Method for expressing human insulin for reducing blood sugar by transgenic lucid ganoderma

A human insulin, transgenic technology, applied in other methods of inserting foreign genetic materials, gene therapy, pharmaceutical formulations, etc., can solve the problems of skin allergy and neuralgia, gene deletion, insufficient secretion of main and companion insulin, and reduce blood sugar levels. Effect

Inactive Publication Date: 2006-12-20
林忠平
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0020] 1. Insulin resistance with insufficient insulin secretion
[0021] 2. Insulin insufficiency mainly with or without insulin resistance
[0023] 1. Deletion of functional genes of islet β cells
[0024] 2. Deletion of the gene for insulin action
[0027] 5. Caused by drugs and chemical poisons
However, since insulin injection does not imitate the physiological process of insulin secretion by pancreatic β cells, it can only control the development of the disease, but not improve the disease, and may cause side effects such as optic atrophy, skin allergies and neuralgia; Pancreas transplantation often causes strong immune rejection

Method used

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  • Method for expressing human insulin for reducing blood sugar by transgenic lucid ganoderma
  • Method for expressing human insulin for reducing blood sugar by transgenic lucid ganoderma
  • Method for expressing human insulin for reducing blood sugar by transgenic lucid ganoderma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Cloning of embodiment 1 InsK gene:

[0075] According to the situation of codon usage in plants provided by Murray E.E. et al., it is speculated that plants prefer codons. The double strand of the human proinsulin analogue gene InsK is divided into 8 segments, so that one of the 5' ends of the two strands is slightly shorter and can be used as a primer for PCR reactions, and the other 3' end is slightly longer, overlapping each other and covering the entire length of the gene. Pay attention to try to end with G and C when segmenting.

[0076] serial number

sequence

No.1

5'-GATCCATGTT CGTTAACCAA CACC-3'

No.2

5’-TTTGCGGATC TCACCTTGTT GAGGCTCTTT ACCTTGTTTG CGGAGAGAGG GGATTC-3’

No.3

5’-TTCTACACCC CAAAGACCGG AGGACCATCT GGAGGAGGAA TCGTTGAGCA ATG-3’

No.9

5’-CTGCACCTCT ATCTGCTCTC TTTACCAACT TGAGAACTAC TGCAACAAGG ACGAGCTTTA AG-3’

No.10

5'-AATTCTTAAA GCTCGTCCTT GTTGCAGTAG TTCTC-3'

No.6

5'-AAGTTGGTAA A...

Embodiment 2

[0149] Example 2 Construction of Ganoderma lucidum expression vector pbinsK:

[0150] The pL221 plasmid was completely digested with HindIII and BamHI, a 1.4kb fragment containing the PLeGPD promoter was excised from it, the small fragment was recovered, and the fragment was ligated to the pGEM11Z-insK plasmid that was also digested with HindIII and BamHI and the large fragment was recovered In , the successfully constructed plasmid was named pLinsK. Use HindIII and SacI to excise a fragment of about 1.6 kb containing GPD-insK from the pLinsK plasmid, and connect this fragment to the pL321b plasmid that is also digested with HindIII and SacI and recover a large fragment, and the successful construction of the plasmid is named for pbinsK. The construction process is attached figure 1 .

[0151] During the operation, plasmid extraction, enzyme digestion, recovery, connection, cell transformation, enzyme digestion identification and PCR identification of recombinant plasmids w...

Embodiment 3

[0153] Example 3 Construction of ganoderma lucidum expression vector p1300-GinsK:

[0154] Completely digest the pbinsK plasmid with HindIII and KpnI, cut out a 3.4kb fragment, recover the fragment, and connect the fragment to the p1300-Super-PAnos plasmid that is also digested with HindIII and KpnI and recover a large fragment, and the construction is successful The plasmid was named p1300-GinsK. The construction process is attached figure 2 .

[0155] During the operation, plasmid extraction, enzyme digestion, recovery, connection, cell transformation, enzyme digestion identification and PGR identification of recombinant plasmids were all carried out according to the steps described in Example 1.

[0156] The initial plasmid p1300-Super-PAnos used in this construction process was donated by the laboratory of Professor StantonB.Gelvin of Purdue University in the United States and kept in this laboratory.

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Abstract

The invention relates to an artificial insulinogen analogue gene, while it adds the internal stay sequence of KDEL to accumulate the protein in the internal network to avoid degrading enzyme of cell. And it uses GPD of high level epiphyte to start the target gene and arranged in the carrier transformed with lucid ganoderma; uses electric hitting method or agricillin or PEG medium gene method to transform lucid ganoderma; the ELISA test has proved that the content of artificial insulinogen analogue is 105.0 mug/g-174.8 mug/g of the fresh weight, and at 4.40%-10.40% of soluble protein. And the invention can effectively reduce blood sugar.

Description

1. Technical field: [0001] The invention relates to artificially synthesizing human insulin gene and modifying the coding region structure. A promoter construct with strong expression in fungi was chosen. Place in a carrier suitable for transformation of Ganoderma lucidum, and genetically transform Ganoderma lucidum by electric shock method, Agrobacterium-mediated method or PEG-mediated method. Ganoderma lucidum expressing human insulin was obtained, and it was proved effective by oral gavage feeding to hyperglycemic rats, thus providing a method for using transgenic Ganoderma lucidum to lower blood sugar. 2. Background technology: [0002] 1. Insulin [0003] Insulin is a small molecular protein secreted by pancreatic β cells, with a molecular weight of about 6000kD. There are two peptide chains in the molecule, called A chain and B chain. The A chain of human insulin has 21 amino acid residues, and the B chain has 30 amino acid residues. The A and B chains are connected...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K36/074A61P3/10C12N15/80C12N15/87
Inventor 林忠平倪挺胡鸢雷孙丽陈溪
Owner 林忠平
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