Construction Of Saccharomyces Cerevisiae Expressing Salmon Calcitonin And Relative Research

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. Calcitonin is a 32-amino-acid-long peptide with an N-terminal disulfide bridge and a C-terminal polyamide residue. 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. To study the oral delivery of salmon calcitonin and to low the price of it, yeast yAGA2-sCT was made with the enterokinase site on it. The bioactivity of yAGA2-sCT was investigated by cell experiment and animal experiment.At first, the salmon calcitonin gene was composed through chemical synthesis and protease approach according to the bias codon of yeast. The production was tested by electrophoresis. The synthetic salmon calcitonin gene was clone to puc18 vector. The forecast of recombinant protein is same to the nature salmon calcitonin. Then, the gene was clone to the vector p-sf to yield p-sct-sf. Plasmid p-sf was modified from pyd1 (INVITROGEN) vector. URA3 gene was added to the two flank of the expression frame as recombinant site. Plasmid p-sf express AGA2 protein, which linked to AGA1 protein on the surface of yeast. By this way, foreign could be expressed on the surface of yeast. Transformant yAGA2-sCT was got by transform p-sct-sf into EBY100. Transformant yAGA2-V5 was also got by transform p-sf into EBY100 to be the control in followed experiment. By indirectly labelled with FITC, yAGA2-V5 and yAGA2-sCT was found to express foreign protein under UV light. Recombinant salmon calcitonin expression was detected by flow cytometer. After 12 hours' induction, 65 % of yAGA2-sCT cell and 52 % of yAGA2-V5 cell began to express salmon calcitonin. Yeast transformants were cut by enterokinase, the solution was short for EKS.Osteoclast was used in Cell experiment. As the final differentiation cell, osteoclast is of the potent of bone resorption. At first, the bone cutter was used to make bovine bone wafers. The yAGA2-V5 EKS or yAGA2-sCT EKS was added to the cell solution with bone wafer in it. Miacalcic (salmon calcitonin injection) was used as positive control. Area of bone lacuna was calculated after a week. The yAGA2-sCT EKS inhibited the formation of bone lacuna, which suggested the recombinant salmon calcitonin inhibited the activity of osteoclast.Animal experiment was conducted according to the England pharmacopoeia. The animal used here is a normal female Wistar rat. There are three kinds of group in the experiment. The negative group: orally administered with 5g/kg yAGA2-V5; the positive group: injected 200mIU/kg Miacalcic; The dosage groups (orally administered 0.1 g/kg, 0.5 g/kg, 5 g/kg yAGA2-sCT). Rats' serum was got from eyes in different time(1, 2, 4, 6, 8, 10 hours)and sent to Navy 401 Hospital of PLA to test. The result is, oral administration of 5g/kg yAGA2-sCT decrease the serum calcium significantly to 2.355±0.012mM(p<0.01).The Tmax is the second hour. The dosage effect and the long-term hypocalcemic effect are found. The long-term hypocalcemic effect of yAGA2-sCT is important to hypercalcemia. Hypercalcemia is a kind of complications with cancer. About 10% advanced cancer suffers have hypercalcemia, which was a deadly factor. For treating hypercalcemia, sCT has been replaced by bisphosphonates in view of their long-term effect. In contrast to bisphosphonates, sCT remains as an alternative in treating hypercalcemia for relieving of osteogenic pain, in particular, when safety considerations have become important.To investigate the effect of yAGA2-sCT to hypercalcemia, the hypercalcemic rats model was made. The female Wistar rats were fed with 1, 25-dihydroxy-cholecalciferol for a week according to the reference. The serum calcium raised from 2.638±0.017 mM to 2.838±0.022 mM, suggested the formation of hypercalcemia. In this experiment, rats was divided to negative group (orally administered with 5g/kg yAGA2-V5), positive group(injected 1.25 mg/kg pamidronate) and dosage group(orally administered 0.1g/kg,0.5g/kg,5g/kg yAGA2-sCT). Serum calcium was got from eyes in 12 hours and sent to Navy 401 Hospital of PLA to test. It was found that oral adminstration of 5g/kg yAGA2-sCT decrease the serum calcium significantly to 2.69±0.025 mM(p<0.01)and oral administration of 0.5g/kg yAGA2-sCT decrease the serum calcium significantly to 2.75±0.016 mM(p<0.05). It suggested the transformant yAGA2-sCT could inhibit hypercalcimia in rats.The safety of transformant was investigated. In acute toxicity experiment, the LD50 could not be finding. In subacute toxicity experiment, the change of rats' weight, the physiologic index, the biochemical criterion and the organ coefficient was investigated. None of this changed significantly. It suggested that the transformant yAGA2-sCT is safe to rats. These results showed, recombinant S. cerevisiae could thus be considered as a new drug delivery system for salmon calcitonin in hypercalcemia therapy. The salmon calcitonin could thus use more popularly in hypercalcimia.