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Design and preparation method and application of novel fasciola hepatica multi-epitope vaccine

A Fasciola hepatica, multi-epitope technology, applied in the field of animal husbandry medicine, can solve problems such as unavoidable repeated treatment, complete deworming of drugs, and increase of drug-resistant worms of Fasciola hepatica

Inactive Publication Date: 2016-08-31
QINGHAI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(3) Drug treatment is difficult to avoid repeated treatment
(4) The drug cannot achieve the effect of complete deworming
[0008] At present, the method of treating Fasciola hepatica mainly adopts broad-spectrum deworming method, which has many disadvantages: such as drug residue in animals, increase of drug-resistant worms of Fasciola hepatica, etc.

Method used

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  • Design and preparation method and application of novel fasciola hepatica multi-epitope vaccine
  • Design and preparation method and application of novel fasciola hepatica multi-epitope vaccine
  • Design and preparation method and application of novel fasciola hepatica multi-epitope vaccine

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0050] Example 1: Molecular structure design of Fasciola hepatica multi-epitope vaccine CTB-SAPE

[0051] According to "The body's immune protection mechanism against Fasciola hepatica" and "Immunological properties of saposin-like protein epitopes", select saposin-like protein Th and B cell epitope FhSAP 21-30 、FhSAP 76-85 、FhSAP 71-80 and FhSAP 81-90 Used in the construction of a new type of Fasciola hepatica multi-epitope vaccine. In the design of the new Fasciola hepatica multi-epitope vaccine, our research group placed 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 was determined as FhSAP 71-80 -FhSAP 81-90 -FhSAP 21-30 -FhSAP 81-90 ; KK is selected as the spacer sequence of the adjacent Th epitope, and GS is selected as the spacer sequence of the adjacent B epitope; the urease epi...

example 2

[0053] Example 2: Construction of recombinant expression vector pETCTB-SAPE (containing fusion gene CTB-SAPE)

[0054] (1) Gene synthesis of polyepitope peptide SAPE nucleotide sequence

[0055] The amino acid sequence of the previously designed multi-epitope peptide SAPE was transformed into the corresponding nucleotide sequence according to the codon preference principle of Escherichia coli, and Nanjing GenScript Biotechnology Co., Ltd. was entrusted to carry out gene synthesis.

[0056] (2) Connection of multi-epitope peptide SAPE gene and pETC expression vector

[0057] The recombinant plasmids pUCSAPE and pETCTB (gifted by Dr. Guo Le) were extracted by plasmid mini-extraction kit (Tiangen), and double-digested with Kpn I / Xho I respectively. The double-digestion reaction system is as follows:

[0058] pUCAPE / pETCTB

[0059] After digestion at 37°C for 2 h, electrophoresis was performed on a 1% agarose gel, and the electrophoresis results were observed. The mult...

example 3

[0062] Example 3: Prokaryotic expression of multi-epitope peptide fusion protein CTB-SAPE

[0063]Transform the correct recombinant expression plasmid pETCTB-SAPE into E.coli BL21(DE3) strain. On the pre-prepared LB plate containing 50 μg / mL Kana, inoculate the loop-streaked genetically engineered strain pETCTB-SAPE / BL21(DE3), place it upside down in a 37°C incubator, and after cultivating overnight for 12-16 hours, pick a single colony. Inoculate in LB medium containing 50µg / mL Amp, culture at 37°C, 180rpm, for 12-16h. Inoculate recombinant bacteria with 2% inoculum in LB medium containing 50µg / mL Kana, shake overnight at 37°C and 180rpm, and inoculate the bacterial solution with 1% inoculum in LB liquid containing 50µg / mL Kana the next morning After shaking the flask at 37°C and 180rpm for 3 hours in the culture medium, IPTG was added to make the final concentration reach 1mmol / L, and the expression was induced at 37°C and 180rpm. The empty vector strain pET28a / BL21(DE3) wa...

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Abstract

The invention provides a novel fasciola hepatica multi-epitope vaccine. An active ingredient of the novel fasciola hepatica multi-epitope vaccine is a polypeptide. The polypeptide is mainly formed by fasciola hepatica sphingolipid activated mucin-like protein-2 Th, a multicopy body of B-cell epitope and a mucosal immunologic adjuvant cholera toxin B subunit. An artificial gene is mainly synthesized through the gene synthesis technology and comprises Th of sphingolipid activated mucin-like protein-2 and a gene sequence of the multicopy body of the B-cell epitope, then the artificial gene is coupled with the gene sequence of the cholera toxin B subunit, and a fusion gene is formed. An escherichia coli prokaryotic expression system is utilized for expressing the fusion gene, and the fasciola hepatica multi-epitope vaccine is obtained after protein purification. The fasciola hepatica multi-epitope vaccine can induce an organism to generate sphingolipid activated protein T cell immune responses and high-titer specific antibody humoral immune responses, and can be used for preventing and treating fasciola hepatica infection related diseases.

Description

technical field [0001] The invention belongs to the field of animal husbandry medicine, and in particular relates to the design, preparation method and application of a Fasciola hepatica multi-epitope vaccine. Background technique [0002] Fasciola hepatica ( Fasciola hepatica ) belongs to the genus Fasciola of the family Fasciolidae in the order Digenea. It mainly parasitizes mammals, especially ruminants. one of the infectious diseases. If the liver and bile ducts of cattle and sheep are parasitized by Fasciola hepatica, the liver tissue is destroyed, causing hepatitis and bile duct hardening. At the same time, the parasites grow and develop in the bile ducts and lay eggs, causing blockage of the bile ducts, affecting digestion and appetite; The toxin penetrates into the blood, dissolves red blood cells, and causes poisoning phenomena such as anemia, weight loss, and edema in livestock; the stimulation of parasites causes the proliferation of the bile duct wall, which ...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61P33/10C12N15/70C12N1/21C07K19/00C07K1/22C12R1/19
CPCA61K39/0003A61K2039/552A61K2039/572A61K2039/575A61K2039/70C07K2319/55C12N9/14C12N2800/101C12N2800/22
Inventor 陈刚汤锋李润乐康明周虎冯琳
Owner QINGHAI UNIVERSITY
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