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TSP4 peptide sequence for cryptosporidium parvum detection and application

A Cryptosporidium parvum and peptide sequence technology, applied in peptides, measuring devices, biological testing, etc., can solve problems such as inability to distinguish oocyst viability

Inactive Publication Date: 2018-11-06
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problem that the existing methods for detecting Cryptosporidium parvum oocysts in feces or environmental samples cannot distinguish the viability of oocysts, and provide an anti-polypeptide monoclonal antibody for the detection of Cryptosporidium parvum sporozoites

Method used

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  • TSP4 peptide sequence for cryptosporidium parvum detection and application
  • TSP4 peptide sequence for cryptosporidium parvum detection and application
  • TSP4 peptide sequence for cryptosporidium parvum detection and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Screening and Synthesis of Cryptosporidium Parvum-Specific Polypeptides

[0037] 1. Screening of Cryptosporidium parvum-specific peptides

[0038] 1) Find multiple target genes on the professional website of Cryptosporidium (http: / / cryptodb.org / cryptodb / ) as alternative antigens;

[0039] 2) Use swissmodel.expasy.org to predict the three-dimensional structure of the amino acid sequence of the target gene, find the relatively strong immunogenicity of the random coil region, and then use iedb.org to predict the prediction of the B cell epitope, and finally Compare the designed peptide with other proteins of Cryptosporidium, and synthesize peptides with strong immunogenicity and specificity;

[0040] 3) In order to enhance the coupling efficiency of polypeptides with KLH and BSA, when designing polypeptides, selectively avoid polypeptides containing cysteine, and artificially add a cysteine ​​after the polypeptide without cysteine ​​at the C-terminus cystine. ...

Embodiment 2

[0055] Embodiment 2 Animal immunization and preparation of monoclonal antibody

[0056] 1. Experimental Animal Immunization Procedures

[0057] Mix six Cryptosporidium-specific peptides coupled with KLH with an equal volume of Freund's adjuvant, and immunize Balb / c mice; the first immunization is mixed with complete adjuvant, and the immunization amount is 100 μg, and the last 4 immunizations are mixed with no The complete adjuvant was mixed, and the immunization amount was 50 μg; the interval between two immunizations was 15 days; three days before cell fusion, 20 μg polypeptide was injected into the tail vein of immunized Balb / c mice for booster immunization.

[0058] 2. Preparation of Monoclonal Antibody

[0059] 1. Preparation of feeder layer cells

[0060] The day before the fusion cells, the culture layer cells were made; a normal BALB / c mouse was taken, killed by pulling the neck, and soaked in 75% alcohol for 5 minutes. Turn to the ultra-clean workbench, cut the abdom...

Embodiment 3

[0102] Example 3 Indirect Immunofluorescence Detection of Clinical Samples

[0103] The six monoclonal antibodies prepared in the above examples were used as primary antibodies in fluorescence detection, and the specific steps are as follows:

[0104] 1) Add a certain amount of sodium hypochlorite to the collected clinical samples for sterilization, and then use sucrose floating method to remove most of the bacteria and impurities;

[0105] 2) Add the collected concentrate to the decyst solution (containing 0.75% taurocholate sodium salt, 0.25% trypsin) and act at 37°C for 1 hour. The purpose is to incubate the oocysts of Cryptosporidium parvum The four sporozoites inside are released to detect whether the prepared monoclonal antibody can act on the sporozoites;

[0106] 3) Centrifuge at 5000g for 10 minutes to remove the decapsulation solution;

[0107] 4) Add 4% paraformaldehyde (2g formaldehyde dissolved in 50ml PBS solution) to the centrifuged mixture, let it act for 30m...

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Abstract

The invention discloses a TSP4 peptide sequence for cryptosporidium parvum detection and application. The method comprises the following steps: coupling a cryptosporidium parvum peptide TSP4 and KLH to serve as an antigen, immunizing Balb / c mice, preparing a monoclonal antibody, collecting samples (oocysts), performing excystation with excystation fluid, incubating into sporozoite at a room temperature, permeabilizing with permeabilizing fluid, respectively adding the monoclonal antibody, a secondary antibody, a PBS (Phosphate Buffer Solution), fluorescent dyes and an anti-fluorescence quenching agent, and observing the samples by utilizing a fluorescence microscope. Repeated experiments show that the peptide specific to six kinds of cryptosporidium coupled with the KLH has excellent immunogenicity, and hybridoma cells of ideal titers can be obtained from immune mice; during screening each time, monoclonal antibodies of which the titers reach 1:16000 or higher can be screened to be used for fluorescence detection. The monoclonal antibody prepared by the peptide synthesized from the TSP4 can be sensitively labeled onto the surface of cryptosporidium parvum sporozoite, and any crossreaction is not caused.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and specifically relates to a TSP4 polypeptide sequence and application for detecting Cryptosporidium parvum. Background technique [0002] Cryptosporidium parvum is a major cause of cryptosporidiosis in humans and other mammals worldwide. Cryptosporidium parvum is mainly transmitted by ingesting water or food contaminated by Cryptosporidium through the fecal-oral route. It mainly parasitizes on the edge of the microvilli of the epithelial cells of the gastrointestinal tract, and the symptoms after infecting the host vary in severity. Diarrhea through proliferation in the intestinal epithelium is generally self-limited in immunocompetent individuals but is a major cause of death in immunocompromised individuals. Cryptosporidium parvum oocysts can survive in the environment for a long time and can resist conventional sodium hypochlorite disinfection, which has become a major problem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06G01N33/68G01N33/577G01N33/569
CPCC07K7/06G01N33/56905G01N33/577G01N33/6854G01N2333/44
Inventor 尹继刚张天宇王东强焦新邵青延
Owner JILIN UNIV
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