黄瓜动蛋白特异肽段的原核表达和多克隆抗体制备

 以黄瓜果实总RNA为模板，通过RT-PCR获得了动蛋白基因CsKF1和CsKF2的cDNA序列，进行测序，构建T-easy载体。SMART在线预测CsKF1和CsKF2均含有动蛋白（Kinesin）约340个氨基酸残基的马达区保守序列（moter domain）。体外表达蛋白的马达区和非马达区，将带有His标签的CsKF1-N、CsKF2-C、CsKF1-M、CsKF2-M融合蛋白通过E. coli BL21（DE3）表达，摸索不同诱导条件下目的蛋白的表达和纯化。其中His-CsKF1-N、His-CsKF2-C、His-CsKF1-M原核表达蛋白的分子量分别为25、30和40 kD，均以0.1 mmol · L^-1 IPTG，22 ℃ 6 h诱导表达效果最好。His-CsKF2-M原核表达蛋白的分子量约38 kD，以18 ℃ 8 h诱导表达效果最好。融合蛋白His-CsKF1-N和His-CsKF2-C经过亲和纯化后作为抗原，通过5轮免疫注射兔子后，取心脏血作为抗血清，通过AminoLink Plus kit试剂盒亲和纯化得到多克隆抗体anti-CsKF1-N和anti-CsKF2-C。经过Western blot分析表明多克隆抗体具有特异性，可与抗原特异结合。

Prokaryotic Expression and Polyclonal Antibody Preparation of Kinesin Peptide Segment from Cucumber

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.