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Mycobacterium tuberculosis fusion protein and application thereof in induction of peripheral blood mononuclear cells to generate cytokines

A technology of fusion protein and protein, which is applied in the field of protein in molecular biology technology, can solve the problem of low specificity of PPD, and achieve the effects of strong sensitivity, good stimulation effect, specificity and induction effect

Active Publication Date: 2016-05-25
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PPD can stimulate T cells to produce IFN-γ, IL-2 and other cytokines, but for samples from individuals who have been immunized with BCG (BCG), PPD can also stimulate the extracted PBMC to produce tuberculosis-related cytokines, so the specificity of PPD Low, need to find more specific stimuli

Method used

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  • Mycobacterium tuberculosis fusion protein and application thereof in induction of peripheral blood mononuclear cells to generate cytokines
  • Mycobacterium tuberculosis fusion protein and application thereof in induction of peripheral blood mononuclear cells to generate cytokines
  • Mycobacterium tuberculosis fusion protein and application thereof in induction of peripheral blood mononuclear cells to generate cytokines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Synthesis of target gene of PPE41-ESAT-6-PE25 fusion protein and construction of prokaryotic expression vector

[0040] The genes encoding PPE41, ESAT-6 and PE25 proteins are ppe41 gene ID: 885945, esat-6 gene ID: 862098, and pe25 gene ID: 885703. Design primers using its full sequence or partial functional region sequence expressing antigen activity.

[0041] 1) Use the upstream and downstream primers of PPE41, ESAT-6 and PE25 to amplify the target fragments of PPE41, ESAT-6 and PE25 respectively by PCR, and use TaqDNA polymerase for PCR amplification and recover the above fragments.

[0042] 2) Synthesize two kinds of nucleotide chains with connecting peptide nucleotide sequences by artificial synthesis. One of the nucleotide chains (hereinafter referred to as connecting peptide chain 1) contains partial sequences of PPE41 and ESAT-6 respectively. In the middle is the base sequence of the connecting peptide (SEQ ID NO: 4), and the other nucleotide chain (hereina...

Embodiment 2

[0045] Example 2: Induced expression and purification of fusion protein PPE41-connecting peptide-ESAT-6-connecting peptide-PE25

[0046] Take the above-mentioned Example 1 to identify the correct recombinant expression bacteria containing the PPE41-connecting peptide-ESAT-6-connecting peptide-PE25 target gene fragment (the base sequence is shown in SEQ ID NO: 2) into 20ml of kana-resistant LB In the medium, 37°C, 250rpm, shaking culture for 6h. Autoclave 2L of LB medium, put 500ml in each 1L Erlenmeyer flask, and divide it into 4 bottles. Add 2.5ml of activated bacteria to each bottle, shake culture at 37°C, 250rpm, and shake for about 3 hours to make the OD600 of the bacteria 0.5. Put the Erlenmeyer flask into cold water, cool the shaker, and then induce it after cooling. Add IPTG to each bottle to make the final concentration 0.5mM, put it in a 20°C shaker, 200rpm, and induce for 20 hours. Centrifugation: 4°C, 5000rpm, 45min. After centrifugation, the supernatant was discar...

Embodiment 3

[0047] Example 3: Separation of human peripheral blood mononuclear cells (PBMC) by density gradient centrifugation

[0048] Use fresh heparin or sodium heparin blood collection tube or vacuum blood collection tube to aseptically draw 4ml of peripheral venous blood from the person to be tested, and mix the anticoagulant and blood until use. Prepare two 15ml centrifuge tubes, and add the same volume of normal saline for injection and lymphocyte separation solution to the blood collection volume. After mixing fresh heparin anticoagulant blood with physiological saline, slowly add it to the separating solution at a uniform speed, and centrifuge at 1800g at 22°C for 20 minutes. After centrifugation, PBMCs cells can be seen in the cloud-like layer. Pipette the PBMCs cell layer into a new 15ml centrifuge tube, make up to 12ml with RPMI-1640 culture medium, and centrifuge at 600g for 10min at 22°C. Pour out the supernatant, make up to 5ml with RPMI-1640 culture medium, gently pipette th...

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Abstract

The invention discloses a mycobacterium tuberculosis fusion protein and an application thereof in induction of peripheral blood mononuclear cells (PBMCs) to generate cytokines. The fusion protein includes three proteins PPE41, ESAT-6 and PE25, and the proteins are connected through connecting peptides. Compared with present stimulants, the fusion protein provided by the invention has the advantages of efficient effect, strong sensitivity, high specificity and good stimulation effect. The fusion protein stimulates the PBMCs to generate a large amount of mycobacterium tuberculosis antigen specific IFN-gamma, IL-2, TNF-alpha and other tuberculosis related factors, and the above stimulation induction reaction is free from BCG vaccine interference. The fusion protein can effectively improve the tuberculosis detection rate and is of positive significance to control the tuberculosis. The fusion protein can be applied in researches of the tuberculosis pathopoiesis and immunoprophylaxis mechanisms and control of the tuberculosis as a stimulant.

Description

Technical field [0001] The invention belongs to the protein field in molecular biology technology, and specifically relates to a Mycobacterium tuberculosis fusion protein and its application in inducing peripheral blood mononuclear cells to produce cytokines. Background technique [0002] Tuberculosis (tuberculosis, TB) is a chronic infectious disease caused by Mycobacterium tuberculosis, which can invade many organs, and pulmonary tuberculosis infection is the most common. The transmission of tuberculosis mainly occurs before the patient is detected and treated. On average, a patient with tuberculosis can infect 15 people. Because the newly ill person did not take any preventive measures before the illness was found, the contacts were easily infected by tuberculosis during the close contact with family members, colleagues, classmates, etc. About one-third of the world's population is infected with tuberculosis bacilli. About 9 million new tuberculosis patients are diagnosed eac...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62G01N33/68G01N33/569A61K39/116A61K39/04A61P31/06
Inventor 黄曦李丽炎杨锟田国宝吴敏昊
Owner SUN YAT SEN UNIV
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