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Recombinantly-expressed Akkermansia membrane protein Amuc_1100 and application thereof

A technology of recombinant bacteria and protein, applied in the direction of recombinant DNA technology, peptide/protein components, medical preparations containing active ingredients, etc., can solve the problems of intestinal mucosal function without the immunological effect of Akkermann's membrane protein, and achieve enhanced Intestinal immune level, increase the depth of colonic crypts, and improve the effect of intestinal inflammation

Active Publication Date: 2020-01-17
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report about the effect of recombinant expression of this membrane protein on colitis, nor about the use of the Akkermans membrane protein Amuc_1100 to explore its immunological effects and its effect on intestinal mucosa

Method used

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  • Recombinantly-expressed Akkermansia membrane protein Amuc_1100 and application thereof
  • Recombinantly-expressed Akkermansia membrane protein Amuc_1100 and application thereof
  • Recombinantly-expressed Akkermansia membrane protein Amuc_1100 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Recombinant expression of Akermann membrane protein Amuc_1100

[0026] 1.1 PCR amplification of the Amuc_1100 gene in Akkermansia

[0027] The genome of Akkermansia muciniphila ATCC BAA-835 [Akkermansia muciniphila ATCC BAA-835] (Gene ID: 34174504), independently designed homologous recombination primers P1 and P2 to amplify the extracted bacterial genome by PCR, P1: 5'-CATGGCTGATATCGGATCCATGAGCAATTGGATTACAGACAACAAGC-3'P2: 5'-CGAGTGCGGCCGCAAGCTTTTAATCTTCAGACGGTTCCTGAGCC-3'

[0028] The above primers were synthesized by Nanjing GenScript Company, and the reaction was carried out in 25 μl system (12.5 μl BU-Taq 2×MasterPCR mix (Takara, China), 2 μl template DNA (bacterial genome), 1 μl upstream and downstream primers, 9.5 μl ddH 2 O) Performed in Biometra PCR instrument (Bio-Rad, USA), the reaction program is: 95°C pre-denaturation for 3min, 94°C for 30s, 72°C for 1min, a total of 30 cycles, and finally 72°C extension for 15min.

[0029] The amplified product ...

Embodiment 2

[0036] Example 2 Detection of the effect of oral recombinant expressed Akkerman membrane protein Amuc_1100 on colitis in mice

[0037] Mice (C57BL / 6 strain, purchased from the Center for Comparative Animal Medicine, Yangzhou University) were gavaged with 3ug of the membrane protein Amuc_1100 (protein purity 91%) purified in Example 1 every day, and added a mass percentage in the drinking water after continuous feeding for 7 days. Colitis was induced by 3.5% dextran sodium sulfate (DSS), during which the body weight changes of mice were recorded. After a total of 14 days, the mice were sacrificed by cervical dislocation to observe the changes in the length of the colon, and the colon was stained with HE to evaluate the depth of crypts and histopathological changes.

[0038] It was found that oral administration of membrane protein Amuc_1100 in mice could increase body weight, increase colon length, and improve intestinal inflammation caused by DSS. Control: Negative control, 1...

Embodiment 3

[0039] Example 3 Detection of the effect of oral recombinantly expressed Akkermann membrane protein Amuc_1100 on mouse intestinal stem cells

[0040] Mice (C57BL / 6 strain, purchased from the Center for Comparative Animal Medicine, Yangzhou University) were gavaged with 3ug of the purified membrane protein Amuc_1100 (protein purity 91%) in Example 1 every day, and after continuous feeding for 7 days, added quality The percentage is 3.5% dextran sodium sulfate (DSS) to induce colitis. After a total of 14 days, the mice were sacrificed by cervical dislocation to extract colon RNA, and immunofluorescent staining was performed on the colon to detect the expression of proliferation-related genes.

[0041] Such as Figure 6 As shown, the Wnt3a and cyc genes were detected by fluorescent quantitative PCR, and the gene expression levels of the mice were significantly increased after oral administration of the Akkerman membrane protein Amuc_1100, indicating that the Wnt signaling pathwa...

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Abstract

The invention discloses recombinantly-expressed Akkermansia membrane protein Amuc_1100 and an application thereof. The invention recombinantly expresses the Akkermansia membrane protein Amuc_1100, through animal experiments, we find that oral administration of the Akkermansia membrane protein Amuc_1100 can significantly improve the body weight of mice, increase colon length and crypt depth, and activate proliferation of intestinal stem cells to protect mice from colitis damage. Isolation of lymphocytes from the lamina propria of the colon shows that the proportion of Treg cells is significantly up-regulated and that of Th17 cells is significantly down-regulated, proving that Amuc_1100 can restore colon damage by adjusting the proportion of immune cells Treg / Th17. Therefore, the protein isexpected to be developed into a protein product for preventing or treating colitis.

Description

technical field [0001] The present invention relates to biological prevention and control technology, in particular to recombinantly expressed Akermans membrane protein Amuc_1100 and its application. Oral administration of recombinantly expressed Akermans membrane protein Amuc_1100 to animals can promote the proliferation of intestinal stem cells and effectively relieve intestinal inflammation; At the same time, the up-regulation of the proportion of Treg cells and the decrease of the proportion of Th17 cells were detected by separating the lymphocytes of the colonic lamina propria, which proved its role in regulating immunity in the process of repairing inflammation. Background technique [0002] Akkermansia muciniphila belongs to Verrucomicrobia (Verrucomicrobiae) and Akkermansiaceae (Akkermansiaceae) in taxonomy. It is a Gram-negative bacterium that is strictly anaerobic. , bacteria that do not form spores. Akkermansia is able to use mucin as its sole carbon and nitrogen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C12N15/70C12N1/21C12N5/074A61K38/16A61K35/741A61P1/00A61P29/00
CPCC07K14/195C12N15/70C12N5/068A61K35/741A61P1/00A61P29/00C12N2501/415A61K38/00
Inventor 庾庆华朱琳达赵珈翊陆晓溪陆林豪王敏娟谢霜
Owner NANJING AGRICULTURAL UNIVERSITY
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