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Design and preparation method and application of echinococcus multilocularis subunit vaccine LTB-Emy162

A multilocular hydatid and subunit vaccine technology, applied in the field of biomedicine, can solve the problems of poor immune effect, large human damage, and high recurrence rate, and achieve strong immunogenicity, high safety, and high yield.

Active Publication Date: 2017-04-26
QINGHAI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, surgery is the main treatment for AE, supplemented by drug therapy, but the operation is very harmful to the human body and has a high recurrence rate. Taking albendazole and mebendazole will cause serious adverse reactions
Compared with other vaccines, recently invented subunit vaccines have the advantages of high safety, high yield, high purity, and good stability, but compared with traditional vaccines, the immune effect is poor, requiring special structural design, special delivery system or adjuvant to enhance its immunogenicity
Therefore, there are currently no effective drugs and vaccines that can be used for clinical prevention and treatment of alveolar echinococcosis

Method used

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  • Design and preparation method and application of echinococcus multilocularis subunit vaccine LTB-Emy162
  • Design and preparation method and application of echinococcus multilocularis subunit vaccine LTB-Emy162
  • Design and preparation method and application of echinococcus multilocularis subunit vaccine LTB-Emy162

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Molecular structure design of multilocular Echinococcus subunit vaccine LTB-Emy162

[0044] In the design of Echinococcus multilocularis subunit vaccine, the present invention selects the Emy162 antigen, and uses Escherichia coli heat-labile enterotoxin B subunit (LTB) as an intramolecular immune adjuvant, which is fused to the N-terminal of Emy162 to enhance the efficacy of Emy162. Immunogenicity.

[0045] Results: The molecular structure design characteristics and ideas of the multilocular Echinococcus subunit vaccine LTB-Emy162 are as follows: figure 1 shown.

Embodiment 2

[0046] Example 2: Construction of the recombinant expression vector pCzn1-LTB-Emy162 (containing the fusion gene LTB-Emy162)

[0047] The amino acid sequence of the previously designed multi-epitope peptide LTB-Emy162 was converted into the corresponding nucleotide sequence according to the codon preference principle of Escherichia 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-Emy162 was designed at both ends of the primers, and it was connected into the expression vector pCzn1 through the cloning sites Nde I and Xba I.

[0048] Results: The recombinant plasmid pCzn1-LTB-Emy162 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-Emy162 is consistent, as figure 2 shown. The vector construction map of the recombinant expression vector pCzn1-LTB-Emy162 is as foll...

Embodiment 3

[0049] Example 3: Prokaryotic expression of multi-epitope peptide fusion protein LTB-Emy162

[0050] The correct recombinant expression plasmid pCzn1-LTB-Emy162 was verified to be transformed into Escherichia coli Arctic Express strain. On the pre-prepared LB plate containing 50 μg / mL Amp, inoculate the loop-streaked genetically engineered strain pCzn1-LTB-Emy162 / Arctic Express, place it upside down in a 37°C incubator, and after cultivating overnight, pick a single colony and inoculate it on a plate containing In LB medium containing 50µg / mL Amp, culture overnight at 37°C, 220rpm. Inoculate the recombinant bacteria with 2% inoculum in LB medium containing 50 µg / mL Amp, 37°C, 220 rpm, cultivate until the OD600 of the bacteria is 0.6-0.8 (about 2 hours), add IPTG to make the final concentration reach 1mmol / L, 37 The expression was induced at 220 rpm for 4 hours at ℃, and the carrier strain pCzn1-LTB-Emy162 / Arctic Express induced without IPTG was used as a negative control.

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Abstract

The invention relates to a design and preparation method and an application of an echinococcus multilocularis subunit vaccine LTB-Emy162. The active component of the echinococcus multilocularis subunit vaccine LTB-Emy162 is a polypeptide, which is mainly composed of an echinococcus multilocularis antigen protein Emy162 amino acid sequence and a mucosal immunoadjuvant E. coli heat-labile enterotoxin B subunit (LTB) amino acid sequence. In the invention, the gene sequence of the echinococcus multilocularis subunit vaccine LTB-Emy162 is synthesized through gene synthesis technology, the gene sequence is then linked to an expression vector through dual enzyme-cut, the expression vector then is converted into Arctic Express to perform expression of fusion protein, after purification of the protein, the echinococcus multilocularis subunit vaccine LTB-Emy162 is produced. The echinococcus multilocularis subunit vaccine can induce body to generate T-cell and B-cell immunologic response of the echinococcus multilocularis and humoral immune response of a high-titer specific antibody, and can be applied in prevention and therapy of echinococcus multilocularis infection related diseases.

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] Multilocular echinococcosis, also known as alveolar echinococcosis, is a serious zoonotic disease. The high incidence and epidemic areas of echinococcosis in my country are mainly concentrated in pastoral areas and semi-agricultural and semi-pastoral areas. Qinghai, Gansu and northern Sichuan Other places are more serious, and Qinghai is the hardest hit area for alveolar echinococcosis. The alveolar coccus parasitizes in the liver of the intermediate host in the early stage, causing local liver tissue lesions, hyperplasia, liver fibrosis, atrophy, degeneration and necrosis, but the clinical symptoms are not obvious; in the late stage, liver cancer-like metastasis can be transferred to the lung, brain, breast, etc. For viscera, even with partia...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61P33/10C07K19/00C12N15/62C07K14/435C12N15/70C12N1/21C12P21/02
CPCA61K39/0003A61K39/39A61K2039/55544A61K2039/57A61K2039/575C07K14/28C07K14/43554C07K2319/55
Inventor 汤锋李润乐杨全余格日力樊海宁刘川川冯琳刘文磊杨宝良
Owner QINGHAI UNIVERSITY
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