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Novel recombinant lactobacillus resisting diabetes mellitus type 1 and application thereof

A technology for recombining lactic acid bacteria and Lactococcus lactis, applied in the field of biomedicine, can solve the problem of high safety production cost, save the purification process, realize large-scale production, and achieve good therapeutic effect.

Inactive Publication Date: 2016-02-03
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Through the way of oral live bacterial vaccine, the effect of mucosal tolerance is achieved, which overcomes the defects of poor safety, high production cost and unacceptable vaccination methods of traditional vaccines

Method used

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  • Novel recombinant lactobacillus resisting diabetes mellitus type 1 and application thereof
  • Novel recombinant lactobacillus resisting diabetes mellitus type 1 and application thereof
  • Novel recombinant lactobacillus resisting diabetes mellitus type 1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The cloning of embodiment 1HSP65-6IA2P2 gene

[0055] The universal primers M13F: 5'-TGTAAAACGACGGCCAGT-3' and M13R: 5'-CAGGAAACAGCTATGACC-3' located on both sides of the target gene on the vector were used as upstream and downstream primers respectively, and the two primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd. The PUC19 plasmid was used as a template for PCR, and the gene fragment encoding 6IA2P2 sequence was obtained. Then it was digested with NheI and HindIII, and connected into the PCYT:HSP65-6P277 vector which was also digested with NheI and HindIII, to construct PCYT:HSP65-6IA2P2.

[0056] The PCR reaction was carried out according to the following conditions: in each step of the reaction, the upper and lower primers were mixed at a ratio of 1:1, and the reaction program was set to 94°C, 1s; 50°C, 45s; 72°C, 50s; a total of 30 cycles, 0.8% agarose gelation PCR products were identified by gel electrophoresis (see image 3 ), the band posit...

Embodiment 2

[0058] Example 2 The recombinant plasmid PCYT: HSP65-6IA2P2 was transformed into Lactococcus lactis competent cells by electric shock transformation

[0059] The process of preparing Lactococcus lactis competent cells is roughly as follows: pick a single colony from a fresh GM17 agar plate, inoculate 10 mL of GM17 medium, and culture overnight at 30°C. Then inoculate 1 mL of the overnight culture into 50 mL of preheated GM17 medium containing 1-2.5% glycine, and culture at 30°C. Monitor OD 600nm. When the OD value of the culture is close to 0.2-0.7, take out the culture and cool it on ice for 10min-30min, then centrifuge the culture in a sterilized tube, centrifuge at 5000g at 4°C for 15min, discard the supernatant, and use an equal volume of ice pre-cooled Suspend the cell pellet twice in OSPS buffer and resuspend the cells with 1 / 100 OSPS. Finally, the cells were added to a sterilized 1.5mL centrifuge tube, snap-frozen in a dry ice-ethanol bath, and stored at -70°C or use...

Embodiment 3

[0060] Example 3 Induction of recombinant Lactococcus lactis strain CCTCCNO: M2014609 to express fusion protein HSP65-6IA2P2

[0061] The recombinant Lactococcus lactis CCTCCNO: M2014609 obtained in Example 2 was inoculated in GM17 medium and cultured overnight at 30°C; the overnight culture solution was transferred to GM17 medium at a ratio of 1:100 and expanded at 30°C until the bacteria grew to an OD value of 0.4 At ~0.7, add the inducer Nisin, continue to cultivate for a period of time, and harvest the bacteria to prepare protein samples.

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Abstract

The invention relates to a novel recombinant lactobacillus resisting diabetes mellitus type 1 and application thereof and belongs to the field of biomedicine and discloses a diabetes mellitus type 1 resistant vaccine using Lactococcus lactics as a vector and apreparation method and application thereof. A recombinant Lactococcus lactics vaccine using Lactococcus lactics as a vector and induced to express fusion protein HSP65-6IA2P2 is built by using a micro gene technology platform of the lab. The correct expression of a fusion protein vaccine is detected by Western blot analysis. In-vivo animal tests show that the vaccine can be orally administered to give significant reduction in the occurrence of diabetes in NOD (non-obese diabetic) mice and to prevent diabetes mellitus type 1, and has a promising clinical application prospect in terms of preventing diabetes.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to an anti-type I diabetes vaccine using Lactococcus lactis as a carrier, a preparation method and application thereof. Background technique [0002] Diabetes is a group of metabolic diseases characterized by hyperglycemia. Hyperglycemia is caused by defective insulin secretion or impaired biological action, or both. Long-term high blood sugar can lead to chronic damage and dysfunction of various tissues, especially the eyes, kidneys, heart, blood vessels, and nerves. Clinically, the main manifestations are "three excesses and one deficiency", that is, polyuria, polydipsia, polyphagia, weight loss and other manifestations. Diabetes is the second killer of modern diseases, and its harm to the human body is second only to cancer. Type I diabetes (IDDM) is a type of diabetes caused by the absolute lack of insulin due to the destruction or loss of function of pancreatic β cells. The blood su...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/62A61K39/39A61K39/00A61P3/10C12R1/01
Inventor 吴洁刘坤锋洪流刘晓锐金亮
Owner CHINA PHARM UNIV
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