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Method for expressing human insulin by using plant seed oil body

A human insulin and plant technology, applied in botany equipment and methods, biochemical equipment and methods, plant gene improvement, etc., can solve the problems of mammalian protein, expensive culture equipment, post-translational processing, etc., to improve transcription Efficiency, avoiding gene drift, and reducing production costs

Inactive Publication Date: 2007-09-19
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to solve this problem, people have tried to produce recombinant human insulin using several traditional expression systems including microbial fermentation and mammalian cells, but they all have various limitations
A major problem with bacterial expression systems is their inability to perform the post-translational processing necessary for mammalian proteins, such as glycosylation
Mammalian cells, on the other hand, have limited their application due to their expensive culture equipment and their relatively low yields.
However, there is no report on the expression of human insulin using the plant oil body expression system

Method used

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  • Method for expressing human insulin by using plant seed oil body
  • Method for expressing human insulin by using plant seed oil body
  • Method for expressing human insulin by using plant seed oil body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: Preparation and sequence determination of Insulin gene

[0040] Find the sequence of human proinsulin in GENEBANK, get the sequence of human insulin A chain and B chain, and insert a short peptide sequence between the A and B chains, and then remodel the base sequence of insulin according to the codons preferred by plants (13 of the 63 bases in the A chain were replaced, and 18 of the 90 bases in the B chain were replaced), and the enzyme cutting sites used in this experiment were eliminated according to the codon degeneracy point.

[0041] Use the online software Primerfinder and DNASTAR to design primers. A total of 8 primers were designed and synthesized. The length of each primer ranges from 30-50bp. There is 20 base overlaps between two adjacent primers. Finally, the full-length was synthesized by bridge PCR. insulin gene. PA1-PA4 is a forward primer, and PA5-PA8 is a reverse primer, wherein PA4 and PA5 are complementary chimeric primers (20 complem...

Embodiment 2

[0082] Example 2: Intermediate vector construction for oil body expression

[0083] Using the total DNA of Brassica napus as a template, primers were designed based on the sequence of its oleosin gene promoter. Primer 1 contains a Hind III restriction site, and primer 2 introduces a Pst I restriction site. After PCR amplification, about 900bp oleosin gene promoter (PCR reaction conditions: 94°C, 5 minutes; 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute; after 25 cycles, 72°C for 5 minutes), PCR products were purified with Hind III and PstI After double digestion, it was connected to pUC19 to obtain pUCON, and the sequencing result showed that the cloned product was the promoter of rapeseed oil body protein gene.

[0084] Primers were designed based on soybean 24kD oleosin gene sequence: primer 1 sequence: 5′-GAA TCT AGA GAT GAT GAT GAT AAG ATG ACC ACA CAAGTA CC, primer 2 sequence: 5′-GCC GGT ACC ATC ATC ATC ATC CTTGGT TGT TGC TGT CAC TG. Primer 1 contains a Pst ...

Embodiment 3

[0086] Example 3: Construction of recombinant oil body expression vector containing insulin gene

[0087] The pUC-insulin plasmid DNA was extracted, digested with BamH I and EcoRI, the digested fragment of insulin was recovered, and connected to the intermediate vector p1390ONE for oil body expression to obtain the recombinant plant oil body expression vector p1390ONE-insulin. It was identified by double digestion with BamH I and EcoRI, and it was confirmed that the size of the fragment was about 180bp.

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Abstract

The invention provides a method of expressing human insulin humulin by plant seed oil body. The invention designs human insulin humulin genes according to plant codon bias, mixing the human insulin humulin genes and oil body protein gene to construct expression carrier containing the mixing genes, further conversing the expression to receptor plant to get conversing palnt whose seeds can effectively express human insulin humulin. The invention uses plant bio reactor to produce human insulin humulin avoiding harm of animal pathogen and endotoxin of coliform bacteria, and can simplify departing and purification process of goal proteins to reduce cost and good for industrialization of human insulin humulin.

Description

technical field [0001] The invention relates to a human insulin (insulin) and a method for expressing it in plant oil body, in particular to a method for producing human insulin by using a fusion protein gene expression system composed of plant oil body protein and target protein. Background technique [0002] Diabetes is mostly caused by the destruction of pancreatic β cells by autoimmune reactions, resulting in insufficient insulin secretion. Patients gradually develop a series of serious complications, such as blindness caused by cardiovascular disease, kidney disease and diabetic retinopathy. At present, there is no cure, and the usual treatment is insulin injection. Human insulin is very important for the treatment of insulin-dependent diabetic patients. However, insulin injection may cause side effects such as optic atrophy, muscle atrophy at the injection site, skin allergies and neuralgia, which bring great pain to patients. Insulin inhalation therapy is a new metho...

Claims

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Application Information

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IPC IPC(8): C12N15/17C12N15/82C12N15/29C12N15/09C12P21/02
Inventor 李校堃柯实肖业臣曲勍王晓慧
Owner JILIN AGRICULTURAL UNIV
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