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Designing and preparing method and application of novel alveolar echinococcosis subunit vaccine

A multilocular echinococcosis and subunit vaccine technology, applied in the field of biomedicine, can solve the problems of few antigen studies, no multilocular hydatid immunotherapy method, complicated immune escape mechanism of multilocular hydatid infection host, etc. , to achieve the effect of enhancing immunogenicity

Inactive Publication Date: 2016-11-09
QINGHAI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The search for Echinococcus multilocularis antigens has always been one of the current research hotspots. At present, the research on echinococcosis mainly focuses on the diagnostic antigens of Echinococcosis multilocularis, and there are few researches on the antigens for preventing and inhibiting Echinococcus multilocularis infection; In addition, the immune escape mechanism of the host infected with Echinococcus multilocularis is complicated, and there is no immunotherapy method for Echinococcus multilocularis at present.

Method used

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  • Designing and preparing method and application of novel alveolar echinococcosis subunit vaccine
  • Designing and preparing method and application of novel alveolar echinococcosis subunit vaccine
  • Designing and preparing method and application of novel alveolar echinococcosis subunit vaccine

Examples

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example 1

[0045] Example 1: Molecular structure design of multilocular Echinococcus subunit vaccine CTB-Emy162

[0046] On the basis of obtaining the amino acid sequence of Emy162, the intramolecular mucosal immune adjuvant CTB was added on this basis, and a fusion protein of Echinococcus multilocularis with a scientific and reasonable structure was designed to effectively improve the mucosal immune response in vivo.

[0047] Results: The molecular structure design characteristics and ideas of multilocular Echinococcus subunit vaccine CTB-Emy162 are as follows ( figure 1 ) shown.

example 2

[0048] Example 2: Construction of recombinant expression vector (containing fusion gene CTB-Emy162)

[0049] (1) Gene synthesis of the nucleotide sequence of the surface antigen Emy162 of Echinococcus multilocularis

[0050] The amino acid sequence of Emy162 obtained earlier 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 the gene synthesis. The synthesized Emy162 was subjected to PCR, and the reaction system was as follows:

[0051] Emy162 (0.5mg / mL)

1 µL

Emy162 primer

1 µL

Mix (0.1mg / mL)

10µL

wxya 2 O (No RNase)

8 µL

Total

20 µL

[0052] The PCR reaction conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 sec, annealing at 72°C for 30 sec, extension at 72°C for 40 sec, a total of 30 cycles, and a final extension at 72°C for 10 min. Then use...

example 3

[0062] Example 3 Prokaryotic expression of fusion protein CTB-Emy162 of Echinococcus multilocularis

[0063] The 100% successful recombinant expression plasmid pET28a-CTB-Emy162 was transformed into E.coli BL21(DE3) strain. On the pre-prepared LB plate containing 50ug / mL Kana, inoculate the loop-streaked genetically engineered strain pET28a-CTB-Emy162 / BL21(DE3), place it upside down in a 37°C incubator, and culture it overnight for 12-16 hours, then pick a single Colonies were inoculated in LB liquid medium containing 50µg / mL Kana, cultured at 37°C, 190rpm, for 6-8h. Inoculate recombinant bacteria with 1% inoculum in LB liquid medium containing 50 µg / mL Kana, shake overnight at 37°C and 180 rpm, and inoculate the bacterial solution at 1% inoculum in LB containing 50 µg / mL Kana the next morning In liquid medium, after shaking the flask at 37°C and 190rpm for 9h, add IPTG to make the final concentration reach 1mmol / L, induce expression at 28°C and 190rpm, and use the empty vect...

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Abstract

The invention provides a novel alveolar echinococcosis subunit vaccine. A polypeptide serves as an active ingredient of the alveolar echinococcosis subunit vaccine, which is mainly composed of a surface antigen Emy162 of alveolar echinococcosis and a mucosal immune adjuvant, namely cholera toxin B subunit (CTB). According to the invention, the gene sequence of the alveolar echinococcosis surface antigen Emy162 is synthesized by virtue of a gene synthesis technology, and then the gene is coupled with the gene sequence of the cholera toxin B subunit, so that a fusion gene is formed. The fusion gene is expressed by virtue of an escherichia coli prokaryotic expression system, and protein is purified, so that the alveolar echinococcosis subunit vaccine is obtained. The alveolar echinococcosis subunit vaccine is capable of inducing a body to generate alveolar echinococcosis targeted T cell and B cell immune response and high-titer specific antibody humoral immune response, and the subunit vaccine can be used for preventing and treating alveolar echinococcosis infection related diseases.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a design and preparation method and application of a novel multilocular hydatid subunit vaccine. Background technique [0002] Alveolar Echinococcosis leads to alveolar echinococcosis. Alveolar Echinococcus grows in the intermediate host liver by budding or infiltrating proliferation, produces new vesicles, grows into the liver tissue, and the outer corner of the cyst wall The cortex is very thin and often incomplete, and there is no obvious boundary between the cyst and the surrounding tissue. Continuous leakage of the cyst fluid can contact the liver tissue, causing local liver tissue lesions, hyperplasia, liver fibrosis, atrophy, degeneration, and necrosis. Liver cancer-like metastasis can be transferred to the lung, brain, breast and other organs. It is known as "worm cancer" and has a very poor prognosis. Alveolar echinococcosis occurs in high-altitude areas of the no...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61P33/10C07K19/00C12N15/62C07K14/435C12N15/12C12N15/70C12N5/10C12N1/21C12P21/02
CPCA61K39/0003A61K2039/55594C07K14/28C07K14/4355C07K2319/55C12N15/70C12N2800/101C12N2800/22
Inventor 樊海宁格日力阳旦才让汤锋周虎李润乐
Owner QINGHAI UNIVERSITY
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