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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.
Keywords:
Anti-protease,
CST3, Cysteine proteases, Human chromosome 20, cystatin c
Gene cloning
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