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Helicobacter pylori multi-epitope series fusion protein LHUC as well as preparation method and application thereof

A technology of Helicobacter pylori and fusion protein, applied in the field of biopharmaceuticals, can solve problems such as difficulty in preparation, achieve strong immune effect, enhance immunogenicity, and avoid toxic and side effects

Active Publication Date: 2021-03-09
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 1999, the recombinant H.pylori urease therapeutic vaccine developed by Orovax entered Phase II clinical trials. Although it had good conservative antigens and tolerance, it required adjuvants and multiple doses; The supervised Salmonella expressing urease has entered phase I clinical trials, and it is easy to take and does not produce a clear mucosal immune response; the inactivated whole bacterial antigen vaccine developed by Antex Biologics has entered phase II clinical trials, which has relatively complete immunity, but Preparation is difficult, and safety needs further investigation (Zou Quanming. Research progress of Helicobacter pylori vaccine. Gastroenterology [J], 2007, 12(9):567-570)

Method used

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  • Helicobacter pylori multi-epitope series fusion protein LHUC as well as preparation method and application thereof
  • Helicobacter pylori multi-epitope series fusion protein LHUC as well as preparation method and application thereof
  • Helicobacter pylori multi-epitope series fusion protein LHUC as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] According to the immune protection mechanism of Helicobacter pylori infection, three T cell epitopes (HpaA 154-171 , UreB 237-251 , UreB 546-561 ) and five B cell epitopes (UreB 349-363 , UreB 327-334 、CAT 394-405 、CAT 387-397 and HpaA 132-141 ). By analyzing the antigenicity between each epitope and adding amino acids KK and GS to avoid the interaction between each epitope, optimize the sequence of each epitope ( Figure 1-Figure 2 ), obtain the sequence 1 polypeptide shown in SEQ ID NO:3 as an alternative peptide chain, named as HUC. Finally, the mucosal adjuvant LTB was connected to the N-terminus of the multi-epitope peptide HUC, separated by a flexible peptide PQDPPP in the middle. The isoelectric point, antigenicity, hydrophilicity, stability and advanced structure of the fusion peptide were analyzed by bioinformatics tools such as DNASTAR, Expasy and MOE. According to the predicted results of physical and chemical properties, the molecular mass is 27.8KD...

Embodiment 2

[0045] Example 2 Recombinant plasmid construction

[0046] According to the codon preference of Escherichia coli, the nucleotide sequence corresponding to the amino acid sequence of the vaccine protein was codon-optimized and synthesized by Qingke Biotechnology Co., Ltd. A NdeI restriction site was introduced at the 5' end of the sequence, a HindIII restriction site was introduced at the 3' end, and the sequence was inserted into the pET28a expression vector. The constructed plasmid was transformed into Escherichia coli DH5ɑ, cultured overnight at 37°C on a Kana-resistant plate, and a single clone was picked for colony PCR and plasmid double-enzyme digestion verification. Plasmids were digested and sequenced for verification. Enzyme digestion electropherogram reference Figure 4 .

Embodiment 3

[0047] Example 3 Preparation and Purification of Multiple Tandem Epitope Vaccines

[0048] (1) Verification of multi-tandem epitope vaccine expression

[0049] The recombinant plasmid constructed in Example 2 was transformed into Escherichia coli BL21(DE3), a single clone was picked and inoculated in LB medium containing 50 ug / ml kanamycin, and shaken overnight at 37°C. Inoculate in LB medium containing 50ug / ml kanamycin at a ratio of 1:100, culture with shaking at 37°C, add IPTG to a final concentration of 1mM when the OD 600 value is between 0.6-0.8, and induce 1 and 2 respectively , 3, 4, 5 hours later collect bacteria. Bacteria and protein loading buffer were mixed, boiled in boiling water for 10 minutes and centrifuged, the supernatant was collected and tested by 12% SDS-PAGE, the results were as follows Figure 5 shown.

[0050] (2) Expression and purification of multi-tandem epitope vaccine

[0051] Cultivate 1L of bacteria according to the above method, and after i...

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Abstract

The invention discloses a recombinant helicobacter pylori multi-epitope tandem vaccine and a preparation method thereof. The vaccine is composed of an escherichia coli labile toxin B subunit (LTB) andepitopes selected from helicobacter pylori antigens HpaA, UreB and CAT. The helicobacter pylori multi-tandem epitope vaccine can generate a high-titer antibody sIgA and a specific IgG antibody in serum in gastric mucosa, and the specific antibody IgG can significantly inhibit helicobacter pylori urease activity. The vaccine can be used for preventing or treating helicobacter pylori infection.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a Helicobacter pylori multi-epitope tandem fusion protein LHUC and its preparation method and application. Background technique [0002] Helicobacter pylori (Helicobacter pylori) is a spiral-shaped, microaerophilic Gram-negative bacterium, and it is the only pathogenic microorganism found to be able to colonize the human stomach for a long time. The global infection rate is more than 50%, mainly concentrated in developing countries, while the infection rate is lower in developed countries. The infection rate in my country is 30%-50%, mainly concentrated in children under 10 years old and middle-aged and elderly people between 40-60 years old. It is closely related to gastrointestinal diseases such as gastritis, gastric ulcer, duodenal ulcer, gastric adenocarcinoma and gastric lymphoma. At least 90% of gastric cancer patients are related to Helicobacter pylori infection, so the...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/02A61K39/39A61K39/385A61P31/04C12R1/19
CPCC07K14/195C12N9/0065C12N9/80C12N15/70C12Y111/01006C12Y305/01005A61K39/0208A61K39/39A61K39/385A61P31/04C07K2319/55C07K2319/00A61K2039/6037A61K2039/55544
Inventor 孔令义杨蕾谢文伟赵文锋
Owner CHINA PHARM UNIV
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