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Fixed point-modified swine GLP-2 recombinant lactobacillus plantarum and preparation method and application thereof

A technology of GLP-2 and Lactobacillus plantarum, which is applied in the field of pig-derived GLP-2 recombinant Lactobacillus plantarum and its preparation, can solve the problems of short half-life and achieve high expression, high biological activity and stable expression

Active Publication Date: 2016-05-25
GUANGZHOU WELLTECH BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This results in a short in vivo half-life of biologically active pGLP-2

Method used

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  • Fixed point-modified swine GLP-2 recombinant lactobacillus plantarum and preparation method and application thereof
  • Fixed point-modified swine GLP-2 recombinant lactobacillus plantarum and preparation method and application thereof
  • Fixed point-modified swine GLP-2 recombinant lactobacillus plantarum and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. Site-directed transformation and primer design of p(Gly2)-GLP-2 gene: The porcine GLP-2 gene sequence (AccessionNo.NP_999489.1) was obtained from GenBank, and the site-directed transformation was carried out, and the second gene of pGLP-2 The alanine at the position is replaced by glycine to form p(Gly2)-GLP-2 that is not digested by DPP-IV, prolonging its half-life.

[0035] Wherein, the amino acid sequence of p(Gly2)-GLP-2 is SEQ ID NO:1.

[0036] SEQ ID NO: 1: HGDGSFSDEMNTVLDNLATRDFINWLLHTKITDSL.

[0037] The codon bias tropism of Lactobacillus plantarum was modified on the sequence, and the optimized sequence was SEQ ID NO: 2.

[0038] SEQ ID NO: 2:

[0039] CACGGTGATGGTTCATTCTCAGATGAAATGAACACTGTTTTAGATAACTTAGCTACTCGTGATTTCATCAACTGGTTATTACACACTAAGATCACTGATTCATTA.

[0040] Two pairs of primers were designed and fused to form p(Gly2)-GLP-2 by overlap extension PCR. The 5' end of primer P1 introduces a restriction site XhoI, the 3' end of primer P4 introduces a ...

Embodiment 2

[0053] 1. Expression of the target gene in Lactobacillus plantarum and identification of its expression product: pick the transformed positive recombinant bacteria pSCPSP-p(Gly2)-GLP-2-LP-1 and empty vector pSCPSP-LP-1 and inoculate in MRS liquid medium (containing 20 μg / mL chloramphenicol) was cultured overnight. Then inoculate the above-mentioned overnight cultured bacterial solution into an appropriate amount of MRS solution at a ratio of 2%, culture it at 37°C for 24 hours, and take its supernatant. The supernatant was desalted first, then ultrafiltered with a 1KD ultrafiltration tube and centrifuged at 4°C for 30 min, and the filtrate was discarded. The remaining supernatant was subjected to 10KD ultrafiltration and centrifugation, and the collected filtrate was placed in a vacuum drying oven at 37°C to concentrate 100 times, added 2×Tricine polypeptide loading buffer, and boiled for 10 minutes. Centrifuge at 11000r / min, 4°C for 10min. Take the supernatant of pSCPSP-p(G...

Embodiment 3

[0059]1. The establishment of chronic enteritis model in mice and the treatment of enteritis by p(Gly2)-GLP-2: DSS (dextran sodium sulfate): enteritis research (IBD) level - the gold standard for enteritis research (molecular weight 36,000-50,000Da ). The mechanism of DSS-induced enteritis is that the toxicity of DSS directly acts on the epithelial cells at the colonic crypts, affecting the integrity of the mucosa, making the crypts shallower, and the intestinal villi shrinking, leading to enteritis.

[0060] In view of the fact that the severity of DSS-induced colitis does not depend on the amount of DSS intake, but is determined by the concentration of DSS, when the difference in the amount of DSS that mice drink is small, it does not affect the severity of the induced colitis. This experiment adopts the method of free drinking water modeling. In order to verify the biological activity of the expression product p(Gly2)-GLP-2 in vivo, the model was constructed according to t...

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Abstract

The invention belongs to the biotechnological field, and discloses fixed point-modified swine GLP-2 recombinant lactobacillus plantarum and a preparation method and application thereof. Fixed point modification is performed on swine GLP-2 to form p(Gly2)-GLP-2 which is not digested by DPP-IV, codon preference modification of lactobacillus plantarum (Lp1) is performed, and the half-life period of the p(Gly2)-GLP-2 and the bioactivity valid time of the p(Gly2)-GLP-2 are prolonged; lactobacillus plantarum separated from the intestinal tract of a healthy piglet is selected, the modified swine GLP-2 is cloned to a pSCPSP expression vector and electrically converted to the lactobacillus plantarum to enable the p(Gly2)-GLP-2 to be successfully expressed in the lactobacillus plantarum. The recombinant lactobacillus plantarum has the bioactivity in vitro and vivo, is beneficial for solving the problem that in livestock breeding production, growth arrest and diarrhea and the like of the piglet occur due to intestinal morphology and structure destruction caused by weaning stress, therefore, the health level of the piglet is improved, and the breeding benefit is increased.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a porcine-derived GLP-2 recombinant plant lactobacillus for site-specific transformation and a preparation method and application thereof. Background technique [0002] my country is a big pig producing country. In the pig production process, the survival rate of piglets directly affects the production efficiency. Especially during the weaning period, due to the weaning stress, the intestinal morphology and structure of the piglets will be damaged to varying degrees, and the piglets will have digestive disorders, diarrhea, and growth stagnation. GLP-2 is a polypeptide gastrointestinal hormone, which can specifically promote the proliferation of intestinal epithelial cells and small intestinal crypt cells, increase the thickness of intestinal mucosa, increase the volume of crypts, increase the height of villi, inhibit cell apoptosis, and strengthen The intestinal trac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74A61K48/00A61K38/26A61P1/00A61P1/14A61P43/00C12R1/25A61K35/747
CPCA61K35/747A61K38/26A61K48/0066C07K14/605A61K2300/00
Inventor 黄毓茂赖强钟泽民刘德辉
Owner GUANGZHOU WELLTECH BIOTECH CO LTD
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