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A method for cultivating peanuts and soybeans expressing human amylin and its application
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A breeding method, amylin technology, applied in the field of genetic engineering to achieve the effect of eliminating drug resistance, preventing complications, and enhancing immune function
Inactive Publication Date: 2018-08-14
GUILIN MEDICAL UNIVERSITY
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[0008] So far, no one has used such plant protein-rich legumes and oil crops as natural 'plants' to produce an important protein that regulates sugar metabolism - human amylin, which is expressed Literature and patent reports on oil crop cultivation methods of human amylin
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Embodiment 1
[0035] A method for cultivating peanuts expressing human amylin gene, comprising the steps of:
[0036] 1. Construct a plasmid DNA containing the coding sequence of the human amylin gene, said gene comprising 37 amino acids, a disulfide bond is formed between the second and seventh cysteine residues in the molecule, and the hydroxyl terminal amide The amino acid composition of the 20th-29th position in the middle has species differences. The amino acid sequence is KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY.
[0037] 2. The extraction of human amylin plasmid DNA, its method is:
[0038] The used Escherichia coli strain DH5α containing the target gene contains 35S promoter, target gene hIAPP, terminator NOS and vector CAMV1301. Pick the preserved bacteria on the ultra-clean workbench and inoculate them on the LB solid medium for culture, place the bacteria plate in a constant temperature incubator, and incubate at 37°C for 12 hours until a single colony is formed; pick a single c...
Embodiment 2
[0056] Breeding soybeans expressing the human amylin gene
[0057] Soybean transduction operation: 1-33 hours after soybean self-pollination, after cutting the stigma, drop the plasmid DNA into the cut with a 1 ml syringe and keep the droplet.
[0058] All the other are with embodiment 1.
Embodiment 3
[0060] Effects of Peanuts Expressing Human Amylin on Blood Glucose in Diabetic Mice
[0061] 1 Establishment of diabetic mouse model
[0062] After 8-12 week-old mice were fasted for 12 hours, blood glucose was measured. Divided into two groups during the experiment, negative control group (injection of normal saline only), model group (one-time injection of 120 mg / kg streptozotocin in the tail vein), then the mice were raised in groups, free to eat and drink, and blood glucose was measured two weeks later value. The results showed that the diabetic mouse model was successfully constructed.
[0063] 2. The test materials are peanuts successfully expressing hIAPP gene in the F1 generation. Peanuts were fed to prediabetic mice, diabetic mice and normal rats, respectively. The dosage is 5-25 g / kg / day. The test initially found that the optimal dose was 15 g / kg / day.
[0064] 3. Feeding diabetic mice with peanuts expressing human amylin, once every 2 days, at a dose of 15 g / kg...
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Abstract
The invention relates to a cultivation method and application of peanuts and soybeans for expressing human amylin. The human amylin gene is recycled through KpnI and XbaI digestion and then connected into a plant expression carrier CAMV-1301, and a plant expression carrier pcambia-CAMV35S-hIAPP-NOS-1301 resisting a kanamycin gene is obtained through structuring. According to the cultivation method and the application of the peanuts and the soybeans for expressing the human amylin, the amylin gene is successfully transformed into the selfing line F1 generation of the peanuts and the soybeans through a pollen tube path way, and the transformation plants for expressing the amylin gene are obtained. By means of the cultivation method, the human amylin gene can be introduced into oil crops. People can prevent diabetes and be assisted in treatment of diabetes in diet by eating the peanuts and soybeans containing the important protein hormones, namely, the amyline, for regulating and controlling glycometabolism, and meanwhile the inherent problems of drug resistance and various adverse reactions of common medicine are avoided. The economical burden and psychological burden which are caused by injection medications and oral medications borne by diabetic patients and various adverse reactions can be relieved.
Description
technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for cultivating peanuts and soybeans expressing human amylin and its application. Background technique [0002] Diabetes mellitus (DM) is a common chronic disease caused by endocrine and metabolic disorders. In 2011, there were approximately 366 million DM patients worldwide, with 4.6 million deaths and an economic loss of 465 billion US dollars; by 2030, the number will rise to 552 million. 2007-2008 my country's national survey results show that the prevalence of DM is as high as 9.6%, and the total number of patients is about 92 million. What's more, the prevalence rate of impaired glucose tolerance (IGT) / impaired fasting glucose (IFG) in pre-DM is as high as 15.5%, and the number of pre-DM patients nationwide is estimated to be about 148 million. Compared with previous survey data, the number of young and middle-aged people who are sick has increased more signifi...
Claims
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