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Fusion protein for inducing peripheral blood mononuclear cells to produce tuberculosis specific cytokines

A fusion protein and protein technology, applied in the protein field of molecular biology technology, can solve the problems of low specificity of PPD, and achieve the effects of strong sensitivity, improved specificity and induction effect, and efficient effect.

Active Publication Date: 2016-01-06
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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  • Fusion protein for inducing peripheral blood mononuclear cells to produce tuberculosis specific cytokines
  • Fusion protein for inducing peripheral blood mononuclear cells to produce tuberculosis specific cytokines
  • Fusion protein for inducing peripheral blood mononuclear cells to produce tuberculosis specific cytokines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: the construction of CFP-10-ESAT-6-EspC fusion protein target gene synthesis and prokaryotic expression vector

[0041] The genes encoding CFP-10, ESAT-6 and EspC proteins are respectively cfp-10 Gene ID: 886194, esat-6 Gene ID: 862098, espC Gene ID: 885770. 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, ESAT-6 and EspC to amplify the target fragments of CFP-10, ESAT-6 and EspC by PCR respectively, use TaqDNA 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 ESAT-6 parts at both ends Sequence, the middle is the base sequence of the connecting peptide (SEQ ...

Embodiment 2

[0046] Embodiment 2: Induced expression and purification of fusion protein CFP-10-connected peptide-ESAT-6-connected peptide-EspC

[0047] Take the correct recombinant expression bacteria identified in Example 1 above containing CFP-10-connecting peptide-ESAT-6-connecting peptide-EspC target gene fragment (its nucleic acid sequence is shown in SEQ ID NO: 2) and insert 20ml containing kana resistance In the LB medium, 37°C, 250rpm, shaking culture for 6h. 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 centr...

Embodiment 3

[0048] Example 3: Separation 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 solution 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. Aspirate 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...

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Abstract

The invention discloses fusion protein for inducing peripheral blood mononuclear cells to produce tuberculosis specific cytokines. The fusion protein includes CFP-10 protein, ESAT-6 protein and EspC 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 protein in molecular biology technology, and specifically relates to a fusion protein for inducing peripheral blood mononuclear cells to produce tuberculosis-specific cytokines. Background technique [0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. In recent years, with the increase of floating population, the concurrent infection of human immunodeficiency virus (HIV) and Mycobacterium tuberculosis, and the emergence of multidrug-resistant strains of Mycobacterium tuberculosis , which increases the global tuberculosis epidemic situation and poses new challenges to the global tuberculosis prevention and control. 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...

Claims

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Application Information

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IPC IPC(8): C07K19/00G01N33/68
Inventor 黄曦冯思源杨锟田国宝吴敏昊
Owner SUN YAT SEN UNIV
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