siRNA that inhibits porcine somatostatin receptor 2 gene expression

A somatostatin receptor and gene expression technology, which is applied in the fields of molecular biology and bioengineering, can solve problems such as unseen research reports, and achieve the effects of improving growth performance, good silencing effect, and promoting growth

Inactive Publication Date: 2011-11-30
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
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Problems solved by technology

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  • siRNA that inhibits porcine somatostatin receptor 2 gene expression
  • siRNA that inhibits porcine somatostatin receptor 2 gene expression
  • siRNA that inhibits porcine somatostatin receptor 2 gene expression

Examples

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Embodiment 1

[0040] Example 1 Construction of pCDNA3.1-SSTR2 eukaryotic expression vector

[0041] 1. The first strand of cDNA was synthesized using the normal porcine pituitary total RNA preserved in our laboratory as a template;

[0042] 2. According to the sequence of porcine somatostatin receptor 2 (SSTR2) (GenBank accession number NM001011694), design a pair of primers and introduce restriction restriction sites Eco R I and Hin d III, the sequence of the upstream primer is SEQ ID NO:10, and the sequence of the downstream primer is SEQ ID NO:11. Using the first strand of cDNA as a template, the SSTR2 gene was amplified by PCR. Reaction system: 5 μL of 10×Ex Taq Buffer, 4 μL of 2.5mM dNTP, 1 μL of upstream primer (10 pmol / μL), 1 μL of downstream primer (10 pmol / μL), 3 μL of cDNA template, 1 μL of Ex Taq DNA polymerase (5 U / μL), supplement ddH 2 0 to 50 μL. Reaction conditions: 94°C, 5min; 94°C, 30sec; 56°C, 30sec; 72°C, 1min, a total of 35 cycles; 72°C, 10min extension and then...

Embodiment 2

[0050] Example 2 Construction of pshRNA-copGFP Lentivector lentiviral interference plasmid

[0051] 1. According to the method and principle of porcine somatostatin receptor 2 mRNA sequence design, target sequences were screened out, and siRNA was designed to be reverse complementary to the mRNA of somatostatin receptor 2 gene, named SSTR2-1, SSTR2-2, and SSTR2 respectively -3, its sense strand sequence is as follows:

[0052] SSTR2-1: SEQ ID NO: 1;

[0053] SSTR2-2: SEQ ID NO: 2;

[0054] SSTR2-3: SEQ ID NO:3.

[0055] 2. According to the siRNA target sequence, design the DNA sequence required to construct the siRNA vector that inhibits the expression of the porcine somatostatin receptor 2 gene: SSTR2 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

[0068] Example 3 Screening of Effective Targets of RNA Interference

[0069] 1. Transfection of eukaryotic cells with three lentiviral interference plasmids

[0070] pcDNA3.1-SSTR2 and LV-siRNA1, LV-siRNA2 and LV-siRNA3 were co-transfected into CHO cells for expression. CHO cells were co-transfected with pcDNA3.1-SSTR2 and LV-shRNA-GFP empty plasmids as a control, in order to be consistent with the transfection background of the treatment group, and three replicates were set for each treatment. The ratio of pcDNA3.1-SSTR2 to lentiviral interference plasmid is 1:5.

[0071] For lipofection, follow Lipofectamine TM 2000 Reagent Manual.

[0072] (1) One day before transfection, seed the cells in a new culture dish and blow the cells completely to ensure the uniform distribution of the cells. When transfecting, the cells should ideally cover 60-70% of the dish.

[0073] (2) Add a total of 4 μg of pcDNA3.1-SSTTR2 and lentiviral interference plasmid at a ratio of 1:5 to 250 ...

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Abstract

The invention discloses a siRNA for inhibiting expression of a porcine Somatostatin receptor 2, belonging to the technical field of genetic engineering. The siRNA sense strand provided by the invention has any sequence shown in the SEQID NO:1-3. The invention further discloses shRNA coding genes containing the siRNA and lentivirus interfering plasmids pshRNA-copGFPLentivector constructed with theshRNA coding genes. Through the real-time quantitative Real-time PCR (Polymerase Chain Reaction) examination, it is found that the expression level of a porcine Somatostatin receptor 2mRNA can be remarkably reduced by about 70% above with each siRNA segment and the expression level of SSTR 2 proteins can be reduced by 30% above in ELISA (Enzyme Linked Immunosorbent Assay) examination. The siRNA segment provided by the invention and expression vectors thereof can be used for preparing preparations of inhibiting the porcine Somatostatin receptors 2 and can be used for remarkably reducing the expression level of the SSTR2 (porcine Somatostatin Receptor 2) genes.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and bioengineering, in particular to an siRNA fragment for inhibiting the expression of porcine somatostatin receptor 2 gene. Background technique [0002] shRNA is short hairpin RNA (short hairpin RNA). In the process of RNAi interference, an effective way to generate dsRNA is to express a short hairpin RNA in vivo. This shRNA contains two short inverted repeats (one of which is complementary to the target gene) separated by a loop sequence, consisting of Hairpin structure. In vivo, shRNA can be processed into siRNA (small interfering RNAs, siRNA), thereby degrading the target gene and inhibiting its expression. [0003] RNAi (RNA interference, RNAi) is a process in which double-stranded RNA (double-stranded RNA, dsRNA) molecules shut down or silence gene expression of the corresponding sequence at the mRNA level. Therefore, RNA technology is vividly called gene silencing (gene sile...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/63C12N15/867A61K48/00A61P43/00
Inventor 张永亮杨林习欠云林海丹何玮璇蔡伟光
Owner SOUTH CHINA AGRI UNIV
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