Molecular Designing And Cloning Of Human Insulin Mutant

Diabetes is one of the most serious diseases that jeopardize people's health and life. The glucose in the bodies of patients cannot be fully used. This results in the big amout of conssumption of carbohydrates, fats and proteins. Along with the loss of water, their weight decreases and they become very thin. Serious patients could lose tens of kilograms of weight and lead to lack of strength and energy. More seriously, diabetes can cause a number of complications such as the disease of heart, kidney, blood vessel, nerve and retina, which bring patients much pain. As the only hormone in animal bodies that decreases the blood sugar level, insulin is very significant to the treatment of diabetes. The traditional preparation of marketing insulin is mainly derived from pig and bovine pancreas. Because the two kinds of insulin are different with human insulin in the primary structure, it may cause immune reaction and affect the activity, though it might not affect the activity shortly. Moreover, the low production cannot meet the demand of market. Therefore, nowadays, the production of insulin by genetic engineering becomes a substitution of the traditional method. On the other hand, the main introduction of insulin is subcutaneous injection. It is quite inconvenient to the patients. Thus the development of other introduction of insulin such as inhalation, patch and oral insulin is a hot spot in today's research.The purpose for this research is to design a short-C-peptide human insulin mutant for future research of its activity and to explore the optimal conditions of potato transformation of human insulin gene. A human proinsulin was designed according to the amino acid sequence of human proinsulin generated by Genbank, considering the codon bias of E coli and the property of the expression vector pGEX-3X. An EcoRI site was inserted to the upstream side of the N-end of chain B and a BamHI site was inserted to the downstream side of the C-end of chain A. The C- peptide was substituted by a shorter fragment. The whole gene sequence was designed as six overlapped segments and was synthesized segment by segment. After TA clone it was sequenced and was named InsM2. Additionally, a plasmid constructed bu the Lab p1301P06H was transformed into Agrobacterium tumefaciens and transformed into potatoes mediated by Agrobacterium tumefaciens. The results of this research are as followed:(1) The designing of the human insulin mutant gene was completed.(2) The gene was synthesized fragment by fragment and extended in vitro to a complete gene of InsM2 which is suitable for the expression in E coli using SOE-PCR and Klenow fragment treatment. And the optimal conditions for this PCR is to set the annealing temperature of the step PCR 60℃,65℃,70℃.(3) Purified fragment was gel extracted. It was TA cloned to pMD-19 simple T-Vector by sticky ends. After selection, the positive strain was sequenced. The result showed two nucleotides mutated but the amino acid sequence was correct.(4) Several parts of the Favorita such as stems, leaves, stem apex and shoots out of the tuber, also small potato tubers which had just germinated along with a number of methods such as leaf disc method, vacuum method and ultrasonic method were used for potato transformation. The optimal conditions for Favorita were to vacuumize to 0.04Mpa for 2.5min then back to regular pressure for three times using the stem apex. Then they were co-cultured in the co-culture medium containing AS for 3 days. Through selection the plants with roots was found in selection medium containing Hyg and they are to be detected using the molecular biological method.Conclusion: The human insulin gene mutant designed contains a four-peptide structure to replace the C-peptide that is aβ-turn structure to connect chain A and chain B. It can keep the spatial structure from changing and it shortens the length of connection peptide. Additionally it was synthesized fragment by fragment which decrease the difficulty and cost of synthesis. In the future it can be used to research its activity and even expressed in potatoes. Also, stem apex method plus vacuum treatment can improve the rate of potato transformation mediated by Agrobacterium tumefaciens.