Process for producing human insulin using transgenic tomato

A technology for human insulin and insulin, applied in the fields of insulin, biochemical equipment and methods, genetic engineering, etc., can solve the problems of high cost, low yield of culture equipment, unclear tolerance or immunity, etc., and achieve the effect of avoiding degradation

Inactive Publication Date: 2003-11-26
林忠平
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Problems solved by technology

At the same time, the dendritic cells scattered in the intestinal cells can also take up the antigens in the intestinal lumen and activate the immune response through the circulation of the lymphatic system, but whether this process induces tolerance or immunity is still unclear
[0006] The treatment of oral tolerance has a certain therapeutic effect on the disease because it can elicit an antigen-specific immune response, but it is limited by some practical problems, that is, this therapy requires a large amount of protein in both humans and experi

Method used

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  • Process for producing human insulin using transgenic tomato
  • Process for producing human insulin using transgenic tomato
  • Process for producing human insulin using transgenic tomato

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Experimental program
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Embodiment 1

[0055] Bacterial culture and DNA operation in all embodiments all refer to " Molecular Cloning: Laboratory Manual " (translated by Jin Dongyan etc., Science Press, Beijing (1993)) and " Refined Guidebook of Molecular Biology " (translated by Yan Ziying, etc., Science Press, Beijing (1998)). Tool enzymes in molecular operations such as restriction endonucleases were purchased from Promega Company and New England Biolabs Company. The artificial synthesis of embodiment 1 human insulin:

[0056] According to the situation of codon usage in plants provided by Murray E.E. et al., it is speculated that plants prefer codons. The summary is shown in Table 1.

[0057] Find the sequence of human proinsulin in GENEBANK, get the sequences of human insulin A chain and B chain, and design the replacement bases according to the plant preferred codons. Add the designed connecting short peptide between the B chain and A chain of the designed synthetic human insulin; add the start codon ATG b...

Embodiment 4

[0089] HinidIII and BamHI double enzyme digestion cut out a small fragment of 0.8kb, PCR amplification can also obtain a small fragment of 0.8kb, which proves that the construction of the vector is correct. (the size of P2A12 and CaMV35S is similar) the transformation of embodiment 4 Agrobacterium agrobacterium: the preparation of Agrobacterium agrocompetent state:

[0090] (1) Inoculum of Agrobacterium agrobacterium strain LBA4404 was cultured in YEB liquid medium containing appropriate antibiotics at 28°C until logarithmic phase;

[0091] (2) Centrifuge at 8,000 rpm for 5 minutes at 4°C, and collect the bacteria in a small centrifuge tube;

[0092] (3) resuspend and wash the cells with 600ul ice-cold 500mM CaCl2;

[0093] (4) Centrifuge at 8,000rpm at 4°C for 5 minutes, add 200ul ice-cooled 500mM CaCl2 to the cell pellet, mix well and set aside (24hr to 48hr is the best); transformation of Agrobacterium agrobacterium:

[0094] (1) Add about 1 ug of the constructed plant ex...

Embodiment 8

[0122] PCR identification proved that the insulin gene was integrated into the tobacco genome, and ELISA detection proved that the insulin gene was highly expressed in the transgenic lettuce. Example 8 uses the positional recombination system Cre / lox and FLP / FRT to control the insulin gene in F 2 Delete in generation:

[0123] In parent 1, the insulin gene driven by the fruit-specific promoter was transferred, and the promoter and insulin were flanked by the LoxP element (Cre recombinase recognition element) in the same direction, and the copper-inducible promoter was used to drive the FLP gene. The vector is transferred into parent 1; the embryo-specific promoter-driven Cre recombinase gene is transferred into parent 2, and there is a blocking sequence between the promoter and Cre recombinase, and the two sides of the blocking sequence are in the same direction The FRT element (FLP recombinase recognition element). Cross parent 1 and parent 2, under the induction of 0.5μM c...

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Abstract

The present invention utilizes transgenic tomato as bioreactor to produce human insulin. The human insulin is produced through designing plant preference codon, artificially synthesizing several DNAsegment, splicing, and adding endoplasmic reticulum residence sequence of KDEL in the C terminal to avoid the degradation of insulin in plant cell. The gene is transformed into tomato via agrobacterium mediating process under the driving of CaMV35S promoter and fruit specificity promoter 2A12 to express human insulin in tomato fruit. This kind of tomato capable of producing human insulin may become one kind of convenient oral product for preventing and treating some diabetes of depending insulin and autoimmune dysfunction, and may be used to extract human insulin for making injection.

Description

Technical field: [0001] The invention relates to a method for producing human insulin by transgenic plants and its product. Background technique: [0002] Bioreactors are an area of ​​great concern to many biotechnology R&D departments in the near future. Human growth factors are produced in mammary bioreactors such as dairy cows and dairy goats. In the research of plant bioreactor, the research of using potato tubers to produce hepatitis B vaccine has received extensive attention. Lettuce and tomato, as vegetables or fruits that can be eaten raw, have great application potential in the production of oral polypeptide drugs. Human insulin is very important for the treatment of insulin-dependent diabetic patients. Metabolic regulation in some diabetic patients is associated with autoimmune function. [0003] Oral insulin has been shown to be effective in the treatment of certain types of diabetes [Reddy S et at, Pancreas 2000, 20(1): 55-60; Ploix C et at, Diabetes 1999, 48...

Claims

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Application Information

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IPC IPC(8): A01H1/00A61P3/10C07H21/04C07K14/415C07K14/62C12N15/17C12N15/82
Inventor 林忠平倪挺陈溪胡鸢雷
Owner 林忠平
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