Thymosin beta4 gene expression inhibiting RNA and use thereof

A technology of expression cassettes and expression vectors, applied in DNA/RNA fragments, gene therapy, recombinant DNA technology, etc., can solve the problem of lack of signal peptides, etc., and achieve the effect of increasing the infection rate

Inactive Publication Date: 2009-12-02
PEKING UNIV THIRD HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, the study found that Tβ 4 There is a lack of signal peptide in cDNA. Although there are some secreted proteins without signal peptide in the body, such as interleukin 1 (IL-1), endothelial growth factor, etc., people still tend to think that Tβ 4 Unlikely to be an exocrine protein, but may be required for some essential cellular functions

Method used

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  • Thymosin beta4 gene expression inhibiting RNA and use thereof
  • Thymosin beta4 gene expression inhibiting RNA and use thereof
  • Thymosin beta4 gene expression inhibiting RNA and use thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1, Thymosin β 4 Preparation of shRNA vector

[0073] 1. RNA interference sequence (siRNA) DNA design

[0074] in Thymosin beta 4 On the mRNA (NM_021109, full-length 656bp), a site to be interfered was selected, and a pair of complementary DNA (SDS505) was synthesized as follows according to the accessibility of the mRNA space and the principle that the siRNA 3' melting activity is higher than that of the 5':

[0075] 5'-AAGAGGTTGGATCAAGTTTAA-3';

[0076] 5'-TTAAACTTGATCCAACCTCTT-3'.

[0077] As a control, a pair of complementary DNA (NC) was synthesized as follows:

[0078] 5'-TTCTCCGAACGTGTCACGT-3';

[0079] 5'-ACGTGACACGTTCGGAGAA-3'.

[0080] 2. Synthesis of DNA sequence A and DNA sequence B

[0081] DNA sequence A:

[0082] 5'-GATCCCCAAGAGGTTGGATCAAGTTTAATTCAAGAGATTAAACTTGATCCAACCTCTTTTTTTGGAT-3' (sequence 8 of the sequence listing); wherein the 7th to 27th nucleotides from the 5' end are a DNA strand of SDS505 (sequence 4 of the sequence listing)...

Embodiment 2

[0106] Example 2, pGC-shTβ 4 Effects on U2OS cell function

[0107] In the present embodiment, the data are represented by "mean ± standard deviation". When the data are compared in pairs, the variances are homogeneous, and the SPSS13.0 software is used to do one-way analysis of variance, and the LSD method is compared in pairs (semi-quantitative RT-PCR, MTT and Western The results of the Blot experiment were adopted); when the variances were not homogeneous, the non-parametric rank sum test was used, and the Nemenyitest method was used for pairwise comparison of data (cell immunochemical staining was used to detect Tβ 4 The results of protein expression were analyzed by) or by Games-Howell method in SPSS13.0 software (transwell invasion test cell calculation results); the difference was statistically significant when P<0.05.

[0108] Bone tumor cell line U2OS: Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, catalog number TCHu88.

[01...

Embodiment 3

[0175] Embodiment 3, thymosin beta 4 RNA interference lentiviral vector preparation

[0176] One, according to the RNA interference sequence (siRNA) DNA of step one design of embodiment 1 synthetic DNA sequence C and DNA sequence D are as follows:

[0177] DNA sequence C:

[0178] 5'-CCGGAAGAGGTTGGATCAAGTTTAATTCAAGAGATTAAACTTGATCCAACCTCTTTTTTTG-3' (sequence 10 of the sequence listing); wherein the 5th to 25th nucleotides from the 5' end are a DNA strand of SDS505 (sequence 4 of the sequence listing), and the 26th to 34th from the 5' end The first nucleotide is a loop sequence, and the 35th to 55th nucleotides from the 5' end are another DNA strand of SDS505 (sequence 5 in the sequence listing).

[0179] DNA sequence D:

[0180] 5'-CCGGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTG-3'; a DNA strand in which the 5th to 23rd nucleotides from the 5' end is NC, the 24th to 32nd nucleotides from the 5' end is a loop sequence, and the 33rd nucleotides from the 5' end Anoth...

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Abstract

The invention discloses a thymosin beta4 gene expression inhibiting RNA and use thereof. The shRNA provided by the invention is a single-chain RNA having a stem-and-loop structure consisting of a stem I, a loop and a stem II; the sequence of the stem I is represented by the sequence 1 in a sequence table; and the sequence of the stem II is represented by the sequence 2 in the sequence table. The RNA of the invention can be used to construct recombinant plasmids and recombinant lentiviruses and obviously inhibit the invasion by tumor cells, the propagation of tumor cells and the expression of the thymosin beta4 gene in cells, thereby having medicinal and pharmaceutical applications and profound significance.

Description

technical field [0001] The invention relates to a method for inhibiting thymosin β 4 RNA for gene expression and its applications. Background technique [0002] 1981, Tβ 4 It was isolated from thymus extract by gel chromatography. The study found that Tβ 4 It is a hydrophobic polypeptide composed of 43 amino acid residues, with a molecular weight of 4964Da and an isoelectric point of 5.1, which is highly conserved in mammals. Chemical conformational studies have shown that Tβ 4 It exists and functions in the form of a single chain, and its 31-43 residues and 18-30 residues are an internal repeating region, with 6 amino acids identical. Although there are two highly helical regions at residues 4-12 and residues 32-40, Tβ 4 No beta turns are formed. In 1991, Daniel Safer from the University of Pennsylvania School of Medicine in Philadelphia accidentally discovered Tβ in the process of studying how actin monomers (G-actin) polymerize to form fiber actin (F-actin) 4 Bind...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63C12N5/10C12N1/00C12N7/01A61K48/00A61P35/00C12R1/93C12N15/113
Inventor 秦泽莲黄似建刘畅
Owner PEKING UNIV THIRD HOSPITAL
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