Application of protein and composition thereof in preparation of product used for preventing, diagnosing or treating EHEC (Enterohemorrhagic Escherichia Coli) infection

A composition and protein technology, applied in the direction of peptide/protein components, immunoglobulins from serum, medical preparations containing active ingredients, etc., can solve the problem of incomplete annotation of genome functions, unclarified functions of virulence factors, and encoded protein functions Issues such as not being confirmed

Active Publication Date: 2013-07-24
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
View PDF4 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, due to the complex and diverse genes involved in the pathogenicity island of EHEC pathogens, the functions of many virulence factors have not yet been elucidated.
Although a large number of complete genomes of EHEC pathogenic bacteria have been published in Genbank, the functional annotation of the genome is not complete, and the functions of a large number of encoded proteins have not been confirmed, especially the functional proteins that can be used in the development of diagnostic and preventive drugs have not been fully discovered. , there is still a lot of room to identify and discover new functional molecules with clinical application value

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
  • Application of protein and composition thereof in preparation of product used for preventing, diagnosing or treating EHEC (Enterohemorrhagic Escherichia Coli) infection
  • Application of protein and composition thereof in preparation of product used for preventing, diagnosing or treating EHEC (Enterohemorrhagic Escherichia Coli) infection
  • Application of protein and composition thereof in preparation of product used for preventing, diagnosing or treating EHEC (Enterohemorrhagic Escherichia Coli) infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1, the acquisition of protein and its composition and its recognition to infected EHEC serum

[0066] 1. Amplification of the target gene

[0067] According to the target gene nucleotide sequence of EHEC O157:H7 published by GenBank, primers were designed and synthesized by introducing 6×His tag (CACCACCACCACCACCAC) into the downstream primers considering the requirements of protein purification.

[0068] Table 1 is the primer name, sequence and restriction site

[0069]

[0070] Using the extracted EHEC O157:H7 genome as a template, the upstream and downstream primers of Z0390 and the upstream and downstream primers of Z4191 were used for PCR amplification respectively, and a PCR product of 1137bp and a PCR product of 597bp were obtained.

[0071] The 50 μl amplification reaction system required for the above PCR amplification is: 1 μl of upstream and downstream primers, 1 μl of template genomic DNA, 4 μl of dNTP, 5 μl of 10×Buffer, 1 μl of Taq enzyme, and ...

Embodiment 2

[0103] Embodiment 2, the immunogenicity of protein or protein composition and the protective effect to EHEC infection

[0104] Female BALB / c mice 14-16g were randomly divided into 2 groups, 10 in each group, and the following experiments were repeated three times:

[0105] Z0390 immune group: the purified recombinant protein Z0390-HIS obtained in Example 1 was immunized by intraperitoneal route as antigen;

[0106] Z4191 immunization group: the purified recombinant protein Z4191-HIS obtained in Example 1 was immunized by intraperitoneal route as antigen;

[0107] Z0390 / Z4191 mixed immunization group I: intraperitoneal route immunization with purified recombinant protein Z0390-HIS and purified recombinant protein Z4191-HIS obtained in Example 1 as immunogens; mass ratio of purified recombinant protein Z0390-HIS and purified recombinant protein Z4191-HIS 1:1;

[0108] Z0390 / Intimin mixed immunization group II: intraperitoneal route immunization of the purified recombinant prot...

Embodiment 3

[0127] Embodiment 3, the preparation and application of the antibody or antibody group of anti-protein antigen

[0128] 1. Preparation and identification of protein antigen antiserum

[0129] The purified recombinant protein Z0390-HIS and the purified recombinant protein Z4191-HIS obtained in Example 1 with a concentration of 1 mg / ml and a purity of more than 95% were used as antigens to immunize big-eared white rabbits (male, 2.5 kg). Respectively in the fourth week (25μg / monkey) and eighth week (50μg / bird) booster immunization. When the titer (ELISA detection) reaches and stabilizes at 1:10 5 As mentioned above, blood was collected from the heart of Daerbai, and the serum was separated by centrifugation at 5000g for 10 minutes, and the serum was filtered with a 2 μm disposable filter (Millipore). The filtered serum was purified by affinity chromatography (Protein A Hitrap, GE Company, operation Follow the instructions), collect 1.2 column volumes for anti-Z0390 antibody (p...

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 an application of protein and composition thereof in preparation of product used for preventing, diagnosing or treating EHEC (Enterohemorrhagic Escherichia Coli) infection. The invention provides an application of any substance shown in A-D (described in the specification) in preparation of the product with the following function 1) and or 2): 1) preventing, diagnosing and/or treating diseases infected or initiated by an EHEC pathogenic bacterium; and 2) reducing intestinal infection damage caused by the EHEC pathogenic bacterium. Experiments in the invention proof that proteins Z0390 and Z4191 are acquired by adopting a genetic engineering technology, an immunology function experimental study for evaluating proteins in vitro and vivo proofs that the two proteins and the composition of the two proteins are important core antigens for the EHEC pathogenic bacterium and can serve as an antigen reagent used for infection diagnosis; and O157:H7 infection can be prevented, so that the protein can serve as a vaccine or medicine for treating the diseases initiated by EHEC.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a protein and its composition and its application in the preparation of products for preventing, diagnosing or treating EHEC infection. Background technique [0002] Enterohemorrhagic Escherichia coli (enterohemorrhage E. coli, EHEC) is the most virulent and pathogenic E. coli, and has received widespread attention. EHEC infection can make people suffer from diarrhea, hemorrhagic colitis (HC), and can also cause serious complications such as hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). 5% to 10%. In April 2011, an outbreak of enterohaemorrhagic Escherichia coli O104:H4 infection and hemolytic uremic syndrome (HUS) broke out in Germany, spreading to 16 countries in Europe and North America. In April and May of the same year, a new infection broke out in the Toyama area of ​​Japan. The serotype was O111. Some cases developed neurological symptoms aft...

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): A61K38/16A61K39/40A61P31/04C07K16/06G01N33/569G01N33/68G01N33/577
CPCY02A50/30
Inventor 王慧李涛王琴韩燃李崭陈芳红
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap