and EcoR I enzymes at 37°C for 3 hours, and the 7741bp linear fragment was recovered by gel after digestion. The annealed double-stranded shRNA oligonucleotide strands were ligated with the recovered linear fragment carrier overnight at 16°C. The ligation product was transformed into competent DH-5α Escherichia coli, and the bacterial solution was evenly spread on the agar plate containing ampicillin (100 μg / ml), and cultured overnight in a constant tempera...

Embodiment 3

[0058] Lentiviral packaging and transduction

[0059] One day before transfection and packaging, inoculate 293T cells in a 10 cm dish and culture them with tetracycline-free fetal bovine serum DMEM medium to ensure that the confluence reaches about 60% during transfection. Add 15 μl of Lenti-X Packaging Mix (containing mixed packaging plasmids), 3 μg each of psiRNA1, psiRNA2, psiRNA3, psiRNA4, psiRNA5, psiRNAc recombinant vector and pLVX empty plasmid identified by sequencing, carry out lentiviral packaging in 293T cells, and collect after 48 hours The virus supernatants of each group were transduced into L-02 hepatocytes, and a blank control group was set up. Before transduction, L-02 hepatocytes were divided into 1 × 10 6 / mL density inoculated in 6cm dishes, each well containing 5mL RPM I-1640 medium without antibiotics. When the cell confluency reached 70%, virus transduction was carried out, and after 8 hours, it was replaced with RPM I-1640 complete medium containing 1...

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Abstract

The invention discloses a shRNA expression vector expressed by specificity inhibitor SET protein, a construction method and an application thereof. The shRNA expression vector comprises a fundamental sequence, a resistance gene sequence, a polyclone locus sequence, a promoter sequence and the expressible oligonucleotides chain formwork sequence of SET shRNA. The expressible oligonucleotides chainformwork sequence of SET shRNA is forwardly inserted into the polyclone locus sequence of the expression vector and designed according to the SET mRNA target sequence. In the invention, the lentivirus vector serves as the mediator to construct the targeted human SET gene interference vector, which has the characteristics of high transfection efficiency and stable interference effect, overcomes the defects that the transfection efficiency of the plasmid or retrovirus is low and the interference effect thereof is not stable and provides powerful tools for the filed of scientific research of SET protein functions and for treating diseases related to abnormal expression of SET protein.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a shRNA expression vector capable of stably expressing in mammalian cells and capable of specifically inhibiting the expression of SET protein and an expression method of the vector. Background technique [0002] The oncoprotein SET, also known as I2PP2A and TAF-Iβ, exists widely in cells and is the main intracellular inhibitory protein of serine-threonine protein phosphatase 2A (protein phosphatase 2A, PP2A) in the body. It has specificity and non-competition and thermal stability, while SET is also a histone chaperone. It was identified for the first time in acute undifferentiated leukemia. Recent studies have shown that SET is a multifunctional protein involved in apoptosis, tumorigenesis, transcription regulation, nucleosome assembly, and histone binding. Madeira A et al. reported that SET protein was involved in the process of neuron apoptosis in Alz...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/867C12N15/113C12N15/66A61K48/00A61P1/16A61P35/00A61P43/00
Inventor 刘建军蒋英芝杨细飞李杰周丽
Owner 上海埃秀马生物科技有限公司
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