کلون و بیان فوتوپروتئین اکورین با استفاده از دنباله اینتئین

Aim:the aim of this study is cloning and expression of aequarin photoprotein associated with intein sequence in order to increase the efficiency of one-step purification ,a low cost and non enzymatic procedure (to separate the intein sequence ). Methods: aequarin coding gene that was cloned in pET21-a vector ,amplified with specific primers and PCR.The PCR product and Ptby21 vector (which have intein sequence )digested with specific restiriction enzymes and then the sticky vector and PCR product ligated by T4 ligase enzyme .cloning is confirmed by double digestion and cloning PCR methods. The cloned vector was transformed in the ER2566 expression strain.cloning accuracy was confirmed by electrophoresis and western blotting. Result: PCR product electrophoresis and double digestion showed cloned vector and presence of 625 base pair fragment.SDS-PAGE analysis revealing a 85 KDa protein band ,which includes chitin binding domain sequence (5KDa),intein (55 KDa),and aequarin photoprotein (25KDa).therefore aequarin in order to better purification were cloned and expressed successfully.

کلیدواژه ها:

intein ، Aequarin ، Cloning ، expression ، purification