中文摘要：

目的：克隆水稻YTB osvdac5基因,原核表达后获得纯化的OSVDAC5蛋白,制备相应的抗体。方法：采用Trizol法提取水稻总mRNA,反转录为cDNA,通过PCR扩增得到该基因与原核表达载体连接,构建重组质粒pET-30a-osvdac5,并转入大肠杆菌进行原核表达,SDS-PAGE检测表达产物。通过镍柱纯化获得的单一目的蛋白用于抗体制备,用Western Blot检测抗体的特异性。结果：克隆到原核表达载体中osvdac5基因的ORF为813 bp,编码271个氨基酸。在大肠杆菌中15℃、0.7mmol/L的IPTG浓度诱导17 h是pET-30a-osvdac5融合蛋白表达的优选条件,表达的OSVDAC5蛋白属于包涵体蛋白。镍柱纯化后的OSVDAC5为30 kD左右的单一条带。Western Blot分析表明,抗体能够与30 kD处的OSVDAC5蛋白进行特异性结合。结论：成功克隆了水稻YTB osvdac5基因,原核表达蛋白OSVDAC5制备的多克隆抗体具有一定特异性,能与免疫抗原结合,这为进一步研究OSVDAC5蛋白在植物不同生长发育时期中的表达模式奠定了基础。

英文摘要：

Objective： To clone osvdac5 gene from rice YTB and expression in E.coli,and prepare the polyclonal antibody with the purified OSVDAC5 protein.Method：The total RNA was extracted from YTB seedlings with Trizol,and the cDNA was reverse transcripte.The osvdac5 gene ORF amplified through PCR was linked with pET-30a to construct the recombinant plasmid pET-30a-osvdac5.The OSVDAC5 protein was expressed induced with IPTG in E.coli strain BL21,and checked by SDS-PAGE.The purified OSVDAC5 protein through Nickel column was used to prepare the polyclonal antibody.The specificity of the polyclonal antibody for OSVDAC5 was checked by Western Blot.Result：The length of osvdac5 gene ORF cloned into the prokaryotic expression vector was 813 bp,which containing 271aa.In E.coli strain BL21 the optimal condition for OSVDAC5 protein expression was under 0.7mmol/L IPTG at 15℃ with 17 hours.The OSVDAC5 fusion protein existed as inelusion body,insoluble form.The purified OSVDAC5 protein was a single band with about 30 kD.The results from Western Blot analysis showed that the polyclonal antibody from OSVDAC5 combined specificity with OSVDAC5.Conclusion： Succession of cloning and expression of the osvdac5 gene from YTB.The polyclonal antibody from OSVDAC5 showed definite specificity with OSVDAC5 protein.These laid a foundation for studying the expression mode of OSVDAC5 during different growth and development stage in plant.