Abstract: Skp1 is a key protein of ubiquitin E3 ligase SCF complex. In order to further identify Skp1 function, the open reading frame of Skpl gene which is composed of 468 nucleotide in length encoding 155 amino acids was amplified by PCR using PGADT7-Skp1 as template, and cloned into expression vector pET-28a to generate recombinant plasmid pET Skp1. After verification by colony PCR and sequencing, the recombinant plasmid was transformed into Escherichia coli strain BL21 for protein expression. SDS-PAGE analyses showed that under the condition of 37℃, Skp1 protein was efficiently expressed after IPTG treatment for 3, 6, and 9 h respectively. Treatment for 6,9 h showed no significant impact on the expression of Skp1, thereby treatment of 3 h could be used for further protein expression. The expressed Skp1 protein was purified and used for producing the antiserum in rabbit. After collecting the fifth anti-serum the sensitivity of the anti-serum was analyzed using ELISA assay and showed the sensitivity between 1:128 000-512 000. Western Blot analysis was further used to detect Skp1 protein using total protein extracted from cucumber leafs, and a specific band at 20 kDa was detected which was corresponding to the expected size of Skp1.

Key words: Cucumber, Skp1, Prokaryotic expression, Antiserum

CLC Number: 

• Q78

• S642.03