SiRNA lentivirus vector of human STAT3 gene and construction method thereof

A technology of lentiviral vector and construction method, which is applied in the field of construction of human STAT3 gene SiRNA lentiviral vector, which can solve the problems of unregulated cell growth and uncontrolled proliferation

Inactive Publication Date: 2011-11-23
马玲娣
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Under normal circumstances, the activation of STAT3 plays an important regulatory role in cell proliferation and

Method used

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  • SiRNA lentivirus vector of human STAT3 gene and construction method thereof
  • SiRNA lentivirus vector of human STAT3 gene and construction method thereof
  • SiRNA lentivirus vector of human STAT3 gene and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1 STAT3SiRNA interference carrier construction and identification

[0072] According to the full-length sequence of STAT3 mRNA (the Genebank accession number of the human STAT3 gene mRNA sequence used is NM_139276), use the SiRNA online design software B LOCK-iT TM RNAiDesigner (https: / / rnaidesigner.invitrogen.com / rnaiexpress / rnaiExpress.jsp) designed 4 pairs of double-stranded oligonucleotide sequences containing the STAT3 gene SiRNA sequence (see Table 1 for the oligonucleotide sequence), using any The siRNA double-stranded gene interference was used as a negative control (PSC-NC), and 4 pairs of STAT3 SiRNA oligonucleotide sequences were synthesized (synthesized by Invitrogen, USA).

[0073] Table 1 Double-stranded oligonucleotide containing STAT3 gene siRNA sequence

[0074]

[0075]

[0076] Note: The underlined part is the base complementary region (from 5'→3'), the bold part is the STAT3 siRNA interference target sequence, and the boxed part is...

Embodiment 2

[0085] Example 2 STAT3SiRNA interference effect detection

[0086] The day before infection, 293T cells in good growth state were divided into 5×10 cells per well 5 Cells were inoculated in a six-well plate for infection. The specific method was as follows: wash the cells once with serum-free medium and then add 1.5mL of cell culture medium; dilute 4ug of interference plasmid and negative control plasmid with 250uL medium respectively, and mix gently; Dilute 10uL Lipofectamine 2000 reagent with 250uL cell culture medium, mix gently, and incubate at room temperature for 5 minutes; mix the diluted plasmid and Lipofectamine 2000 dilution, and place at room temperature for 20 minutes; slowly add to each cell well, and mix gently; Place at 37°C, 5% CO 2 After culturing in the incubator for 4-6 hours, replace the complete culture medium without antibiotics and continue culturing overnight. The cells were collected 48 hours after infection, the total RNA was extracted according to ...

Embodiment 3

[0096] Example 3 STAT3SiRNA Western-blot detection

[0097] The virus infection method is the same as before. The specific steps of Western-blot detection are as follows:

[0098] 1) Wash the cell debris with cold PBS, and put the cells on ice;

[0099] 2) Add an appropriate amount of pre-cooled 1×Lysis Buffer containing protease inhibitor (PI);

[0100] 3) Cells were lysed on ice for 10-15 minutes. Transfer the sample into the Ep tube;

[0101] 4) Break the cells with an ultrasonic breaker (200W for 4 times, each time for 5S, with an interval of 2S);

[0102] 5) 4°C, 12000g, centrifuge for 15min;

[0103] 6) Take the supernatant, measure the total protein concentration of the sample, and adjust it to a final protein concentration of 2 μg / μL;

[0104] 7) Take an appropriate amount of sample for SDS-PAGE electrophoresis, control the total protein content in the loading sample to be between 20-60 μg, add the corresponding loading buffer, mix well with a shaker, and cook at...

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Abstract

The invention relates to an SiRNA lentivirus vector of human STAT3 gene and a construction method thereof. The SiRNA lentivirus vector comprises a segment of sequence and a lentivirus vector pLenti6.3/V5-DEST; the nucleotide sequence comprises an interference target sequence SiRNA aming at an mRNA sequence of the human STAT3 gene; the registry number of the mRNA sequence of the human STAT3 gene in the Gene bank is NM_139276; and the interference target sequence SiRNA is selected from SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3 and SEQ ID NO:4. The lentivirus vector expresses sense chains and anti-sense chains of siRNA molecules of the human STAT3 gene when the lentivirus vector is in target cells, so that the mRNA of the human STAT3 is subjected to specific degradation and the expression is silent; and the invention provides an effective method for knocking out STAT3 gene expression in vivo, and opens up a new way for treating tumors.

Description

technical field [0001] The present invention relates to members of the human Signal Transducton and Activators of Transcription STATs (Signal Transducton and Activators of Transcription STATs) family, and more specifically, the present invention relates to a method for constructing a human STAT3 gene SiRNA lentiviral vector. Background technique [0002] The STATs family of signal transducers and activators of transcription is a family of proteins that exist in the cytoplasm of cells and can translocate into the nucleus and bind to DNA after activation. They have dual functions of signal transduction and transcription regulation. Seven members of the STATs family have been discovered: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6, which are encoded by different genes and are widely involved in the regulation of various physiological functions such as cell growth, proliferation, differentiation, and apoptosis. It is closely related to inflammation, tumor and immune re...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66
Inventor 马玲娣文世宏倪诚夏蕾蒋丽佳
Owner 马玲娣
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