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PD-1 gene armored RNA standard substance and applications thereof

A PD-1 and standard material technology, applied in the field of RNA standard material, can solve the problems of non-diagnosed PD-1 nucleic acid, etc., and achieve the effects of easy storage and transportation, ensuring accuracy, and good protection

Inactive Publication Date: 2015-04-22
ETEMUS BIOMEDICINE XIAMEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no diagnostic PD-1 nucleic acid detection reagents and quality control products in China

Method used

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  • PD-1 gene armored RNA standard substance and applications thereof
  • PD-1 gene armored RNA standard substance and applications thereof
  • PD-1 gene armored RNA standard substance and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] 1. Implementation 1: PD-1 primer design, target sequence acquisition and identification, expression vector connection

[0065] 1. Materials

[0066] Normal human blood PBMC and T carrier were purchased from Beyontian Biotechnology Co., Ltd.

[0067] 2. Method

[0068] (1) PD-1 primer design and target selection

[0069] According to the PD-1 gene sequence published by GeneBank (GenBank: AF363458.1), the CDS region of the PD-1 gene was selected, and the target was compared with the sequence of the PD-1 gene published in GeneBank by Blast, and determined to be the human PD-1 gene. Primers were designed using Oligo software. The target sequence (that is, the W gene sequence) is shown in SEQ ID NO.1 in the sequence listing.

[0070] (2) Obtain the expression vector containing MS2 phage maturation enzyme protein gene and capsid protein gene:

[0071] According to MS2 phage gene sequence, design a pair of specific primers, described a pair of specific primers are as foll...

Embodiment 2

[0098] Two. Example 2: Preparation of armored PD-1 gene standard substance

[0099] 1. Quantitative determination of the RNA standard substance particles of the armored PD-1 gene

[0100] With the pseudovirus particle that embodiment 1 obtains, utilize ultraviolet spectrophotometer to measure 260nm place OD value detection nucleic acid absorption value and determine nucleic acid content, carry out 10 12 , 10 11 , 10 10 1-fold concentration dilution for RNA extraction. A group of samples were directly subjected to PCR reaction without reverse transcription; another group of samples were subjected to reverse transcription, followed by PCR amplification, and the amplification results were detected by electrophoresis. After the samples were diluted, one group was directly subjected to PCR reaction without reverse transcription, and the test results were all negative, indicating that there was no DNA template contamination in the prepared virus-like particle suspension and in th...

Embodiment 3

[0106] Three. Embodiment 3: Preparation of RNA standard substance dilution gradient standard substance of armored PD-1 gene

[0107] 1. Establishment and identification of standard substance dilution gradient

[0108] The standard substance obtained in Example 1 was measured by an ultraviolet scenery photometer, and the value of the standard substance pseudovirus particle was converted according to the OD value, and the initial concentration of the virus particle was determined to be 10 13 Power, use TE buffer to carry out 10-fold dilution gradient, prepare 10 12 、10 11 、10 10 、10 9 、10 8 Dilution gradient, RNA extraction, reverse transcription and fluorescent quantitative PCR for gradient detection. The standard curve was analyzed by linear regression, and its R value was >0.99. Fluorescent results such as Figure 12 show, Figure 12 The curves in the graph are expressed as follows: 1 is 10 12 Pseudovirions, 2 out of 10 11 Pseudovirions, 3 out of 10 10 Pseudovirio...

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Abstract

The invention provides a PD-1 gene armored RNA standard substance and applications thereof. The RNA standard substance is prepared through the following steps: firstly, obtaining a W-based gene sequence for PD-1 nucleic acid detection, and then obtaining an expression vector containing MS2 phage mature zymoprotein genes and capsid protein genes, namely, a PET32a-CP vector; then, obtaining a prokaryotic expression vector PET32a-CP-PD-1 of W genes and CP genes; transferring the prokaryotic expression vector into a prokaryotic expression strain, carrying out inducible expression, carrying out frozen low-temperature freeze thawing, centrifuging, and purifying, so that an expression product particle is obtained; and carrying out DNase I digestion and purification, and then diluting the obtained product, so that the PD-1 gene armored RNA standard substance is obtained. The substance disclosed by the invention has the characteristics of stability, no biological infectivity, ribonuclease resistance, and the like, and is used as a standard substance in the nucleic acid detection of immune suppression factors PD-1.

Description

technical field [0001] The invention relates to an RNA standard substance of an armored PD-1 gene and an application thereof. Background technique [0002] PD-1, the programmed death receptor 1, is an important immunosuppressive molecule. It was originally discovered on programmed death T cells. PD1 is mainly expressed in CD4+T cells, CD8+T cells, NK T cells, B Cells and activated monocytes are mainly induced by T cell receptor (TCR) or B cell receptor (BCR) signaling, and TNF can enhance the expression of PD1 on the surface of these cells. PD-1 has two ligands, PD-L1 and PD-L2. When PD-1 binds to PD-L1 or PD-L2, it can inhibit the proliferation of activated T cells and the production of cytokines. In particular, it inhibits the function of the corresponding CD8+ T cells. Therefore, Quezada and Don M. Benson Jr conducted research on the PD1 signaling pathway and found that inhibiting the PD1 signaling pathway can reactivate the host immune response to resist tumor develop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/12C12N15/10C12N15/70
CPCC12Q1/6888C12Q2600/166
Inventor 连文昌阮润生黄丽萍李剑
Owner ETEMUS BIOMEDICINE XIAMEN
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