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Transformant for reducing weight and fat and preparation method thereof

A transformant, oxyntomodulin technology, applied in the field of biomedicine, can solve the problems of poor acceptance by obese patients, short half-life in the body, and high production costs, and achieve the effects of overcoming high production costs, improving curative effect, and low cost

Inactive Publication Date: 2009-12-23
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method of production and administration has the disadvantages of high production cost, short half-life in vivo and inconvenient administration, and is not easily accepted by obese patients

Method used

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  • Transformant for reducing weight and fat and preparation method thereof
  • Transformant for reducing weight and fat and preparation method thereof
  • Transformant for reducing weight and fat and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0032] The preparation of example 1OXM gene transformation bifidobacterium

[0033] 1. Construction method of OXM Bifidobacterium-Escherichia coli recombinant expression plasmid:

[0034] 1.1 Chemical synthesis of extracellular exo-xylanase (exo-xylanase) secretory signal peptide nucleic acid sequence Xs (SEQ NO.1); using pBAD / gIII plasmid DNA as a template with primers 1 (SEQ NO.4) and 2 (SEQ NO.5) The pBDA promoter DNA fragment was amplified under the conditions of denaturation at 94°C for 45 seconds, annealing at 54°C for 45 seconds, extension at 72°C for 1 minute, repeating 30 cycles, and extension at 72°C for 5 minutes. Mix the resulting pBDA DNA fragment with the secretory signal peptide nucleic acid sequence Xs, add high-fidelity DNA polymerase and dNTP, denature at 94°C for 1 minute, anneal at 60°C for 2 minutes, and repeat 8-10 cycles, then use primer 1 and Primer 3 (SEQ NO.6) was used to amplify the full-length nucleic acid fragment of the pBDA / Xs fusion gene by PCR...

example 2

[0078] The preparation of example 2 transformant oral liquid of the present invention

[0079] 1. Preparation of Seed Bacteria

[0080] 1.1 Amplification culture: take the OXM gene stably transformed bifidobacterium bacterial liquid identified by PCR detection, Gram staining and induction expression experiment, inoculate in 10ml improved MRS medium (containing 0.05% cysteine, 100ug / ml ampicillin), anaerobic culture at 37°C; when the OD695 absorbance value of the bacterial suspension reaches 0.6, centrifuge at 4500 rpm for 5 minutes to harvest the bacteria;

[0081] 1.2 Frozen storage:

[0082] 1.2.1 Freeze storage method 1: add an appropriate amount of MRS culture medium containing 15-50% glycerol or 10-20% dimethyl sulfoxide (DMSO) antifreeze, and freeze at -20 to -80°C after aliquoting as seeds bacteria.

[0083] 1.2.2 Cryopreservation method 2: add glycerin, 10% (w / v) skimmed milk powder, 3% (w / v) lactose and 3% (w / v) final concentration of glycerin Freeze-dry the freez...

example 3

[0088] Example 3 freeze-dried powder

[0089] Get the bacterium precipitation that 2.1 method harvests in the example 2, add the xylo-oligosaccharide solution resuspended bacterium that final concentration is 20% (w / v), then add the glycerol that final concentration is 15% (w / v), 10% ( w / v) of skimmed milk powder, 3% (w / v) of lactose and 3% (w / v) of trehalose, formulated to not less than 10 per gram 8 The mixture of viable bacteria is divided into packages and then freeze-dried on a freeze-dryer, sealed and stored at 4-8°C or -20°C.

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Abstract

The invention relates to a transformant for reducing weight and fat, which is obtained by converting recombinant expression plasmids of a human gastric acid regulin gene into probiotic bacteria of human beings or animals, wherein the recombinant expression plasmids of the human gastric acid regulin gene are obtained by cloning a genetic fragment for coding human gastric acid regulin to an expression area of prokaryotic expression plasmids taking an ara operon as a promoter; and the probiotic bacteria of human beings or animals are bifidobacteria or lactic acid bacteria. The transformant can induce, express and excrete gastric acid regulin inside and outside intestinal tracts through revulsant L, namely, arabinose or low-polyxylose and plays a role of reducing weight and fat, therefore, the transformant can be prepared into an oral live bacterial preparation for reducing weight and fat.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a transformant with the oxyntomodulin gene as an exogenous gene. Background technique [0002] Obesity is a health issue of great concern in the world today. In developed countries in Europe and America, more than half of the people are overweight, and 20-30% of them are obese patients. Obesity not only affects movement and quality of life, but also can cause a series of diseases such as cardiovascular disease, type 2 diabetes and kidney disease, which are extremely harmful to human health. Statistics show that for every unit increase in body mass index (BMI), the risk of developing hypertension increases by 10%. [0003] Although there are many causes of obesity, factors such as genetics, endocrine disorders, diet structure and lifestyle can all induce obesity. However, the vast majority of obese patients are simply obese due to poor diet and living habits. At present, the treatme...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74A61P3/04A61P3/06C12R1/01C12R1/225C12R1/46
Inventor 曾位森罗深秋龙若庭
Owner SOUTHERN MEDICAL UNIVERSITY
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