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

Chimeric protein, method of production and use thereof, and also a nucleic acid molecule, expression cassette, expression vector, host cell, composition for the diagnosis of leishmaniasis, kit for the diagnosis of leishmaniasis and method of diagnosis of leishmaniasis in vitro

A technology of chimeric proteins and nucleic acid molecules, applied in the field of chimeric proteins, can solve problems such as limitations and improve the quality of disease diagnosis

Pending Publication Date: 2021-12-10
FUNDACAO OSWALDO CRUZ FIOCRUZ
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, to date, the clinical use of these molecules, especially for canine VL diagnosis, has remained limited and / or has not resulted in a significant improvement in the quality of disease diagnosis
[0051] As can be seen, no prior art literature teaches a single tool for VL diagnosis in humans or dogs

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
  • Chimeric protein, method of production and use thereof, and also a nucleic acid molecule, expression cassette, expression vector, host cell, composition for the diagnosis of leishmaniasis, kit for the diagnosis of leishmaniasis and method of diagnosis of leishmaniasis in vitro
  • Chimeric protein, method of production and use thereof, and also a nucleic acid molecule, expression cassette, expression vector, host cell, composition for the diagnosis of leishmaniasis, kit for the diagnosis of leishmaniasis and method of diagnosis of leishmaniasis in vitro
  • Chimeric protein, method of production and use thereof, and also a nucleic acid molecule, expression cassette, expression vector, host cell, composition for the diagnosis of leishmaniasis, kit for the diagnosis of leishmaniasis and method of diagnosis of leishmaniasis in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0144] Bioinformatics methods, chemical synthesis and subcloning of chimeric genes

[0145] Prediction of the presence of linear B-cell epitopes in the sequences of selected proteins (Lci2, Lci3 and Lci12) was performed by the program BCPred12 (EL-MANZALAWY; DOBBS; HONAVAR, 2008). A chimeric gene sequence optimized for expression in Escherichia coli was designed using the Gendesigner program (WELCH et al., 2011) and provided by GenScript (GenScript, Piscataway, New Jersey, USA) (Q1 protein) and Thermo (Life Tech, Paulo, Brazil) (Q5 protein) for commercial synthesis.

[0146] These genes have been cloned into the commercial vector pUC57 flanked by restriction sites for the enzymes XbaI / HindIII. For subcloning into the bacterial expression vector pRSETa (Thermo Life Tech, Paulo, Brazil), the chimeric gene was recovered by double digestion with restriction enzymes XbaI / HindIII and subcloned into the same site of the pRSETa vector.

[0147] After the first subcloning of the Q...

Embodiment 2

[0149] Expression and purification of recombinant proteins

[0150] To express the chimeric protein, the plasmid construct derived from the pRSETa vector was transformed into competent E. coliRosetta TM 2 cells (Merck Millipore) were then selected at 37°C on solid LB medium (Luria Bertani) supplemented with ampicillin (100 μg / ml) and chloramphenicol (34 μg / ml).

[0151] Clones of transformed cells were grown in liquid LB medium with the same concentration of antibiotics, and the expression of recombinant protein was induced by adding IPTG to a final concentration of 0.1 mM and an optical density (D.O) at 600 nm of 0.6 to 0.8.

[0152] After staining the gel with Coomassie Blue R-250, the results were visualized using a polyacrylamide gel (SDS-PAGE 15%). To obtain recombinant protein, the cell pellet obtained after induction was resuspended and equilibrated in 20 mL of lysis buffer (100 mM sodium phosphate, 10 mM Tris, 8 M urea, 20 mM imidazole-pH 8.0) and passed through at 4 ...

Embodiment 3

[0155] Western blot analysis

[0156] For Western blot analysis, chimeric proteins were fractionated on 15% SDS-PAGE gels and transferred to PVDF membranes (Immobilon-P ), blocked in TBS buffer (20 mM Tris, 500 mM NaCl, pH 7.5) supplemented with 5% skim milk and 1% Tween-20.

[0157] The membrane was then incubated with antibody / serum against the target protein at final dilutions of 1:3000 and 1:1000 in TBS buffer containing 5% milk and 1% Tween-20. After washing with 1% TBS / Tween-20, a new incubation was performed with peroxidase-labeled rabbit anti-IgG (Jackson Immunoresearch Laboratories) at 1:1 in TBS buffer containing 5% milk and 1% Tween-20. 10000 dilution.

[0158] After further washing, the membrane was exposed to a solution of 1.2 mM luminol, 0.4 mM iodophenol and 0.03% hydrogen peroxide for 2 minutes. These films were then dried and exposed to autoradiographic film for 1 and 5 minutes before the film was developed.

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

PropertyMeasurementUnit
Sensitivityaaaaaaaaaa
Login to View More

Abstract

The present invention relates to chimeric proteins, and the uses and method of production thereof, comprising protein fractions native to Leishmania infantum for the diagnosis of visceral leishmaniasis. The invention also relates to the nucleic acid, expression cassette, expression vector, host cell, composition for the diagnosis of visceral leishmaniasis, kit for the diagnosis of visceral leishmaniasis, method of diagnosis of visceral leishmaniasis and vaccine composition.

Description

technical field [0001] The invention relates to the fields of diagnostic medicine and biotechnology. In particular, the present invention relates to polypeptides, more particularly chimeric proteins, for diagnostic applications of Leishmania infantum in humans, dogs and other vertebrate hosts. Background technique [0002] Leishmaniasis is an infectious parasitic disease caused by flagellar protozoa belonging to the family Trypanosomatidae and the genus Leishmania. [0003] These protozoa have a heterogeneous life cycle that alternates between vertebrate hosts (including humans and other wild and / or domestic mammals such as dogs) and vector dipterous insects, which belong to the genus Phlebos ( Phlebotomus) and Lutzomyia (PACE, 2014). [0004] Currently, there are approximately 30 known species of Leishmania, 10 of which are found in the Old World and 20 of which are found in the New World, of which about 20 are capable of causing skin and / or visceral Leishmaniasis. [0...

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 Applications(China)
IPC IPC(8): C07K14/44C12N15/62C12N15/63A61K39/008C12N1/00G01N33/569
CPCA61K39/008C07K14/44C12N15/63Y02A50/30C12N15/70C07K2319/00C07K2319/21G01N33/56905G01N2469/20C07K2317/24G01N33/569
Inventor O·P·D·M·内托A·M·雷森德D·D·H·C·塔瓦雷斯W·J·T·多斯桑托斯A·L·D·C·内托F·B·马加莱斯M·B·多·纳西门托
Owner FUNDACAO OSWALDO CRUZ FIOCRUZ
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