Mixed polypeptide for producing tuberculosis associated cell factors by inducing peripheral blood mononuclear cell

A technology of cytokines and peripheral blood, applied in the field of protein detection, can solve the problem of low specificity of PPD, and achieve the effect of strong sensitivity, high specificity and good stimulation effect

Inactive Publication Date: 2016-05-04
GUANGZHOU DEAOU MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Tuberculin pure protein derivative (PPD) is an active substance obtained from the culture filtrate of Mycobacterium tuberculosis, which can stimulate T cells to produce cytokines such as IFN-γ and IL-2, but for individuals who have been immunized with BCG (BCG) PPD can also stimulate the extracted T cells to produce tuberculosis-related cytokines, so the specificity of PPD is low, and it is necessary to find a more specific stimulator

Method used

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  • Mixed polypeptide for producing tuberculosis associated cell factors by inducing peripheral blood mononuclear cell
  • Mixed polypeptide for producing tuberculosis associated cell factors by inducing peripheral blood mononuclear cell
  • Mixed polypeptide for producing tuberculosis associated cell factors by inducing peripheral blood mononuclear cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1. Epitope Analysis and Polypeptide Synthesis of Specific Mixed Polypeptides

[0060] Referring to the GenBank database, using bioinformatics technology to analyze and predict antigenic epitopes, a mixed polypeptide (SEQ ID NO: 1-18) is selected, each polypeptide contains 20-25 amino acids, and there are 8-25 amino acids between adjacent two polypeptides. 10 amino acid repeats. There is no rigid regulation on the mixing ratio. Preferably, the 18 polypeptides are mixed with equal mass.

[0061] The amino acid sequence is further obtained according to the calculation: wherein the mixed polypeptide SEQIDNO: 1-18 is coded by the nucleotide sequence SEQIDNO: 19-36 respectively. Polypeptide solid-phase synthesis (SPPS) was used to generate the desired polypeptide according to the instructions of the synthesizer, and the product was freeze-dried for future use.

Embodiment 2

[0062] Example 2. Separation of human peripheral blood mononuclear cells (PBMC) by density gradient centrifugation

[0063] Use fresh heparin lithium or heparin sodium blood collection tubes or vacuum blood collection tubes to aseptically extract 4ml of peripheral venous blood from the person to be tested, and invert the blood to mix the anticoagulant and blood. Prepare two 15ml centrifuge tubes, and add physiological saline for injection and lymphocyte separation solution equal to the volume of blood collection respectively. After mixing the fresh heparin anticoagulated blood with normal saline, slowly add it into the separation liquid at a uniform speed, and centrifuge at 1800g for 20min at 22°C. After centrifugation, PBMC cells can be seen to exist in the cloud-like layer. Aspirate the PBMC cell layer into a new 15ml centrifuge tube, make up to 12ml with RPMI-1640 culture medium, and centrifuge at 22°C and 600g for 10min. Pour off the supernatant, make up to 5ml with RPMI-...

Embodiment 3

[0065] Example 3. Mixed polypeptides induce human peripheral blood mononuclear cells to secrete IL-2 levels

[0066] The heparin sodium anticoagulated peripheral venous blood samples of 20 patients with positive sputum smear or sputum culture test and 20 patients with negative PPD skin test were respectively selected, and PBMC were separated by density stratification, and diluted with serum-free medium after cell counting. to 2.5×10 6 cells / ml, the cells were inoculated into 96-well plates for culture, and each well was inoculated with 2.5×10 5 For each cell, three detection wells were set up for each sample, which were blank control wells (add 50 μl serum-free medium), experimental wells (add 50 μl mixed polypeptide with a concentration of 2 μg / ml, and use serum-free medium for dilution) and positive wells. Control wells (add 50 μl phytohemagglutinin PHA), 37°C, 5% CO 2 Conditioned incubation for 18-24 hours. After incubation, the culture supernatant of each well was colle...

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Abstract

The invention discloses a mixed polypeptide for producing tuberculosis associated cell factors by inducing a peripheral blood mononuclear cell. The mixed polypeptide has polypeptide sequences represented by SEQ ID NO:1-18. Compared with common stimulating proteins CFP-10, ESAT-6 and Rv1985c or a mixture of the three proteins, the mixed polypeptide has relatively efficient induction performance and the relatively good specificity. The mixed polypeptide only reacts with immunologic memory T cells produced after human body is infected by mycobacterium tuberculosis, the produced cell factors are tuberculosis associated factors including IFN-gamma, IL-2, TNF-alpha and the like with mycobacterium tuberculosis antigen specificity, and meanwhile, the stimulus response is not interfered by BCG. The specific antigen polypeptide provided by the invention is significant for the research of tuberculosis pathopoiesia and immunoprophylaxis mechanisms and therefore has positive significance to the control of tuberculosis.

Description

technical field [0001] The invention relates to the field of protein detection, in particular to a mixed polypeptide for inducing peripheral blood mononuclear cells to produce tuberculosis-related cytokines. Background technique [0002] Tuberculosis is a common chronic infectious disease caused by Mycobacterium tuberculosis, which can invade many organs, with pulmonary tuberculosis infection being the most common. At present, nearly 1 / 3 of the world's people have been infected with Mycobacterium tuberculosis. Statistics show that in 2013, 1.5 million people died of tuberculosis and 9 million new cases. Tuberculosis has become one of the main diseases that cause adult deaths from infectious diseases all over the world. . China is one of the 22 countries with a high burden of tuberculosis in the world, and the number of active tuberculosis patients ranks second in the world. The transmission of tuberculosis mainly occurs before the patient is discovered and treated. One tub...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N5/078G01N33/68G01N33/569
CPCC07K14/00C12N5/0634C12N2501/998G01N33/56911G01N33/68
Inventor 黄曦胡鹏男李妍
Owner GUANGZHOU DEAOU MEDICAL TECH CO LTD
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