Ali Karami, Darioush Ghasemi, Zahra Safiari

Standard Scientific Research and Essays 10/2013; 10:216-220

ABSTRACT Background: Cystatines have regulative and preventive roles on Cysteine Proteases which existing in all biologic fluids of the human body. The protein-coding genes in humans and mice are located on chromosomes 20. The purpose of the present study is cloning and sequencing of hman Cystatin Gene of human normal tissues isolated from Iranian samples for future expression and production of recombinant protein to design of ELISA kit for cystatin detection in patients as a substitute for current creatinin. Methods: In this research, total RNA was extracted from human cord, thyroid, breast and blood tissues and then cDNA was synthesized by RT-PCR method. The resulted fragment was cloned in pET28a vector and transformed into the E.coli DE3-BL21 and finally DNA sequence of the gene was revealed by sequencing and compared with the reference sequence. Result: Amplification of cDNA from different tissue revealed that best result came from new born cord sample comparing to other human tissues like kidney. Cloned gene was verified by restriction digestion, PCR and sequencing. Conclusion: Analysis of sequence from cloned gene revealed that there is only single deletion before primer location with 99% alignment to the reference gene. Amino acid analysis shows that protein is correct and can be used for expression and design of Elisa kit