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Recombinant food lactic acid bacteria for preventing chronic heavy metal poisioning and preparation method and use thereof

A lactic acid bacteria, food-grade technology, applied in the field of genetic engineering, can solve problems such as teratogenicity, hindering intestinal absorption of iron, and damaging blood vessels

Active Publication Date: 2013-08-14
广州市暨源生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

4) Cadmium entering the body can damage blood vessels, lead to tissue ischemia, and cause multi-system damage; it can also interfere with the metabolism of trace elements such as copper, cobalt, and zinc, hinder intestinal absorption of iron, and inhibit the synthesis of hemoglobin and alveolar macrophages. The metabolic process of oxidative phosphorylation, and then cause lung, kidney, liver damage, but also has carcinogenic, teratogenic and mutagenic effects
However, traditional lactic acid bacteria vectors all carry one or more genes encoding resistance to specific antibiotics (such as erythromycin, chloramphenicol, etc.). The transfer of sex factors will have serious consequences for biosafety

Method used

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  • Recombinant food lactic acid bacteria for preventing chronic heavy metal poisioning and preparation method and use thereof
  • Recombinant food lactic acid bacteria for preventing chronic heavy metal poisioning and preparation method and use thereof
  • Recombinant food lactic acid bacteria for preventing chronic heavy metal poisioning and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Acquisition of the gene encoding α-galactosidase in Lactobacillus plantarum

[0093] Plant Lactobacillus plantarum (Orla-Jehsen) Holland, ATCC11095, Guangdong Microbial Culture Collection Center, number: GIM1.191) bacterial strain on GM17 plate [composition: 3.725% M17 broth (M17Broth, purchased from Biokar Diagnostics, France), 0.5% glucose, 1.5% agar powder] for strain activation, and anaerobic culture at 37°C for 48h. 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 synthesized by China Shanghai San...

Embodiment 2

[0094] Embodiment 2: Construction of recombinant food-grade lactic acid bacteria expression vector pMG36m

[0095] 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

[0096] Example 3: Construction of recombinant expression plasmid pMG36m-GSMT

[0097] Send the DNA fragment encoding the fusion protein composed of glutathione S-transferase, small molecule ubiquitin-related modifier mature peptide and human metallothionein type IA to Guangzhou Jetway Biotech Co., Ltd. Ltd., China) for total gene synthesis. This DNA fragment was used as a template, GSMT-F (SEQ ID NO.14) and GSMT-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, follow the standard PCR reaction conditions (94°C denaturation for 4 minutes and then cycle, the cycle parameter is 94°C denaturation 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 180 seconds, a total of 25 cycles) to amplify the target fragment. The target DNA fragment was detected and recovered by electrophoresis and named as GSMT (SEQ ...

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Abstract

The invention relates to recombinant food lactic acid bacteria containing human IA type metallothionein fusion protein and a preparation method thereof, in particular to a method for performing fusion expression by using glutathione S-transferase, small ubiquitin-related modifier mature peptide and human IA type metallothionin. In the invention, the gene of the fusion protein is cloned into a lactic acid bacterium-based food expression vector pMG36m and then transferred to the lactococcus lactis MG1363, and then melibiose is used as a carbon source to perform recombinant screening. The recombinant lactic acid engineering bacteria can continuously express the fusion protein of the human IA type metallothionin in a fermentation process, and the protein is capable of bonding heavy metal Pb, Cd and Hg in living environments. The invention also relates to the use of the recombinant food lactic acid bacteria in preparation of medicines, foods or cosmetics for preventing human chronic metal poisioning.

Description

technical field [0001] The invention belongs to the field of genetic engineering. More specifically, the present invention relates to a recombinant food-grade lactic acid bacterium containing human type IA metallothionein fusion protein, its preparation method and the use of the recombinant food-grade lactic acid bacterium in the preparation of drugs, food or cosmetics for preventing chronic heavy metal poisoning in humans. in the application. Background technique [0002] With the development of industrialization of various industries in China, heavy metal pollution has become a problem of great concern to the society. A large number of toxic heavy metal ions pollute through water, air, soil and the biological chain to animal and plant products and water sources on which human beings depend for survival, bringing potential harm to people's health. Generally speaking, heavy metal pollution refers to the pollution of heavy metal ions such as lead, mercury, cadmium, and arse...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74A61K35/74A61K8/99A61P39/02A23C9/12C12R1/01C12R1/25
Inventor 苏志坚黄亚东项琪张齐好冯娅肖巧学
Owner 广州市暨源生物科技有限公司
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