水稻Argonaute 2蛋白的原核表达与多克隆抗体制备

为了制备水稻Argonaute 2(AGO2)的多克隆抗体,该研究采用RT-PCR扩增OsAGO2蛋白165~401aa片段和440~570aa片段的编码序列,并构建了2个原核表达载体。诱导表达重组蛋白后注射家兔,制备了相应的多克隆抗体,最后利用Western blot初步分析水稻AGO2蛋白的表达模式。结果表明:成功构建2个表达载体,通过诱导获得了分子量约为30kD和23kD的重组蛋白。其中,以440~570aa片段为抗原所制备的多克隆抗体免疫印记效果较好。Western blot表明在水稻花药、愈伤组织及小穗中检测到OsAGO2表达。该研究为进一步深入探讨水稻OsAGO2基因的特性与功能奠定了基础。

Prokaryotic Expression and Polyclonal Antibody Preparation of Argonaute 2 in Rice (Oryza sativa L.)

To prepare the polyclonal antibody of rice Argonaute 2 (OsAGO2),the coding regions of 165~401 aa and 440~570 aa of this protein was amplified by RT-PCR in this study,followed by cloned into the prokaryotic expression vector pET-23d.Afterwards,the recombinant OsAGO2 proteins were expressed and used as antigen to immune rabbits.The expression pattern of OsAGO2 was detected by Western blot analysis using antibodies prepared.The results demonstrated that two prokaryotic expression vectors were obtained,and the 30 kD and 25 kD of recombinant proteins were expressed successfully.The antibody prepared by 440~570 aa region of OsAGO2 showed higher specificity tested by immunoblotting.Western blot analysis showed that the OsAGO2 was expressed in tissues of anther,callus and spikelets.This work would contribute to study the properties and function of OsAGO2 in rice.