关键词: 黄瓜, 果实, 动蛋白, 肽段, 原核表达

Abstract: Using the total RNA of cucumber（Cucumis sativus L.）fruits as the template，we cloned two novel Kinesin genes CsKF1 and CsKF2 by RT-PCR. The results from deduced protein sequence analysis indicated that both CsKF1 and CsKF2contain moter domain of kinesin family. For protein expression in vitro，the coding regions for KF1-N，KF2-C，KF1-motor and KF2-motor were amplified by PCR and inserted into either the pET28a vector or the pET30a vector and then transformed into E. coli strain BL21（DE3）. SDS-PAGE analysis results showed that the recombinant plasmid His-CsKF1-N and His-CsKF1-N，His-CsKF2-C，His-CsKF1-M could be expressed a 25 kD，30 kD，40 kD molecule mass of fusion protein with 0.1 mmol · L^-1 isopropyl thiogalactoside for 6 h at 22 ℃. But His-CsKF2-M could be expressed a 38 kD molecule mass of fusion protein with 0.1 mmol · L^-1 isopropyl thiogalactoside for 8 h at 18 ℃. The His-tagged fusion proteins were purified using a Ni²⁺-chelating Sepharose Fast Flow（Amersham Biosciences）column. Polyclonal anti-CsKF1-N，anti-CsKF2-C antibodies were raised in rabbits using the purified His-CsKF1-N and His-CsKF2-C protein as the antigens. Antiserum was then affinity purified using the AminoLink Plus kit with immobilized antigens according to the manufacturer’s instructions. Western blot analysis show that the polyclonal antibodies could specifically identify the corresponding antigen peptides His-CsKF1-N，His-CsKF2-C.

Key words: cucumber, fruit, Kinesin, peptide segment, prokaryotic expression