水稻Osε-cop1基因的原核表达及多克隆抗体制备

Prokaryotic Expression and Polyclonal Antibody Preparation of Osε-cop1 in Rice (Oryza sativa L.)

ε-COP蛋白是真核生物分泌途径中COPⅠ有被小泡的一个亚基。本研究利用PCR技术扩增水稻ε-cop基因(Osε-cop1)的ORF(开放阅读框),并克隆到原核表达载体pET-23d上,将表达载体pET-Osε-cop1转入大肠杆菌BL21(DE3),以1.0mmol/L的IPTG(isopropyl β-D-thiogalactoside)诱导表达重组蛋白,然后以重组蛋白作为抗原免疫家兔,制备多克隆抗体。SDS-PAGE电泳分析结果表明,成功诱导表达了分子量约为35kD的重组蛋白,Western blot检测表明,免疫家兔的抗血清与水稻幼穗总蛋白杂交信号较好。Osε-COP1抗体的制备有助于研究该基因及COPI小泡在水稻中的功能。

The ε-COP is one of the subunits of COP [ vesicle in secret pathway of eukaryotic cells. The ORF （open reading frame） orε-cop gene in rice （Ose-copl） was obtained by PCR and cloned into the prokaryotic expression vector pET-23d. The resulting plasmid was then introduced into E. coli strain BL21 （DE3） and induced to ex- press the recombinant Oss-COPI protein by 1.0 mmol/L isopropyl [3-D-thiogalactoside （IPTG）. Afterwards, the re- combinant Osε-COP1 protein was used as antigen to immune rabbits. The results demonstrated a 35 kD protein was expressed successfully inducing by IPTG. Western blot analysis showed that the antiserum immunological recog- nized rice Osε-COP1 protein with high specificity, indicating that the polyclonal antibody was prepared successful- ly. This work would contribute to study the function ofOsε-copl gene and COP I vesicle in rice.