Recombination, Expression And Purification Of Hepatocellular Carcinoma-Associated Antigen Kinectin

Objective: To study a method to recombinate and express the gene segment of HCC-associated antigen-kinectin in vitro. Find out the suitable conditions under which the fusion protein can be expressed most efficiently. Then provide a basis for the production and application of this antigen.Methods: The gene segment of Kinectin was amplified by PCR (Polymerase Chain Reaction), and then inserted into the vector pMAL-C2 at the downstream of the gene encoding the MBP protein. The recombinant vector was transfected into E.coli DH5.The blue-white selection assay, PCR, incision enzyme assay and DNA sequencing were performed to find out the right recombinant clone. The right clone was transfected into the expression host (E.coli TB1), and the engineering bacterias were induced by IPTG in different conditions,such as different induction time,different induction temperature,etc.The products was analyzed throught the SDS-PAGE methods to find out the best induction conditions under which the engineering bacterias can obtain the highest expression efficiency.After the induction bacterias broken by ultrasound, purifing the supernatant throught the Amylose-resin column, and find out the better purification conditions to obtain the more purity MBP-kinectin.Result:The gene segment of kinectin have been successfully inserted into the vector pMAL-C2 at the multiple clone site, and the interest protein was expressed when the engineering bacterias induced by IPTG. When the engineering bacterias induced in 34℃,the production of the fusion protein is highter and form less inclusion body than in 37℃. After the IPTG is added,the production of the fusion protein increase in the first 3 hours,but drop after it. The...