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siRNA for silencing expression of Rongchang pig CD14 as well as screening method and application of siRNA

A screening method, technology of Rongchang pig, applied in the direction of DNA/RNA fragments, DNA preparation, recombinant DNA technology, etc.

Inactive Publication Date: 2018-12-11
SOUTHWEST UNIVERSITY
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Problems solved by technology

But for ribonucleic acid interference, not all siRNAs randomly designed for the target gene messenger ribonucleic acid sequence can cause effective gene silencing, and the selection of the interference target sequence is crucial to the interference efficiency

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  • siRNA for silencing expression of Rongchang pig CD14 as well as screening method and application of siRNA
  • siRNA for silencing expression of Rongchang pig CD14 as well as screening method and application of siRNA
  • siRNA for silencing expression of Rongchang pig CD14 as well as screening method and application of siRNA

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Embodiment Construction

[0059] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0060] An siRNA for silencing Rongchang pig CD14 expression in the present invention is firstly established by first establishing porcine alveolar macrophages (porcine alveolarmacrophages cells, PAM) stably expressing CD14-enhanced green fluorescent protein EGFP fusion protein of Rongchang pig CD14 gene; and then according to CD14 gene sequence, design and synthesize four different siRNAs targeting CD14, and use high-efficiency transfection reagents to transfect the four siRNAs into stably expressed cell lines; siRNA that inhibits CD14 gene expression.

[0061] Based on the above experimental data, it was found that the third (669) siRNA could significantly inhibit the expression of CD14 gene.

[0062] The specific operation steps are as follows:

[0063] Step 1. Construction of recombinant expression plasmid pCD14-EGFP

[0064] According ...

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Abstract

The invention discloses an siRNA for silencing the expression of a Rongchang pig CD14. Firstly, a porcine alveolar macrophage of a CD14-enhanced green fluorescent protein (EGFP) fusion protein for stably expressing a Rongchang pig CD14 gene is established; next, four siRNAs having different loci and targeting the CD 14 gene are synthesized; then, the four siRNAs are transfected into a CD14-EGFP stable cell line by applying an efficient transfection reagent; and finally, the siRNAs capable of efficiently inhibiting the expression of the Rongchang pig CD14 gene are screened, namely the siRNAs inan article 3 (669) are capable of obviously inhibiting the expression of the Rongchang pig CD14 gene, and therefore, a theoretical basis is provided for researches related to inflammatory diseases caused by treating endotoxin shock, endoxemia and gram-negative bacteria by targeting the Rongchang pig CD14 gene.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering and biotechnological medicine, and in particular relates to a small ribonucleic acid molecule (siRNA) for silencing the expression of Rongchang pig CD14, and also relates to a screening method and application of the siRNA for silencing the expression of Rongchang pig CD14. Background technique [0002] Leukocyte differentiation antigen 14 (cluster of differentiation antigen 14, CD14) is a high-affinity receptor for lipopolysaccharide (LPS), anchored to monocytes, On the surface of myeloid cell membranes such as macrophages, CD14 is divided into two types: membrane-bound CD14 (membrane CD14, mCD14) and soluble CD14 (soluble CD14, sCD14). [0003] mCD14 is a membrane protein with a molecular weight of 55kD, which is mainly distributed on the surface of monocytes, macrophages and dendritic cells, and a small amount of mCD14 is also found on the surface of activated neutrophils; sCD14 i...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10A61K31/713A61P39/02A61P31/04
CPCA61P31/04A61P39/02C12N15/1138C12N2310/14C12N2320/11
Inventor 焦寒伟万堃黄庆洲伍莉陈吉轩帅学宏赵宇王红均
Owner SOUTHWEST UNIVERSITY
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