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Method for distinguishing smooth type lipopolysaccharide and rough type lipopolysaccharide

A technology of lipopolysaccharide and rough type, which is applied in the field of identifying smooth lipopolysaccharide and rough type lipopolysaccharide, can solve the problems of cumbersome steps and unsatisfactory accuracy of chemical identification methods

Inactive Publication Date: 2014-11-05
HAINAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The purpose of the present invention is to provide a method for identifying smooth lipopolysaccharide and rough lipopolysaccharide, which solves the problems of cumbersome steps and unsatisfactory accuracy in the chemical identification method in the prior art

Method used

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  • Method for distinguishing smooth type lipopolysaccharide and rough type lipopolysaccharide
  • Method for distinguishing smooth type lipopolysaccharide and rough type lipopolysaccharide
  • Method for distinguishing smooth type lipopolysaccharide and rough type lipopolysaccharide

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Embodiment 1

[0057] The present invention is a kind of method that can quickly distinguish smooth type lipopolysaccharide and rough type lipopolysaccharide, concrete implementation according to the following steps:

[0058] Step 1, according to the sequence of membrane-bound leukocyte differentiation antigen 14 (mCD14) gene, according to the basic principle of small interfering ribonucleic acid molecule (siRNA) design, synthesize a pair of small interfering ribose against membrane-bound leukocyte differentiation antigen 14 (mCD14) Nucleic acid molecule (siRNA-224.3), the siRNA sequence is shown in the sequence list 1 in the attachment, and you can choose to perform specific technical operations through Shanghai Gemma Technology Co., Ltd.;

[0059] Step 2, packing the synthesized siRNA-224.3 into a lentiviral vector

[0060] 2.1) Oligo Design (preparation of oligonucleotides): TTCAAGAGA is selected for the loop structure in the Lentivirus-shRNA template to avoid the formation of a terminati...

Embodiment 2

[0107] According to the step of embodiment 1, implement according to the following specific parameters:

[0108] Step 1. Synthesize a pair of small interfering ribonucleic acid molecule siRNA-224.3 directed at membrane-bound leukocyte differentiation antigen 14 according to the sequence of the membrane-bound leukocyte differentiation antigen 14 gene and according to the basic principles of small interfering RNA molecule design;

[0109] Step 2, packing the synthesized siRNA-224.3 into a lentiviral vector

[0110] 2.1) Prepare oligonucleotides: TTCAAGAGA is selected as the loop structure in the Lentivirus-shRNA template; T is added to the 5' end of the sense strand template to complement the sticky end formed after digestion with Hpa I; AGCT is added to the 'end, which is complementary to the sticky end formed after Xho I digestion; T4 ligase is ligated to form a recombinant vector;

[0111] 2.2) Anneal the Lentivirus-shDNA template, refer to Table 1 to configure the annealing...

Embodiment 3

[0144] According to the step of embodiment 1, adopt following concrete parameter to implement:

[0145] Step 1. Synthesize a pair of small interfering ribonucleic acid molecule siRNA-224.3 directed against membrane-bound leukocyte differentiation antigen 14 according to the sequence of the membrane-bound leukocyte differentiation antigen 14 gene and in accordance with the basic principle of small interfering RNA molecule design;

[0146] Step 2, packing the synthesized siRNA-224.3 into a lentiviral vector

[0147] 2.1) Prepare oligonucleotides: TTCAAGAGA is selected as the loop structure in the Lentivirus-shRNA template; T is added to the 5' end of the sense strand template to complement the sticky end formed after digestion with Hpa I; AGCT is added to the 'end, which is complementary to the sticky end formed after Xho I digestion; T4 ligase is ligated to form a recombinant vector;

[0148] 2.2) Anneal the Lentivirus-shDNA template, refer to Table 1 to configure the annealin...

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Abstract

The invention discloses a method for distinguishing smooth type lipopolysaccharide and rough type lipopolysaccharide. The method comprises the following steps of: firstly synthesizing a small interfering RNA (siRNA-224.3) capable of effectively interfering mCD14 gene expression, and a negative control siRNA-NC, and preparing recombinant lentiviral vector; secondly, infecting the mice macrophage RAW264.7 with the lentiviral vector, and screening the positive clone by the puromycin, and building steady cell lines siRNA-224.3 and siRNA-NC; and finally, using an enzyme-linked immuno-sorbnent assay to detect the nitric oxide, the tumor necrosis factor alpha and the interleukin 6 in cell supernatant of the mice mononuclear macrophage steady cell line which is respectively stimulated by the smooth type lipopolysaccharide and the rough type lipopolysaccharide to express the small interfering RNA, by comparing the difference of the detection, distinguishing the smooth type lipopolysaccharide and the rough type lipopolysaccharide. In comparison with the prior art, the method provided by the invention is more convenient and more accurate.

Description

technical field [0001] The invention belongs to the technical field of biotechnology application and relates to a method for distinguishing smooth lipopolysaccharide and rough lipopolysaccharide. Background technique [0002] Leukocyte differentiation antigen 14 (cluster of differentiation antigen, CD14) is a differentiation antigen that exists on the surface of monocytes, macrophages and other cells, and is one of the important receptors that mediate the biological effects of lipopolysaccharide (LPS) in vivo . Leukocyte differentiation antigen 14 (CD14) includes two forms: membrane-bound leukocyte differentiation antigen 14 (membrane bound CD14, mCD14) and soluble leukocyte differentiation antigen 14 (soluble CD14, sCD14): membrane-bound leukocyte differentiation antigen 14 (mCD14) is the molecular weight It is a glycoprotein of 55 kilodaltons (KD), and its C-terminus is anchored to the surface of the cell membrane by means of a glycosyl phosphatidylinositol (GPI) structur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/53
Inventor 杜丽王凤阳焦寒伟雷明
Owner HAINAN UNIV
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