CTL epitope peptide of cryptosporidium parvum and application and vaccine of CTL epitope peptide

A technology of Cryptosporidium parvum and epitope peptides, applied in the fields of application, medical preparations containing active ingredients, peptide sources, etc., can solve the problems that hinder the research and application of Cryptosporidium parvum epitope vaccines, and achieve huge development and application potential effect

Active Publication Date: 2019-06-28
ZHOUKOU NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the most studied CTL epitopes of Cryptosporidium parvum are human HLA-B and HLA-C genotypes, while other human genotypes such as HLA-A*0201 molecules widel

Method used

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  • CTL epitope peptide of cryptosporidium parvum and application and vaccine of CTL epitope peptide
  • CTL epitope peptide of cryptosporidium parvum and application and vaccine of CTL epitope peptide
  • CTL epitope peptide of cryptosporidium parvum and application and vaccine of CTL epitope peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Selection and synthesis of CTL epitope polypeptide of Cryptosporidium parvum Gp15 protein.

[0033]Scan the main pathogenic protein and surface protein GP15, GP40, CSL, GP900, HSP70, CP15 and CP23 of Cryptosporidium parvum, select a polypeptide with amino acid sequence such as SEQ ID No.1, named P1, and its nucleotide sequence Research was carried out as shown in SEQ ID No. 2 in the Sequence Listing. Polypeptide P1 is derived from GP15 protein. Polypeptide P1 is synthesized by solid-phase peptide synthesis method (Fmoc / tBu strategy). After synthesis, it is purified by HPLC with a purity of 98%.

[0034] The present invention first uses the bioinformatics analysis method to predict the H-2Kb, H-2Db restricted polypeptide, and screens a large number of polypeptides from these epitopes for testing, and finally screens out 8 polypeptides with strong predicted affinity ( peptide1-peptide8, peptide2 are polypeptides with amino acid sequences such as SEQ ID No.1, named P1), a...

Embodiment 2

[0036] Detection of Binding Ability of Cryptosporidium parvum CTL Epitope Polypeptide to Mouse H-2kb and Human HLA-A*0201 Molecules by Protein Refolding Experiment

[0037] 1. Preparation of mouse MHC class I molecule (H-2Kb) and light chain β2m (mouseβ2m, mβ2m) inclusion body protein

[0038] The DNA molecule ( The nucleotide sequence is inserted between the prokaryotic expression plasmid pET-21a (+) restriction site Nde I and Xho I (as shown in SEQ ID No.4 in the sequence table) to obtain a recombinant expression plasmid named pET-21a ( +) / H-2kb.

[0039] The constructed pET-21a(+) / H-2Kb recombinant expression plasmid was transformed into Escherichia coli BL21(ED3) competent cells, cultured overnight at 37°C, and recombinant transformants were screened by ampicillin (Amp) resistance. Single colonies of positive recombinant bacteria (named BL21(ED3) / H-2Kb) were picked and inoculated in 100 mL of LB medium containing Amp. After cultured with shaking at 37°C,

[0040] Accor...

Embodiment 3

[0054] The specific CTL immune response of Cryptosporidium parvum CTL epitope polypeptide immunized mice was detected by ELISPOT method.

[0055] The MHC I / antigenic peptide complex is a key factor in the activation of specific CTL immune responses. C57BL / 6N mice have a clear MHC genetic background and can be used as an experimental animal model for studying cellular immune responses to Cryptosporidium parvum. Rasmussen et al. showed that C57BL / 6N mice (whose MHC genotype is: H-2Kb, H-2Db) were inoculated 10 6 After one Cryptosporidium parvum oocyst, the infected state remained throughout the experimental period, indicating that this strain of mice is related to the resistance and susceptibility of Cryptosporidium parvum. And the H-2Kb restricted epitope has similar anchor residues with the epitope of HLA-A*0201 molecule widely expressed in the human population. Therefore, using C57BL / 6N mice as hosts to screen avian influenza virus CTL epitopes will provide a reliable basis...

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Abstract

The invention relates to a CTL epitope peptide of cryptosporidium parvum. The peptide has the amino acid sequence of SEQ ID NO.1. The invention further provides a nucleic acid for coding the CTL epitope peptide of cryptosporidium parvum. The nucleic acid has the nucleotide sequence of SEQ ID NO.2. The CTL epitope peptide P1 of cryptosporidium parvum has quite high affinity to H-2Kb molecules or HLA-A*0201 molecules, and it means that the CTL epitope peptide has the corresponding immunology functions. A new direction is provided for preparing a cryptosporidium parvum vaccine. The peptide presents excellent irritation reactions in the polypeptide specificity CTL immune reaction in C57BL/6N mouse splenocytes and has huge development and application potential in the cryptosporidium parvum specific immunotherapy field.

Description

technical field [0001] The invention belongs to the technical field of biomolecules and relates to a CTL epitope polypeptide of Cryptosporidium parvum and its application and vaccine. Background technique [0002] Cryptosporidium parvum belongs to Class Sporozoa of Phylum Apicomplexa, was first reported and named by Tyzzer in 1912, and is an important zoonotic protozoa. Among the 31 zoonotic Cryptosporidium species reported, Cryptosporidium parvum can infect humans and most mammals, and Cryptosporidium parvum has the most extensive hosts, mainly including humans, bovines, and various rodents Mammals, camelids, equines, canids, non-human primates and marine mammals and fish. According to incomplete statistics, from 2004 to 2010, there have been hundreds of fulminant and sporadic Cryptosporidium cases caused by water-borne or food-borne transmission in many countries around the world. Cryptosporidium parvum can survive in the environment for a long time, and general sewage t...

Claims

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Application Information

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IPC IPC(8): C07K14/44C12N15/30A61K39/00A61P33/02
CPCY02A50/30
Inventor 樊淑华王永立杨蟠刘晓萌吴常景朱文帅李俐俐陈龙
Owner ZHOUKOU NORMAL UNIV
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