siRNA for reducing expression of THEM6 gene, recombinant vector and application thereof

A recombinant vector and gene expression technology, which can be used in retro RNA viruses, DNA/RNA fragments, applications, etc., and can solve problems such as the lack of research on the biological function of THEM6 gene.

Active Publication Date: 2019-07-26
青岛言鼎生物医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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Method used

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  • siRNA for reducing expression of THEM6 gene, recombinant vector and application thereof
  • siRNA for reducing expression of THEM6 gene, recombinant vector and application thereof
  • siRNA for reducing expression of THEM6 gene, recombinant vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] This example is used to illustrate the high expression of THEM6 in breast cancer cell lines.

[0045] RNA extraction and reverse transcription quantitative PCR (RT-qPCR).

[0046] 1. Extraction of total RNA: this embodiment is carried out at low temperature. Cell culture was carried out in a 6-well plate, remove the medium, rinse with PBS 3 times, add Trizol 1000μl to each well, shake for 10min; collect into 1.5ml centrifuge tube, add 200μl chloroform to each tube, mix vigorously for 30sec, let stand for 15min , centrifuge at 12000rpm at 4°C for 15min; gently pipette 400μl of the upper layer into another new centrifuge tube, add an equal volume of isopropanol, gently invert and mix, centrifuge at 12000rpm at 4°C for 10min; discard the supernatant, add 1ml of 75% alcohol to wash the precipitate centrifuge at 12,000 rpm at 4°C for 10 min; discard the supernatant as much as possible, and dry it at room temperature for 10 min, add 10 μl of RNase-free water to each tube, di...

Embodiment 2

[0054] This example is used to illustrate that knocking down the expression of THEM6 gene can inhibit the proliferation and / or growth of breast cancer.

[0055] 1. Preparation of RNAi lentiviral clones

[0056] 1. Target design

[0057] For the THEM6 gene sequence, according to the RNAi sequence design principles, design three RNAi target sequences, the nucleotide sequences of three siRNAs of the THEM6 gene, and the nucleotide sequences of the negative control (NC). The corresponding names of the three siRNAs and the negative control are THEM6-si-1a, THEM6-si-1b, THEM6-si-2a, THEM6-si-2b, THEM6-si-3a, THEM6-si-3b, NC-si -a and NC-si-b are sequences designed for the negative control group. See Table 2 for details.

[0058] Table 2 Nucleotide sequences of 3 RNA interference targets (siRNA) and negative controls

[0059]

[0060]

[0061] Table 3 shows the nucleotide sequences of the three shRNAs used in the examples. The shRNAs used in the examples are THEM6-sh-1a, THE...

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Abstract

The invention belongs to the technical field of molecular biology and genetic engineering, and relates to siRNA for reducing the expression of a THEM6 gene, a recombinant vector and application thereof. The siRNA specifically reduces the expression of the THEM6 gene. Since the siRNA is capable of specifically reducing the expression of the THEM6 gene, the siRNA can be applied to the preparation ofa drug for treating and/or preventing breast cancer.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and genetic engineering, and more specifically, relates to a siRNA that reduces the expression of THEM6 gene, a shRNA corresponding to the siRNA, a DNA encoding the shRNA, a recombinant vector including the DNA, and a recombinant vector including the recombinant The recombinant lentivirus of the vector includes the siRNA, shRNA, DNA encoding shRNA, the host cell of the recombinant vector and the recombinant lentivirus, and their applications. Background technique [0002] Breast cancer is one of the most common malignant tumors in women worldwide, and its incidence rate and mortality rate rank first among all types of cancer. Although the incidence of breast cancer in my country is relatively low, the population base is large, the overall number of patients is large, the incidence rate is increasing year by year, and the age of onset is younger. Breast cancer exhibits great complexity ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/867C12N5/10A61P35/00
CPCA61P35/00C12N15/11C12N15/113C12N15/86C12N2310/14C12N2740/15043
Inventor 张虎杜欣娜杨留才胡明李春明
Owner 青岛言鼎生物医疗科技有限公司
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