SiRNA inhibiting BMP15 gene expression and application thereof

A gene expression and expression carrier technology, applied in the field of molecular biology and bioengineering, to achieve the effect of reduced expression, good silencing effect, and reduced protein expression

Inactive Publication Date: 2013-03-13
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But there is no research report on buffalo bone morphogenetic protein 15 siRNA

Method used

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  • SiRNA inhibiting BMP15 gene expression and application thereof
  • SiRNA inhibiting BMP15 gene expression and application thereof
  • SiRNA inhibiting BMP15 gene expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of pCDNA3.1- BMP15 eukaryotic expression vector

[0039] 1. Synthesize the buffalo bone morphogenetic protein 15 (BMP15) sequence (GenBank accession number NM_001031752), and connect it to the vector pUC19, restriction site Bam HI / Eco RI. double digestion ( Eco R I and BamHI) and sequencing proved that the synthesized fragments were correct (see figure 2 ).

[0040] 2. Construction of pCDNA3.1-BMP15 eukaryotic expression plasmid

[0041] (1) Enzyme digestion reaction

[0042] reaction system: Eco R I and BamHI double-digest the PCR product. Eco R I and BamHI double-digest pcDNA3.1(-), enzyme digestion system: 10×Buffer K 2 μL, Eco R I 1 μL, BamHI 1 μL, plasmid 6 μL, distilled water 10 μL, total volume 20 μL. Reaction conditions: act at 37°C for 1.2h, and perform 1% agarose gel electrophoresis. The digested products were purified with a DNA purification kit, and finally the DNA was eluted with 30 μL of water and stored at -20°...

Embodiment 2

[0047] Example 2 Construction of pshRNA-copGFP Lentivector lentiviral interference plasmid

[0048] 1. According to the buffalo bone morphogenetic protein 15 mRNA sequence design method and principle, the target sequence was screened out, siRNA was designed, and it was reversely complementary to the buffalo bone morphogenetic protein 15 gene mRNA, named BMP15-1, BMP15-2, BMP15- 3. The sense strand sequence is as follows:

[0049] BMP15-1: SEQ ID NO: 1;

[0050] BMP15-2: SEQ ID NO: 2;

[0051] BMP15-3: SEQ ID NO:3.

[0052] 2. According to the siRNA target sequence, design the DNA sequence required to construct the siRNA vector that inhibits the expression of buffalo bone morphogenetic protein 15 gene: BMP15 shRNA template, introduced at both ends Bam H I and Eco The R I enzyme cutting site was sent to Shanghai Bioengineering Technology Service Co., Ltd. for synthesis. The specific sequence is as follows (F indicates the sense strand, R indicates the antisense strand): ...

Embodiment 3

[0065] Example 3 Screening of Effective Targets of RNA Interference

[0066] 1. Transfection of eukaryotic cells with three lentiviral interference plasmids

[0067] pcDNA3.1-BMP15 and LV-siRNA1, LV-siRNA2 and LV-siRNA3 were co-transfected into CHO cells for expression. CHO cells were co-transfected with pcDNA3.1-BMP15 and LV-shRNA-GFP empty plasmids as a control, in order to be consistent with the transfection background of the treatment group, and 6 replicates were set for each treatment. The ratio of pcDNA3.1-BMP15 to lentiviral interference plasmid is 1:1.

[0068] Lipofection was carried out according to the instruction manual of Polyfect (purchased from QIAGEN).

[0069] (1) One day before transfection, inoculate cells in a new six-well culture dish, and completely blow off the cells to ensure that the cells are evenly distributed. When transfecting, the cells should ideally cover 40-90% of the dish.

[0070] (2) Add pcDNA3.1-BMP15 and lentivirus interference plasm...

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Abstract

The invention discloses an SiRNA inhibiting the gene expression of buffalo bone morphogenetic protein 15 (BMP15), and belongs to the technical field of gene engineering. The positive-sense strand of the siRNA has any sequence indicated by SEQ ID NO: 1-3. The invention also discloses an shRNA encoding gene containing the siRNA and slow virus interference plasmid pshRNA-copGFPLentivector constructed by the same. Through real-time quantitative Real-time PCR (Polymerase Chain Reaction) detection, people find that siRNA segments can respectively reduce the BMP15 mRNA expression quantity by 29.25%, 57.75%, and 82.5%; and the BMP15 protein expression quantity detected by ELISA (enzyme-linked immuno sorbent assay) detection is also correspondingly reduced. The siRNA segments and the expression vector thereof can be used to prepare a preparation inhibiting the expression of buffalo BMP15, and can significantly reduce the BMP 15 gene expression quantity.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and bioengineering, in particular to an siRNA fragment for buffalo inhibiting gene expression of bone morphogenetic protein 15 (BMP15). Background technique [0002] In 1998, Andrew Fire of the Carnegie Research Institute of Washington and Craig Mello of the University of Massachusetts Cancer Center first discovered RNA interference (RNA interference, RNAi). RNAi is a post-transcriptional gene silencing process triggered by double-stranded RNA (double-stranded RNA, dsRNA) molecules. It is a ubiquitous monitoring mechanism in eukaryotes to resist virus invasion, inhibit transposon activity, and regulate gene expression. RNAi is a phenomenon or mechanism in which double-stranded RNA induces the degradation of its homologous mRNA, resulting in post-transcriptional silencing of the target gene. [0003] siRNA is the most important discovery in the mechanism of RNAi. Biological studies hav...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N15/867A61K48/00A61P15/08A01K67/027
Inventor 习欠云张永亮施振旦肖敏焦莉石德顺刘庆友
Owner SOUTH CHINA AGRI UNIV
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