Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A Mycobacterium tuberculosis fusion protein for inducing cytokine production in peripheral blood mononuclear cells

A technology of fusion protein and tuberculosis, applied in the field of protein in molecular biology technology, can solve the problem of low specificity of PPD, and achieve the effect of strong sensitivity, high efficiency and high specificity

Active Publication Date: 2019-06-25
SUN YAT SEN UNIV
View PDF2 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A Mycobacterium tuberculosis fusion protein for inducing cytokine production in peripheral blood mononuclear cells
  • A Mycobacterium tuberculosis fusion protein for inducing cytokine production in peripheral blood mononuclear cells
  • A Mycobacterium tuberculosis fusion protein for inducing cytokine production in peripheral blood mononuclear cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: CFP-10-EsxG-EsxH fusion protein target gene synthesis and construction of prokaryotic expression vector

[0041] The genes encoding CFP-10, EsxG and EsxH proteins are respectively cfp-10 Gene ID: 886194, wxya Gene ID: 886604, wxyaGene ID: 886603. Primers were designed using the full sequence or the partial functional region sequence expressing the antigenic activity.

[0042] 1) Use the upstream and downstream primers of CFP10, EsxG and EsxH to ​​amplify the target fragments of CFP-10, EsxG and EsxH by PCR respectively, use Taq DNA polymerase to perform PCR amplification and recover the above fragments.

[0043] 2) Two kinds of nucleotide chains with connecting peptide nucleotide sequences were synthesized by artificial synthesis, and one of the nucleotide chains (hereinafter referred to as connecting peptide chain 1) contained CFP-10 and EsxG partial sequences at both ends, respectively. The middle is the base sequence of the connecting peptide (SEQ I...

Embodiment 2

[0046] Example 2: Induced expression and purification of fusion protein CFP-10-linked peptide-EsxG-linked peptide-EsxH

[0047] Take the correct recombinant expression bacteria identified in the above example 1 containing the CFP-10-connecting peptide-EsxG-connecting peptide-EsxH target gene fragment (the nucleic acid sequence is shown in SEQ ID NO: 2) into 20 ml containing kana-resistant LB medium, 37°C, 250rpm, shaking culture for 6 h. Autoclave 2L of LB medium, put 500ml in each 1L Erlenmeyer flask, and divide into 4 bottles. Add 2.5ml of activated bacterial solution into each bottle, shake and culture for about 3 hours at 37°C and 250 rpm, so that the OD600 of the bacterial solution is 0.5. Put the Erlenmeyer flask into cold water, cool down the shaker, and induce after cooling. Add IPTG to each bottle to make the final concentration 0.5mM, put it into a shaker at 20°C, 200rpm, and induce for 20 hours. Centrifugation: 4°C, 5000rpm, 45min. After centrifugation, discard ...

Embodiment 3

[0048] Example 3: Isolation of Human Peripheral Blood Mononuclear Cells (PBMC) by Density Gradient Centrifugation

[0049] 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 medium at a uniform speed, and centrifuge at 1800g for 20min at 22°C. After centrifugation, PBMCs cells can be seen to exist in the cloudy 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 off the supernatant, make up to 5ml with RPMI-1640 ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses mycobacterium tuberculosis fusion protein for inducing peripheral blood mononuclear cells to produce cytokines. The fusion protein includes CFP-10 protein, EsxG-6 protein and EsxH protein which are connected through connecting peptides. Compared with an existing stimulus, the fusion protein is more efficient in effect, higher in sensitivity, higher in specificity and good in stimulating effect. Meanwhile, the fusion protein stimulates PBMC to produce lots of tuberculosis related factors such as IFN-gamma, IL-2 and TNF-alpha with mycobacterium tuberculosis antigenic specificity, and the stimulus-induced reaction is not interfered with by bacillus calmette guerin vaccine. By means of the fusion protein, the relevance ratio of tuberculosis is more effectively improved, and the fusion protein has positive significance in control over tuberculosis. The fusion protein as a stimulus can be applied to research on a tuberculosis pathopoiesia and immunity prevention mechanism and further control over tuberculosis.

Description

technical field [0001] The invention belongs to the field of proteins in molecular biology technology, and in particular relates to a mycobacterium tuberculosis fusion protein for 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, with pulmonary tuberculosis infection being the most common. About 1 / 3 of the world's population is infected with Mycobacterium tuberculosis, about 9 million new tuberculosis patients are diagnosed every year, and about 3 million people die from tuberculosis every year. Tuberculosis has become one of the main diseases that cause adult deaths from infectious diseases all over the world. According to the "Global Tuberculosis Report 2014" published by WHO on October 22, 2014, 9 million people were infected with tuberculosis and 1.5 million died in 2013, and about 95% of tubercu...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62G01N33/68G01N33/569A61K39/116A61K39/04A61P31/06
Inventor 黄曦李金爱杨锟田国宝吴敏昊
Owner SUN YAT SEN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com