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Recombination food-grade lactic acid bacterium capable of being used for preventing or treating human ulcerative colitis as well as preparation method and application of recombination food-grade lactic acid bacterium

A lactic acid bacteria, food-grade technology, applied in the field of genetic engineering, can solve problems such as biosafety consequences

Inactive Publication Date: 2012-01-25
JINAN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional lactic acid bacteria vectors all carry one or more genes encoding resistance to specific antibiotics (such as erythromycin, ampicillin, etc.). The transfer of factors will have serious consequences for biosafety

Method used

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  • Recombination food-grade lactic acid bacterium capable of being used for preventing or treating human ulcerative colitis as well as preparation method and application of recombination food-grade lactic acid bacterium
  • Recombination food-grade lactic acid bacterium capable of being used for preventing or treating human ulcerative colitis as well as preparation method and application of recombination food-grade lactic acid bacterium
  • Recombination food-grade lactic acid bacterium capable of being used for preventing or treating human ulcerative colitis as well as preparation method and application of recombination food-grade lactic acid bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1: Acquisition of the gene encoding α-galactosidase in Lactobacillus plantarum

[0092] Plant lactobacillus (Lactobacillus plantarum (Orla-Jehsen) Holland, ATCC11095, Guangdong Provincial Microbial Culture Collection Center, number: GIM1.191) bacterial strain on GM17 plate [composition: 3.725% M17 broth (M17 Broth, purchased from Biokar Diagnostics , France), 0.5% glucose, 1.5% agar powder] for strain activation, 37 ° C anaerobic culture for 48 hours. Then, pick a single clone and transfer it to GM17 liquid medium [ingredients: 3.725% M17 broth, 0.5% glucose], anaerobic static culture at 37°C until OD 600 =0.6, the bacteria were collected by centrifugation and genomic DNA was extracted using a bacterial genome extraction kit (TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit Ver.2.0, purchased from Dalian Bao Biological Engineering Co., Ltd., Dalian, China). Using the genomic DNA as a template, add mel AF (SEQ ID NO.12) and mel AR (SEQ ID NO.13) (both synthe...

Embodiment 2

[0093] Embodiment 2: Construction of recombinant food-grade lactic acid bacteria expression vector pMG36m

[0094] The wild-type Lactococcus lactis MG1363 (purchased from NIZO Co. Ltd., Catalog: ELS09000-01, Netherlands) containing the pMG36e plasmid was cultured on an EGM17 solid culture plate containing erythromycin antibiotics (ingredients: 3.725% M17 broth, 0.5% Glucose, 1.5% agar powder, and erythromycin with a final concentration of 10 μg / ml) were streaked for culture. After 36 hours of anaerobic culture at 37°C, pick a single colony from the plate medium and inoculate it into EGM17 liquid medium containing erythromycin antibiotics (ingredients: 3.725% M17 broth, 0.5% glucose, final concentration of 10 μg / ml red Mycin), 37 ℃ anaerobic static culture to OD 600 = 0.6. The bacteria were collected by centrifugation and the plasmid pMG36e was obtained by using a plasmid extraction kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.2.0, purchased from Dalian Bao Biological En...

Embodiment 3

[0095] Example 3: Construction of recombinant expression plasmid pMG36m-ULHA

[0096] Send the DNA fragment (sequence: SEQ ID NO.11) encoding the fusion protein composed of Usp45 protein signal peptide, secretagogue peptide and human acidic fibroblast growth factor to Guangzhou Jetway Biotech Co., Ltd. .Ltd., Guangzhou, China) for total gene synthesis. This DNA fragment was used as a template, ULHA-F (SEQ ID NO.14) and ULHA-R (SEQ ID NO.15) were used as upstream and downstream primers, and then dNTP (each 2.5 μmol L -1 ) 10 μl, 10 μl of 10x PCR pfu buffer and 1 μl of pfu Taq DNA polymerase (5U / μl), add water to a total volume of 100 μl, and follow the standard PCR reaction conditions (94°C denaturation for 4 minutes before entering the cycle, the cycle parameter is 94°C Denaturation for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 60 seconds, a total of 35 cycles) to amplify the target fragment. The target DNA fragment was detected and recovered by ele...

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Abstract

The invention relates to a recombination food-grade lactic acid bacterium for secreting and expressing a fusion protein containing a human acidic fibroblast growth factor, a preparation method of the recombination food-grade lactic acid bacterium, and in particular relates to a method for carrying out fusion expression by using a Usp45 protein signal peptide, a secretion-promoting protein peptide and the human acidic fibroblast growth factor. According to the invention, genes for coding the fusion proteins are cloned to a lactic acid bacterium constitutive expression vector pMG36m, then transformed into lactococcus lactis NZ9000, and subjected to recombinant screening by using melibiose as a carbon source. The recombination lactic acid engineering bacterium can continuously secret the fusion protein containing the human acidic fibroblast growth factor in a fermentation expression process. The invention relates to an application of the recombination food-grade lactic acid bacterium in the preparation of cosmetics and medicines or foods for preventing and treating human ulcerative colitis.

Description

technical field [0001] The invention belongs to the field of genetic engineering. More specifically, the present invention relates to a preparation of recombinant food-grade lactic acid bacteria containing human acidic fibroblast growth factor, its preparation method and the use of said recombinant food-grade lactic acid bacteria in the preparation of cosmetics or for the prevention and treatment of human ulcerative colitis. Applications in medicine or food. Background technique [0002] Ulcerative colitis (UC), also known as nonspecific ulcerative colitis, is a chronic inflammatory bowel disease of unknown cause. The pathogenesis of the disease is mainly located in the mucosal layer of the colon, often dominated by ulcers, mostly involving the rectum and distal colon, and can also spread throughout the entire colon [Lian Jun et al., 2009, Journal of Xinjiang Medical University, 32(11): 1619] . The clinical manifestations of UC patients include persistent or recurrent dia...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74A61K8/99A61Q19/00A61K35/74A61P1/04A23C9/12C12R1/01C12R1/25A61K35/744
Inventor 黄亚东苏志坚项琪张齐好冯娅
Owner JINAN UNIVERSITY
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