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Recombination human insulin and its coding gene and application

A technology of recombinant human insulin and coding gene, applied in insulin, application, gene therapy and other directions, can solve the problems of cumbersome process and achieve the effect of good application prospect

Inactive Publication Date: 2008-03-26
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Now the production of insulin is through multiple steps of gene engineering, such as mass expression, purification, cyanogen bromide cracking, denaturation, renaturation, etc., and the process is cumbersome.

Method used

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  • Recombination human insulin and its coding gene and application
  • Recombination human insulin and its coding gene and application
  • Recombination human insulin and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, acquisition of recombinant human insulin gene and engineering bacteria expressing it

[0043] 1. Synthesis of recombinant human insulin gene

[0044] Replace 37 base pairs in the human insulin gene sequence according to the preferred codons of lactic acid bacteria, and add the coding sequence of the connecting peptide (RRSPNGKR) between the A and B chain sequences 5'-CGTAGATCTCCTAATGGAAAACGT-3', RR and KR for insulin The amino acids at both ends of the original C peptide, SPNG is conducive to the formation of a β-turn structure, and the spatial structure simulated by the recombinant protein is very similar to the natural insulin structure.

[0045] Design eight splicing primers (shown in Table 2, 1-4 are positive strand splicing primers in Table 2, and 5-8 are negative strand splicing primers) according to its positive and negative strand sequences, add 5'-Pi with T4 phosphokinase , then heated to 95°C, slowly annealed to room temperature, ligated overnig...

Embodiment 2

[0080] Embodiment 2, engineering bacterium ATCC27092-pSW501 is used as the animal effect experiment of oral vaccine

[0081] 1. The culture and collection of engineering bacteria ATCC27092-pSW501 live bacteria

[0082] The engineering bacterium ATCC27092-pSW501 was inoculated to 1% by volume (10 7 cfu / ml) inoculum amount inoculated in MRS medium, cultured statically at 30°C until OD 600 0.3-0.4. The bacteria were collected as an oral therapeutic vaccine for non-obese diabetic (NOD) mice, and Lactobacillus casei ATCC27092 was cultured to OD in the same way 600 0.3-0.4, collect the bacteria as a control.

[0083] 2. Oral effect experiment of engineering bacteria ATCC27092-pSW501 on non-obese diabetic (NOD) mice

[0084] Eighteen 4-week-old non-obese diabetic (NOD) mice (purchased from the Institute of Experimental Animals, Chinese Academy of Medical Sciences) were randomly divided into a PBS group, a control group and an experimental group, with 6 mice in each group. The PB...

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Abstract

The present invention discloses one kind of recombinant human insulin and its coding gene and application. The recombinant human insulin is following protein: 1. the protein comprising the amino acid residue sequence of Sequence 1 in the sequence list; and 2. the derivative protein with the sequence of Sequence 1 through the substitution, deletion and / or addition of amino acid residue(s) and possessing the function of human insulin. The recombinant human insulin has a connecting peptide introduced into the chain A and B sequences of natural insulin to favor to forming beta angle structure, a spatial structure similar to that of natural insulin and insulin activity. The present invention also provides one kind of engineering lactic acid bacteria with the recombinant human insulin gene and capable of surviving in intestinal tract for over 48 hr to stimulate the immune system of NOD mouse to generate specific recombinant human insulin antibody, raise the IL-4 level and induce immunologic tolerance generation.

Description

technical field [0001] The present invention relates to a recombinant human insulin and its coding gene and application, in particular to a recombinant human insulin and its coding gene and its application in the preparation of lactic acid bacteria oral vaccine for treating type I diabetes. Background technique [0002] Insulin (Insulin, INS) is an important endocrine hormone and the main autoantigen that causes the autoimmune disease type 1 diabetes mellitus (T1DM). At present, there is no effective preventive method for T1DM, and the patients can only inject insulin three times a day to keep blood sugar stable. [0003] Studies have shown that oral administration of insulin to non-obese diabetic (NOD) mice, a model of T1DM, can reduce or delay its pathogenesis. Receptivity-related effector T cells are activated, triggering oral tolerance, thereby protecting insulin from damage (Weiner HL. Oral tolerance: immune mechanisms and the generation of Th3-type TGF-beta-secreting ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/62C12N15/17C12N1/21A61K38/28A61K48/00A61P3/10
Inventor 钟瑾陈思维还连栋
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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