Application of recombinant lactobacillus and SNase in preparing drug for preventing or treating I type diabetes mellitus

A technology for recombinant lactic acid bacteria and type 1 diabetes, which is applied in the direction of drug combination, microbial-based methods, microbial measurement/testing, etc., can solve the problem of no report of hypoglycemia, avoid tedious and complicated purification steps, and save production costs Effect

Active Publication Date: 2016-07-20
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The above-mentioned pCYT:SNase is a conventional carrier, and there is no report on hypoglycemia

Method used

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  • Application of recombinant lactobacillus and SNase in preparing drug for preventing or treating I type diabetes mellitus
  • Application of recombinant lactobacillus and SNase in preparing drug for preventing or treating I type diabetes mellitus
  • Application of recombinant lactobacillus and SNase in preparing drug for preventing or treating I type diabetes mellitus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Extraction of pCYT:SNase plasmid and verification by agarose gel electrophoresis

[0037] Use the plasmid mini-extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd.) for plasmid extraction. The process is as follows: Add 500 μL of balance solution BL to the adsorption column CP3 (the adsorption column is placed in a collection tube), centrifuge at 12,000 rpm for 1 min, pour it out and collect For the waste liquid in the tube, put the adsorption column back into the collection tube; take 5mL of overnight cultured bacterial solution, add it to the centrifuge tube, centrifuge at 12000rpm for 1min, and suck out the supernatant as much as possible; add 250 μL of solution P1 (with RNaseA and lysozyme added in advance), use a pipette to thoroughly suspend the bacterial pellet, and place it in a 37°C water bath for 40 minutes; add 250 μL of solution P2 to the centrifuge tube, and gently turn it up and down 6-8 times Fully lyse the bacteria; add 350 μL of s...

Embodiment 2

[0038] Example 2: Induction of recombinant Lactococcus lactis strains L. lactis NZ9000pCYT:SNase expresses SNase.

[0039] Lactococcus lactis L. lactis NZ9000pCYT: SNase, prepared according to the method provided in the literature "LuisG.Bermúdez-Humaránetal.Controlledintra-orextracellularproductionofstaphylococcalnucleaseandovineomegainterferoninLactococcuslactis[J].FEMSMicrobiologyLetters,224(2003):307-313", named UCpCYT:Nase in the literature. The recombinant lactic acid bacteria L. lactis NZ9000pCYT: SNase, namely Lactococcus lactis NZ9000pCYT: SNase Lactococcus lactis NZ9000pCYT: SNase is deposited in the China Center for Type Culture Collection, address: Wuhan, China, Wuhan University; the preservation number is CCTCCNO: M2016084, and the preservation time is March 4, 2016.

[0040] Lactococcus lactis L. lactis NZ9000pCYT: SNase was inoculated in GM17 medium, cultured overnight at 30°C; overnight culture solution was transferred to GM17 medium at 1:100, expanded ...

Embodiment 3

[0041] Embodiment 3: recombinant Lactococcus lactis L. lactis NZ9000pCYT: Pharmacodynamic evaluation of SNase anti-type 1 diabetes vaccine.

[0042] Twenty-four 4-week-old female NOD / LtJ mice (purchased from Beijing Huafukang Biotechnology Co., Ltd., license number: SCXK (Beijing) 2012-0004) were randomly divided into two groups, respectively L. lactis NZ9000 group and L. lactis NZ9000pCYT: SNase group, 12 in every group, when the mice were five weeks old, they began to give oral administration once a day in the first week, and once a week from the second week after the administration until the mice were small. The administration was terminated when the mice were 20 weeks old. Before gavage, adjust the concentration of live bacteria in each group to 2×10 10 CFU / ml, the dosage is 4×10 9 CFU / 200μL / time / only.

[0043] (1) Recombinant Lactococcus lactis L. lactis NZ9000pCYT: Effect of SNase Vaccine on Morbidity in NOD Mice.

[0044] From the beginning of the experiment,...

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Abstract

The invention relates to application of recombinant lactobacillus and SNase in preparing a drug for preventing or treating I type diabetes mellitus. The purpose is to provide new application of a vaccine with recombinant lactobacillus L.lactis NZ9000 p CYT: SNase as a carrier in resisting I type diabetes mellitus. The recombinant lactobacillus can express destination protein staphylococcal nuclease, SNase after being induced by lactose. The L.lactis NZ9000 p CYT: SNase living bacterium vaccine is used for immunity of female NOD/LtJ mice four weeks old in an administering oral medication mode, and a series of pharmacodynamic indexes are evaluated, it is found that morbidity of NOD mouse diabetes can be obviously reduced by orally taking L.lactis NZ9000 p CYT: SNase, blood sugar and body weight of the NOD mice can be controlled well, and the survival rate of the NOD mice can be increased remarkably. Therefore, the recombinant lactobacillus L.lactis NZ9000 p CYT: SNase can obviously restrain occurrence and development of NOD mouse diabetes and can be used for developing drugs for I type diabetes mellitus.

Description

technical field [0001] The invention relates to a recombinant lactic acid bacterium with an anti-type 1 diabetes effect, as well as derivatives containing the recombinant lactic acid bacterium and similar pharmaceutical preparations. The invention relates to the related fields of pharmacy and medicine. Background technique [0002] Diabetes mellitus (DM) is an endocrine and metabolic disease characterized by high blood sugar and multiple complications caused by absolute or relative insulin deficiency, and is one of the most common chronic diseases. Diabetes is usually divided into two types, type 1 diabetes (Type1diabetesmellitus, T1DM) and type 2 diabetes (Type2diabetesmellitus, T2DM). T1DM is generally considered to be a genetically based, multifactorial, organ-specific autoimmune disease. The islet β-cells of the patient are immune damaged by T lymphocytes, resulting in absolute deficiency of insulin secretion. At the same time, T1DM is also the result of the comprehens...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/46A61K35/744A61P3/10C12Q1/02G01N33/573C12R1/225
CPCA61K35/744A61K38/465C12Q1/02C12Y301/31001C12N1/205C12R2001/225G01N33/573
Inventor 吴洁郎君超刘坤锋
Owner CHINA PHARM UNIV
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