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A design, preparing method and applications of an echinococcus multilocularis multi-epitope vaccine LTB-AE

A multilocular echinococcosis, LTB-AE technology, applied in the field of biomedicine, can solve the problems of unsatisfactory hydatid disease effect, multilocular hydatid escaping, difficult expression, extraction, purification, etc.

Active Publication Date: 2017-08-15
QINGHAI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Sugimoto C found that the surface antigen TSP3 of Echinococcus multilocularis has good immunogenicity; but in the latest study by Oku Y et al., they found that TSP3 and TSP3 fusion FBP changed the immune type from Th1 to Th2 in the late stage of immunization of mice , leading to the occurrence of immune escape of Echinococcus multilocularis, and the inhibition rate is only 60%. Therefore, the preparation of Emy162 subunit genetic engineering vaccines of Echinococcus multilocularis has problems such as difficult expression, extraction, and purification.
Therefore, the effect of traditional medicines and vaccines in the prevention and treatment of echinococcosis in clinical application is not satisfactory.

Method used

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  • A design, preparing method and applications of an echinococcus multilocularis multi-epitope vaccine LTB-AE
  • A design, preparing method and applications of an echinococcus multilocularis multi-epitope vaccine LTB-AE
  • A design, preparing method and applications of an echinococcus multilocularis multi-epitope vaccine LTB-AE

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Molecular Structure Design of Multilocular Echinococcus Multi-epitope Vaccine LTB-AE

[0041] According to "The body's immune protection mechanism against Echinococcus multilocularis" and "Epitope vaccine construction theory", the epitope Emy162 of the antigenic protein Emy162 of Echinococcus multilocularis was used 36-48 、Emy162 7-13 and TSP3 epitope TSP3 33-42 、TSP3 80-90 It is used for the construction of multi-epitope vaccine against Echinococcus multilocularis. In the design of the multi-epitope vaccine for Echinococcus multilocularis, the present invention puts the Th cell epitope at the front of the B cell epitope, which is helpful for the effective presentation of the B cell epitope, and then through bioinformatics DNAstar and RANKPEP Software analysis and evaluation, the epitope sequence is determined as TSP3 80-90 -Emy162 36-48 - TSP3 33-42 -Emy162 7-13 ; KK is selected as the spacer sequence of the adjacent Th epitope, and GS is selected a...

Embodiment 2

[0043] Example 2: Construction of recombinant expression vector pCzn1-LTB-AE (containing fusion gene LTB-AE)

[0044] The amino acid sequence of the previously designed multi-epitope peptide LTB-AE was converted into the corresponding nucleotide sequence according to the codon preference principle of E. coli, and the full-length splicing primer was designed based on the method of PAS (PCR-based Accurate Synthesis) , A protective base synthesis gene LTB-AE was designed at both ends of the primers, and connected into the expression vector pCzn1 through the cloning sites Nde I and Xba I.

[0045] Results: The recombinant plasmid pCzn1-LTB-AE to be detected was digested with Nde I and Xba I, reacted at 37°C for 2 hours, and detected by 1% agarose gel electrophoresis. The theoretical size of the gene LTB-AE is consistent, as figure 2 shown. The vector construction map of the recombinant expression vector pCzn1-LTB-AE is as follows: image 3 shown. The obtained recombinant plas...

Embodiment 3

[0046] Example 3: Prokaryotic expression of multi-epitope peptide fusion protein LTB-AE

[0047] The correct recombinant expression plasmid pCzn1-LTB-AE was verified to be transferred into Escherichia coli Arctic Express strain. On the pre-prepared LB plate containing 50μg / mL Amp, inoculate the loop-streaked genetically engineered strain pCzn1-LTB-AE / ArcticExpress, place it upside down in a 37°C incubator, and after culturing overnight, pick a single colony and inoculate it on a plate containing 50μg / mL Amp in LB medium, 37°C, 220rpm, culture overnight. Inoculate the recombinant bacteria with 2% inoculum in LB medium containing 50 µg / mL Amp, 37°C, 220 rpm, culture until the OD600 of the bacteria is 0.6-0.8 (about 2 hours), add IPTG to make the final concentration reach 1mmol / L, The expression was induced at 37°C and 220rpm for 4 hours, and the carrier strain pCzn1-LTB-AE / Arctic Express induced without IPTG was used as a negative control.

[0048] Results: Compared with the ...

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Abstract

The invention relates to a design, preparing method and applications of an echinococcus multilocularis multi-epitope vaccine LTB-AE. An active component of the multi-epitope vaccine LTB-AE is a polypeptide. The polypeptide mainly comprises an echinococcus multilocularis multi-epitope peptide AE, and a mucosal immune adjuvant that is an escherichia coli heat-labile enterotoxin B subunit (LTB). A gene sequence of the multi-epitope vaccine LTB-AE is synthesized mainly by a gene synthesis technique, and is connected to an expression vector through double digestion, then the expression vector is converted into arctic express to perform expression of a fusion protein, and after the protein is purified, the multi-epitope vaccine LTB-AE is obtained. The multi-epitope vaccine can induce a body to generate immune responses and high-titer specific antibody humoral immune responses to echinococcus multilocularis T cells and B cells, and can be used for preventing and treating echinococcus multilocularis infection related disease.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a design, preparation method and related application of a multilocular echinococcus vaccine. Background technique [0002] Echinococcosis is a zoonotic parasitic disease, also known as echinococcosis, including larval disease caused by Echinococcus granulosus: Cystic echinococcosis (CE ) and larval disease caused by Echinococcus multilocularis: alveolar echinocococosis (AE). Echinococcus multilocularis grows in the form of budding or infiltrating in the liver of the intermediate host, producing new vesicles that grow into the liver tissue, the outer horn of the cyst wall is very thin and often incomplete, and there is no obvious boundary between the cyst and surrounding tissues , continuous leakage of cystic fluid can contact with liver tissue, causing local liver tissue lesions, hyperplasia, liver fibrosis, atrophy, degeneration and necrosis, advanced liver canc...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61P33/10
CPCA61K39/0003A61K2039/572A61K2039/575A61K2039/70
Inventor 格日力汤锋李润乐樊海宁刘川川冯琳刘文磊杨宝良杨全余
Owner QINGHAI UNIVERSITY
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