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CTLA-4 gene armored RNA standard substance and applications thereof

A technology of CTLA-4 and CTLA-4C, which is applied in the field of RNA standard substances, can solve the problems of non-diagnosed CTLA-4 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 CTLA-4 nucleic acid detection reagents and quality control products in China

Method used

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  • CTLA-4 gene armored RNA standard substance and applications thereof
  • CTLA-4 gene armored RNA standard substance and applications thereof
  • CTLA-4 gene armored RNA standard substance and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0061] Embodiment 1: CTLA-4 primer design and target sequence acquisition and identification, expression vector connection

[0062] 1. Materials

[0063] Normal human blood PBMC, T vector was purchased from Beyontine Biotechnology Co., Ltd., and the pET32a-CP plasmid was preserved by the R&D Center of our company.

[0064] 2. Method

[0065] (1) CTLA-4 primer design and target selection

[0066] According to the CTLA-4 gene sequence published by GeneBank (GenBank: AF414120), the CDS region of the CTLA-4 gene was selected, and the target was compared with the sequence of the CTLA-4 gene published in GeneBank by Blast, and it was determined to be the human CTLA-4 gene. Primers were designed using Oligo software. The target sequence (ie CTLA-4 C gene sequence) is shown as SEQ ID NO.1 in the sequence listing.

[0067] (2) Obtain the expression vector containing MS2 phage maturation enzyme protein gene and capsid protein gene:

[0068] According to the MS2 phage gene sequence,...

Embodiment approach 2

[0093] Two. Embodiment 2: Preparation of Armored CTLA-4 Gene Standard Substance

[0094] 1. Quantitative determination of standard substance particles of armored CTLA-4 gene

[0095] 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 the vir...

Embodiment 3

[0100] Three, embodiment 3: the preparation of the RNA standard substance dilution gradient standard substance of armored CTLA-4 gene

[0101] 1. Establishment and identification of standard substance dilution gradient

[0102] 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 Ps...

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Abstract

The invention provides a CTLA-4 gene armored RNA standard substance and applications thereof. The RNA standard substance is prepared through the following steps: firstly, obtaining a C-based gene sequence for CTLA-4 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-CTLA-4 of CTLA-4 C-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 CTLA-4 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 CTLA-4.

Description

technical field [0001] The invention relates to an RNA standard substance of armored CTLA-4 gene and its application. Background technique [0002] Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is expressed on the surface of activated T cells and acts as a co-stimulatory signal for lymphocyte activation. Plays a negative regulatory role in T cell activation. It acts in opposition to the lymphocyte activator CD28. However, CTLA-4 and CDA28 molecules are very similar in gene structure, chromosome location, sequence homology and gene expression. The two have 31% homology in amino acids. Has the same same receptor B7 molecule. However, the binding affinity between CTLA4 and B7 is 10-20 times higher than that between CD28 and B7, which means that only a small amount of CTLA-4 can block the binding of CD28 and B7, thereby inhibiting the activation of T cells. When CTLA-4 is blocked, it can increase the activation or proliferation of T cells. In 2010, an anti-CTLA-4 m...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/70C12Q1/68
Inventor 连文昌阮润生黄丽萍李剑
Owner ETEMUS BIOMEDICINE XIAMEN
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