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

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

Example Embodiment

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

[0062] 1. Material

[0063] Normal human blood PBMC and T vector were purchased from Biyuntian Biotechnology Co., Ltd., and pET32a-CP plasmid was kept by the company's R&D center.

[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 CTLA-4 gene was selected, and the sequence of CTLA-4 gene was published in GeneBank using Blast comparison to determine the human CTLA-4 gene. Oligo software was used for primer design. The target sequence (ie CTLA-4 C gene sequence) is shown in SEQ ID NO.1 in the sequence table.

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

[0068] According to the MS2 phage gene sequence, a pair of specific primers was designed, the pair of specific pr...

Example Embodiment

[0093] 2. Embodiment 2: Preparation of armored CTLA-4 gene standard material

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

[0095] The pseudoviral particles obtained in Example 1 were used to measure the OD value at 260nm using an ultraviolet spectrophotometer to detect the nucleic acid absorption value to determine the nucleic acid content, and proceed to 10 12 , 10 11 , 10 10 Double the concentration dilution for RNA extraction. Carry out a group of samples without reverse transcription to directly carry out PCR reaction; after reverse transcription of another group of samples, carry out PCR amplification and electrophoresis to detect the amplification results. After the samples were diluted, one group was directly subjected to PCR without reverse transcription, and the test results were all negative, indicating that there was no DNA template contamination in the preparation of virus-like particle suspension and virus-like parti...

Example Embodiment

[0100] 3. Example 3: Preparation of a dilution gradient standard of RNA standard material of armored CTLA-4 gene

[0101] 1. Establishment and identification of the dilution gradient of reference materials

[0102] The standard substance obtained in Example 1 was measured by an ultraviolet spectrophotometer, and the value of the standard substance pseudoviral particles was converted according to the OD value, and the initial concentration of viral particles was determined to be 10 13 To the power, use TE buffer to perform a 10-fold dilution gradient to prepare 10 12 , 10 11 , 10 10 , 10 9 , 10 8 Dilution gradient, RNA extraction, reverse transcription and fluorescence quantitative PCR for gradient detection. Analyze the standard curve by linear regression, and its R value> 0.99. Fluorescence results such as Picture 12 display, Picture 12 The curves in are expressed as follows: 1 is 10 12 Pseudovirus particles, 2 is 10 11 Pseudovirus particles, 3 is 10 10 Pseudovirus particles, 4 ...

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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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