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Researching on Transferring Human Insulin Gene into Chicken

Author: SuoJianFei
Tutor: LeiXueQin
School: Henan University of Science and Technology
Course: Animal Genetic Breeding and Reproduction
Keywords: Human insulin Clone Eukaryotic expression vector Transgenic chickens
CLC: S831
Type: Master's thesis
Year: 2010
Downloads: 45
Quote: 0
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Abstract


Diabetes is a common endocrine and metabolic diseases caused by the body relative or absolute lack of insulin. Diabetes has become the third place following the cardio-cerebral vascular tumor diseases of serious harm to human health. With the development of human society, nutrition, obesity, lack of exercise, such as aging and more serious problem, which will greatly increase the incidence of diabetes. The number of patients with diabetes in China only as high as more than 30 million, and the year is still 20% increments. Insulin is a hormone the body to lower blood sugar only clinical drug of choice for treatment of human type I diabetes, also will increase market demand for medical insulin. Insulin production directly from the initial extracted from the pancreas of humans and animals, the development of the use of different biological types of genetic engineering to produce insulin. Eggs as bioreactors for its production cycle is short, low-cost, the protein unique advantage of easy separation and purification, and future research directions in the production of pharmaceutical proteins. First, the experiment is to extract total RNA from pancreatic cells from human fetal pancreas and design a pair of primers based on the published sequence of the human insulin gene in Genbank., Primer5.0 biology software, using RT-PCR method to obtain a 350bp human insulin gene fragment (INS), the sequencing results showed that the cloned human insulin gene sequences and Genbank consistent released. Then construct pUC57-INS, for the preservation of the human insulin gene and amplification. Finally, the INS fragment subcloned to contain green fluorescent protein marker gene eukaryotic expression vector pEGFP-N1 After PCR, enzyme digestion and sequencing method to prove the human insulin gene eukaryotic expression vector pEGFP-N1-INS successfully constructed used to prepare the transfer of research of the human insulin gene chicken. This test uses the method of liposome encapsulation and chick embryo microinjection will build good pEGFP-N1-INS transfected into chick embryo to prepare a transgenic chicken. Has to collect of 220 Henan the Romer Lvkedanji eggs, 20 set untreated eggs for hatching of the blank control group, found that egg fertilization rate of 85%. 82 dead embryos, transgenic manipulation of 200 eggs in the hatching process different embryonic chicks, 24, 94 the transgenic immature eggs in the operation. Green fluorescence detection of the fusion protein (human insulin with green fluorescent protein) has 82 dead embryos and found four dead embryo tissue green fluorescent protein positive rate of 4.88%. Then use a pair of specific primers primer5.0 biology software design, dead embryo tissue fluorescence detection positive PCR detection, two positive, human insulin gene detection rate of 2.44%. Also three live chickens, for the protection of the test material, only the target protein detection in the blood, the result is not found protein-positive individuals. The test instructions in turn prepared to turn the human insulin gene chicken is feasible. Efficient production of medicinal insulin for the use of transgenic chickens laid the foundation for the benefit of humanity biological products.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Poultry > Chicken
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