| Human proinsulin (HPI) is an important single chain non-glycosylated protein, which contain 109 amino acids with a molecular weight about 12 kD. HPI is the precursor protein of insulin, the only hypoglycemic hormone in human body. There are many studies have found that HPI can effectively reduce glucagon stimulation of glycogenolysis in vivo or in vitro, and it can combine the insulin receptor to exert biological effects. Long-term imbalance of insulin levels in the body easily lead to diabetes, and the number of worldwide diabetes has been increasing with the continuous development of human society and the improvement of people’s living standards. China has become the world’s most populous country in diabetic patients, and diabetes prevalence rate is still 9.7% in 2010 with the data from the survey of diabetes in people aged over 18 by the Chinese Society of Endocrinology and China Center for Disease Control and Prevention. Currently the main treatment for diabetes is subcutaneous insulin injection, but this can bring the patient great mental pain, and also easily lead to hyperinsulinemia, hypoglycemia, weight gain and other complications. The treatment of diabetes is expensive, and the cost of each America diabetic is about $11744 per year. More and more researches is beginning to focus on the development of oral insulin preparations, but oral protein drugs must be overcome the two problems, which are the damage to protein by the extreme environments in gastrointestinal and the low drug absorption.Bacillus subtilis is a nonpathogenic gram-positive probiotics, and it can form resistant dormant body called spore under the conditions of the extreme threatening environment. Spores can remain active in the acid gastric environment, endure saliva and bile attack, and smoothly through the animal digestive tract directly to the small intestine, and it has gradually become the extreme environment (such as gastrointestinal tract) effective carrier of heterologous protein or a biologically active molecule. Therefore, the polypeptide proinsulin with an enterokinase site (Asp-Asp-Asp-Asp-Lys) was displayed on the spore surface according to spore resistance and the characteristics of human intestina digestion, and this could construct the recombinant spores with the nutrition and hypoglycemic function, in which the polypeptide proinsulin could be freely digested and absorbed into the gut. The main contents and results of this research are as follows:(1) Specific primers with restriction enzyme sites Nco I and Sac I were designed by using the software Primer 5.0, and the human proinsulin gene (hpi) was obtained by PCR amplification. Then hpi gene was successfully identified by sequencing and database alignment.(2) The enterokinase site (Asp-Asp-Asp-Asp-Lys) was added in front of the human proinsulin gene to construct the integrated type recombinant plasmid pJS700-HPI by building the plasmid pET30a-HPI. Then the recombinant plasmid was transformed into the Bacillus subtilis. Through PCR analysis, amylase activity analysis, the recombinant Bacillus subtilis was obtained. The recombinant spores was induced in DSM culture medium, and then the proinsulin dispayed on the spores was further confirmed by Western blot and immunofluorescence analysis.(3) The five instar molting silkworm was microinjected with glucose solution. The blood glucose of part silkworm sample was measured at different time points by using glucose oxidase method, which indicated the silkworm can absorb and metabolic the glucose. The remaining silkworm continued injection of glucose solution. Three days later, the weight, length and total blood sugar of silkworm was measured, and the result showed the high blood sugar (9 mg/ml) can obviously inhibit the growth and development of silkworm. Compared with the normal group silkworm (blood sugar about 6 mg/ml), the weight of high blood sugar silkworm loss about 0.35 g.(4) The midgut total protein of normal and hyperglycemic silkworms was comprehensively analyzed by using the two-dimensional eletrophoresis technology, and the protein differently expressed in midgut was identified by mass spectrometry. GO analysis was used to identify the metabolism and catalytic properties of those protein, which was verified at the transcriptional level. The result shows the silkworm IDGF was close with glucose metabolism, and western blot experiment of midgut further demonstrated the IDGF plays an important role in inhibition the normal growth process of silkworm by high-glucose diet.(5) A large number of recombinant spores was obtained by fermentation, and then oral experiments was carried out with the silkworm hyperglycemic model. Three days later, the silkworm serum was collected to test the glucose level, and the result showed the recombinant spores can reduce the blood glucose concentration of silkworm from 9 mg/ml to about 6 mg/ml, which indicated the recombinant spores can significantly recude the glucose level of silkworm hemolymph.(6) The diabetic mice model was constructed by intraperitoneal injection of streptozotocin solution, and the intragastric administration experiment was carried out by using the recombinant spores. After the experiment the blood glucose was analyzed by the eyeball blood. Results showed that oral administration of recombinant bacillus can significantly reduce the blood glucose levels of diabetic mice to about 2.5 mg/ml, and increase the content of serum insulin in diabetic model mice to 6.72μIU/ml, which indicated the recombinant spore has a certain degree of hypoglycemic effect. |
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