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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。
OBJECTIVE: To clone the gene of rice YTB osvdac5, obtain the purified OSVDAC5 protein after prokaryotic expression, and prepare the corresponding antibody. Methods: The total mRNA of rice was extracted by Trizol method and reverse transcribed into cDNA. The recombinant plasmid pET-30a-osvdac5 was constructed by PCR and cloned into prokaryotic expression vector. The recombinant plasmid pET-30a-osvdac5 was transformed into E. coli for prokaryotic expression. The expression product is detected. Single target protein purified by nickel column was used for antibody preparation, and the specificity of the antibody was detected by Western Blot. Results: The ORF of osvdac5 gene cloned into prokaryotic expression vector was 813 bp, encoding 271 amino acids. The optimal conditions for the expression of pET-30a-osvdac5 fusion protein at 17 ℃ for 17 h induced by IPTG at 0.7 mmol / L in E. coli were expressed as inclusion protein. The nickel column purified OSVDAC5 is a single band around 30 kD. Western Blot analysis showed that the antibody specifically binds to the OSVDAC5 protein at 30 kD. CONCLUSION: The rice YTB osvdac5 gene has been cloned successfully. The polyclonal antibody of prokaryotic expression protein OSVDAC5 has certain specificity and can bind with immune antigen, which lays the foundation for further study on the expression pattern of OSVDAC5 protein in different plant growth and development stages.