Construction Of A Multi-copy Integration Vector For Saccharomyces-cerevisiae Expressing Salmon Calcitonin And Safety Evaluation

Copp discovered calcitonin as a kind of hypocalcemic principal in 1960's. It was secreted by parafollicular cells of the thyroid in mammals or ultimobranchial body in fish. Calcitonic ( CT ) is a polypeptide with 32 amino acid residues secreted by parafollicular cells of the thyroid in mammals and ultimobranchial body in fish. It plays an important role in controlling calcium and phosphorus turnover. It plays an important role in controlling calcium and phosphorus turnover. The peptide was shown to potently inhibit bone resorption by directly acted on osteoclast. Calcitonin's potent anti-resorptive effect has led to its use in treating Paget's disease of bone, osteoporosis,and hypercalcemia. Among all species, salmon calcitonin is of the highest potent which is about 30-40 times to human calcitonin. Salmon calcitonin is used in medical treatment by injection or nasal administration. The high price is the other limitation of salmon calcitonin. Proteins and peptide drugs have been the most rapidly growing class of drugs in the last two decades. Despite rapid advances in proteins and peptides, the efficacy of therapeutic proteins and peptides is dependent upon injections, which are associated with poor patient acceptance due to pain and occasional local adverse effects[.Therefore, safe and effective noninvasive administration routes are required as alternatives to injection. Oral administration is considered one of the most attractive methods due to its convenience. As orally administered protein and peptides drugs are very poorly absorbed, partly due to enzymatic degradation in the gastrointestinal tract so that the use of carrier systems is preferable. Recently, many studies have indicated that Saccharomyces cerevisiae are suitable vehicle for peptides drugs.In 2001, Blanquet draw the concept-"biodrug"which was successfully demonstrated by using recombinant S. cerevisiae to express the plant cytochrome P450 73A1 (an important enzyme in various detoxication systems) in an artificial gastrointestinal tract. In addition, freeze-dried S.cerevisiae showed a high tolerance to the digestive conditions of the artificial gastrointestinal environment. The use of recombinant S. cerevisiae as a peptide drug delivery system may be effective. Salmon Calcitonin (sCT) gene was designed and synthesized. The multi-copy integration vector p-r-sCT was constructed by inserting sCT gene and a 2.2kb rDNA fragment from Saccharomyces cerevisiae into pYD1 . Synthesized Salmon Calcitonin gene was cloned and expressed in Saccharomyces cerevisiae yAGA2-r-sCT via p-r-sCT. Fluorescent pictures indicated that Salmon Calcitonin was expressed in yAGA2-r-sCT and the quantity of expression was detected by flow cytometry. The resorption activity of osteoclasts was inhibited by recombinant human-salmon calcitonin protein released from the cell surface of yAGA2-r-sCT. Blood calcium determination in rats showed the activity of yAGA2-r-sCT in vivo.Oral administration of 2 g/kg lyophilized yAGA2-r-sCT to hypercalcemic rats decreased serum calcium from2.818±0.232 mM to 2.475±0.0191mM.Oral administration recombinant Saccharomyces cerevisiae expressing salmon calcitonin made a new method for salmon calcitonin delivery system. Transgenic Saccharomyces cerevisiae was evaluated for its acute (7 days) and subacute toxicity (8 weeks) by the oral route in KM mouse and Wistar rats respectively. In the acute toxicity test, every group did not produce any hazardous symptoms or deaths (LD50>10000 mg/kg). The subacute treatment with transgenic Saccharomyces cerevisiae (2 and 0.5 g/kg) failed to change body weight gain, food and water consumption.Organ/weight ratios, haematological and biochemical indices analysis showed no significant differences (P>0.05) in any of the parameters examined. Nothing pathological changes was observed in slice. These results clearly suggest that recombinant Saccharomyces cerevisiae expressing salmon calcitonin no negative influences on the animal, it is safe and nontoxic.