Cell Study Of Calcitonin Gene Transfer

With the world population become aged, osteoporosis have been seen one of the hot interesting of whole globe public sanitation. Osteoporosis is charactered by low bone density and microstructure destroy of bone tissue, thus increased the bone brittleness and fracture danger. It have high mortality and invalid rate, so, it is a serious dangerous common disease for the health of old man.The basic and deposit calcitonin (CT) plasma concentration level is decreased among the primary osteoporosis. CT is a natural peptide and one of the most effective drugs that can cure the osteoporosis at present. Now, CT, which we have used to treat the osteoporosis , is extracted and purified from other animals. The feeble antigen, high price, finitied resource have limited it to be used for long time in vivo.Gene therapy is a technique for correcting defective genes responsible for disease development. Osteoporosis disease is related with many genes .Up to now, there is no article which is care about hCT as the gene therapy target gene. There are few researchers who studies in osteoporosis gene therapy field. If we could take advantage of the gene therapy technique to treat the osteoporosis disease, we can get great economic and social benefit.We construct a CT expression vector by using gene engineering, then transfect the target cell-NIH3T3 with lipofectamine?2000. At last, we have got the cell clone of CT persistent high expression.Materials and Methods1. Obtain the target gene and clone of calcitonin and murine Ig kappa signal peptide (MIKSP): A gene coding for human calcitonin (hCT) and murine Ig kappa signal peptide (MIKSP) was divided into six fragments, then synthesize it by changing template PCR technique and cloned it into pMD18-T vector. The hCT and MIKSP gene was assembled through three round of PCR from a total of only six 40-60bp oligos . The synthesized gene was inserted into pMD18-T vector , and then transferred into E.coli JM109 .Finally , the nucleotide sequence of synthesized hCT and MIKSP gene was determined with double enzyme digestion and the DNA sequencing. The recombined plasmid was named pMD18-T-hCT.2. To construct the human calcitonin and secretory eukaryotic expression Vector containing MIKSP gene: The target segment was digested from pMD18-T-hCT with Hindlll and Xba I restriction enzymes, and ligated into pcDNA3.0 pre-cut with Hindlll and Xba I enzymes. Double enzyme digestion confirmed the recombined plasmid, and the recombined plasmid was named pcDNA3.0-hCT.3. The secretion and expression in the NIH3T3 cell of the human Calcitonin: (l)The establishment and cultivation of positive cell clones: We have transfected theNIH3T3 cell with pcDNA3.0-hCT and pcDNA3.0 respectively by lipofectamine?2000 and selected the positive cell line by G418 .After 2 weeks, we harvest the cell and keep to cultivate it for 4 weeks, then reproduce it as usual.(2) Identification of positive cell clones: (l)RT-PCR: After cultivate the positive cells 4 weeks, we harvest the cell and extract the total RNA to appraise the expression from mRNA level by RT-PCR. ?Radioimmunity test: We appraise the expression and expression rate of CT by measurement the CT concentration in the culture medium.Results:1. The result indicates that the nucleotide sequence of the hCT and MIKSP gene is the same as the sequence designed. Human calcitonin and MIKSP gene was cloned. And for the further study, a stop codon (TAA) was introduced to the expected 3' terminus before the endonuclease cut site of the inserted gene.2. The target segment was digested from pMD18-T-hCT with Hindlll and Xba I restriction enzymes, and ligated into pcDNA3.0 pre-cut with Hindlll and Xba I enzymes. Double nzyme digestion confirmed the recombined plasmid, and the recombined plasmid was named pcDNA3.0-hCT.3 The secretion and expression in the NIH3T3 cell of the human Calcitonin: After cultivate the vector cell line 4 weeks, we harvest the cell and extract the total RNA .We have got the 212bp target gene fragment by two steps RT-PCR. We have measured the CT concentration of culture medium by radioimmunity test. The results showed that, the concentration of hCT in the positive cell culture medium is remarkable higher than other groops.Conclusions:1. The result demonstrates that a reliable method of synthesis of oligo-peptide gene (or cDNA) and cloning is provided;2. We have successfully cloned the human Calcitonin and MIKSP Gene;3. The secretory eukaryotic expression vector pcDNA3.0-hCT was screened successfully;4. We have got the positive cell clone of CT persistent high expression;5. This research is a basic work for the further study on hCT-associated disease .