Cloning And Expression Of The Human Calcitonin

Calcitionin is an important hormone, which excretes from thyroid C cell. Calcitionin can be used to cure many diseases, including osteoporosis and so on. The applications of calcitionin to clinical treatment are extensive because its tiny secondary effect and conspicuous therapeutic effects. Calcitionin has been ratified to go on the market by FDA.Taking calcitionin from zoogenic tissues by pheresis restricts its production on account of the limited source of the zoogenic tissues. Otherwise by the use of solid phase and liquid phase methods to synthesis calcitionin has many weaknesses such as too much steps, low purities and pick-up rates, which can lead to high price. We can obtain calcitionin with great yield, high activity, long half life and low-grade antigenicity to supply for clinical application, by the means of bioengineering.In this study, according to the favors of the Bacillus coli codon, we designed the whole gene orders of human calcitionin and synthesised this sequence by chemosynthesis methods. Then we cloned it into pET-28a (+).After getting the correct sequence, we cloned it into pET-32a (+).It expressed soluble protein detected by SDS-PAGE.We purified fusion human calcitionin from supernatant of E. coli ultrasonic fragmentation by means of metal chelated chromatogram and gel filtration.Then fusion human calcitionin was cleaved by chemical method,and human calcitionin was purified by means of metal chelated chromatogram and ion exchanged chromatogram.The character of recombinant human calcitionin is closed to natived human calcitionin,which was conformed by SDS-PAGE and isoelectric focusing.We amidated human calcitionin by amidating enzyme which was purified by our laboratory,and purified amidated human calcitionin from amidating system by means of gel filtration.The result of amidating was conformed by RP-HPLC.In this study, an idea of producting human calcitionin by bioengineering was put forward.The foundation of recombinant human calcitionin being applicated as a kind of remedy at an early date was laid.