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IL-35 recombinant lactic acid bacteria for improving colitis

A technology for recombining lactic acid bacteria and lactic acid bacteria, applied in the biological field, can solve problems such as restricting long-term use

Inactive Publication Date: 2019-04-16
郝凤奇
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These therapeutic drugs all have some side effects, thus limiting their long-term use

Method used

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  • IL-35 recombinant lactic acid bacteria for improving colitis
  • IL-35 recombinant lactic acid bacteria for improving colitis
  • IL-35 recombinant lactic acid bacteria for improving colitis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The preparation of embodiment 1 IL-35 recombinant lactic acid bacteria

[0025] IL-35 is a heterodimeric protein composed of Ebi3 and IL-12a p35 subunits; the gene sequence encoding the peptide from alanine 23 to proline 228 of Ebi3 was selected (Ala23-Pro228, 621 bp), With the gene sequence (Arg23-Ala215, 582 bp) encoding the peptide of the 23rd arginine to the 215th alanine of the IL-12a p35 subunit, the two are connected by a conventional linker protein gene (45 bp); The recombinant IL-35 gene with a full length of 1248 bp was obtained by artificial synthesis or PCR technology, named rIL-35, and the gene sequence table is shown in SEQ ID1.

[0026] A lactic acid bacteria recombinant expression vector carrying the rIL-35 gene was constructed using conventional molecular cloning techniques; in this example, Sac II and Sma I restriction sites were added upstream and downstream of the rIL-35 gene, and the pW425N lactic acid bacteria expression vector was used to finally ...

Embodiment 2

[0027] Example 2 IL-35 recombinant lactic acid bacteria increase intestinal IL-35 levels in mice

[0028] Twenty C57BL / 6J mice aged 6-8 weeks were randomly divided into 4 groups, 10 mice in each group (half male and half male). Respectively PBS group, Lb. acidophilus group (empty bacteria group), Lb. acidophilus / pW425N group (empty vector group), Lb. acidophilus / rIL-35 group and heat-inactivated Lb. acidophilus / rIL-35 group ( Lb. acidophilus / rIL-35 i).

[0029] The PBS group was administered with 0.2 ml PBS, and the other groups were administered with 0.2 ml bacterial solution. Except for the PBS group, the number of bacteria was 10 each time 9 cfu / ml. After gavage 3 times a week, after 3 weeks of continuous gavage, samples were taken every 7 days and recorded as 7d, 14d and 21d. The sampling site was colon tissue, and IL-35 ELISA kit was used to detect the level of IL-35 in colon tissue according to the instructions. The result is as figure 1 As shown, a...

Embodiment 3

[0030] Example 3 IL-35 recombinant lactic acid bacteria relieve experimental colitis

[0031] Experimental colitis in mice was induced by the classic DSS method. 3%DSS free drinking water was used to establish the model, and the mice were divided into the following experimental groups: normal drinking water group (Ctr), 3%DSS group, 3%DSS+ Lb. acidophilus / pW425N group, 3%DSS+ Lb. acidophilus / rIL-35 group, 3%DSS+Lb.acidophilus / rIL-35 i group, 3%DSS+IL35 group (IL-35 protein 3μg / mouse / day via tail vein injection). During the modeling period, the mice were weighed every day and the weight loss rate was calculated. The occult blood in the feces of the mice was evaluated according to the instructions using the fecal occult blood kit. The diarrhea of ​​the mice was observed every day, and the disease of the mice was calculated according to Table 1. Disease active index (DAI) score. On the seventh day of the experiment, the mice were sacrificed, the colons were removed, and ...

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Abstract

The invention discloses a recombinant rIL-35 gene with the nucleotide sequence shown as SEQ ID NO.1 in the Sequence Listing; a lactic acid bacteria expression vector carrying the rIL-35 gene with thegene shown in the SEQ ID NO.1 of the Sequence Listing inserted; a preparation method of an escherichia coli-lactic acid bacteria shuttle expression vector carrying the rIL-35 gene. The preparation method comprises: 1) synthesizing the gene shown in the SEQ ID NO.1 of the Sequence Listing and adding Sac II and Sma I restriction enzyme cutting sites upstream and downstream; 2) conducting digestion on the gene described in step 1) and pW425N with the Sac II and the Sma I; inserting the gene shown in the SEQ ID NO.1 of the Sequence Listing into pW425N. The invention further discloses IL-35 recombinant lactic acid bacteria for converting the lactic acid bacteria expression vector carrying the rIL-35 gene and application of the IL-35 recombinant lactic acid bacteria in preparing drugs for improving or alleviating the colitis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an IL-35 recombinant lactic acid bacterium for improving colitis and a preparation method thereof. Background technique [0002] Ulcerative colitis is an idiopathic colonic inflammatory disease, which is different from various infectious colonic diseases. Although the symptoms of the lesions are similar, so far the research has not been able to find which pathogenic bacteria is the cause of this type of colonic disease. The causative agent of mucous membrane inflammation. The disease is mainly centered on chronic, non-specific inflammatory lesions in the node, rectal mucosa and submucosa, mucosal epithelium and glands are destroyed, hyperplasia or atrophy, inflammatory cells infiltrate, and crypt abscesses are formed, resulting in mucosal hyperemia, erosion, Ulcers and proliferative changes. The clinical manifestations also reflect the characteristics of inflammatory bo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/24C12N15/74C12N15/66C12N1/21A61K35/747A61P1/00C12R1/23
CPCA61P1/00A61K35/747C12N15/66C12N15/74C07K14/54
Inventor 郝凤奇田苗苗王建勇李景梅
Owner 郝凤奇
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