黑尾叶蝉RNAi途径关键蛋白AGO2多克隆抗体制备及应用

Preparation and application of polyclonal antibody of key factor AGO2 in RNAi pathway of Nephotetlix cincticeps

【目的】制备效价高且特异性强的黑尾叶蝉RNAi途径关键蛋白Argonaute 2(AGO2)多克隆抗体,为进一步研究AGO2蛋白在叶蝉RNAi免疫途径中的调控功能及作用机制提供技术支持。【方法】通过RT-PCR从黑尾叶蝉基因组扩增出AGO2基因的功能片段,与原核表达质粒pET-28a重组构建重组质粒,然后转化大肠杆菌BL21感受态细胞进行诱导表达;以纯化的AGO2融合蛋白免疫雄性新西兰大白兔获得高效价的多克隆抗体,并以多克隆抗体为一抗,采用Western blotting检测AGO2蛋白在健康和带毒黑尾叶蝉中的表达情况。【结果】RT-PCR扩增获得的黑尾叶蝉AGO2基因片段为1635 bp,构建的pET-AGO2重组质粒能在BL21感受态细胞中成功表达出AGO2融合蛋白,其最适表达条件:温度28℃,IPTG浓度0.5 mmol/L,诱导时间5 h,摇床转速220 r/min。以纯化AGO2融合蛋白免疫雄性新西兰大白兔获得的多克隆抗体效价为1∶106,抗体浓度约350μg/mL。Western blotting检测结果表明,AGO2蛋白在健康和水稻矮缩病毒(RDV)感染黑尾叶蝉中均有表达,但与健康黑尾叶蝉相比,AGO2蛋白在RDV感染黑尾叶蝉中的表达水平更高,即RDV侵染能提高AGO2蛋白的表达水平。【结论】制备获得的黑尾叶蝉RNAi途径关键蛋白AGO2多克隆抗体具有高纯度和高效价,且特异性强,可用于检测AGO2蛋白在病毒感染黑尾叶蝉中的表达水平,进而揭示AGO2蛋白在介体昆虫RNAi途径中的功能机制。

【Objective】To provide technical support for elucidating further the regulatory function and mechanism of Argonaute2(AGO2)protein in the RNAi immune pathway of Nephotetlix cincticeps,N. cincticeps RNAi pathway key protein AGO2 polyclonal antibody with high titer and strong specificity was prepared.【Method】The AGO2 gene fragment was amplified by RT-PCR from N. cincticeps genome,and recombinant plasmid pET28a-AGO2 was constructed with prokaryotic expression plasmid pET-28a. pET28a-AGO2 was expressed in Escherichia coli BL21 competent cell. The purified AGO2 fusion protein was immunized into New Zealand rabbits to obtain polyclonal antibody with high titer. With the high titer polyclonal antibodies against AGO2,the level of AGO2 expression was confirmed by Western blotting in healthy and infected N. cincticeps.【Result】The N. cincticeps AGO2 fragment with 1635 bp was successfully amplified by RT-PCR,and the reconstructed pET-AGO2 recombinant plasmid could express AGO2 fusion protein in BL21 competent cell. The optimal conditions of AGO2 expression was temperature at 28 ℃,IPTG concentration 0.5 mmol/L,induction time 5 h and rotate speed at 220 r/min. Purified AGO2 fusion protein immunized New Zealand rabbits obtained polyclonal antibody with a titer of 1∶106,and antibody concentration was about 350 μg/mL. Western blotting analysis showed that AGO2 protein was expressed in healthy and rice dwarf virus(RDV)infected N. cincticeps. Compared with healthy N. cincticeps,AGO2 protein expression level in RDV infected N. cincticeps was higher,which indicating that RDV infection could increase the expression level of AGO2 protein.【Conclusion】The prepared N. cincticeps RNAi pathway key protein AGO2 polyclonal antibody has high purification and titer,and its specificity is also strong. It can detect the expression level of AGO2 protein in infected N. cincticeps,and reveal the fuction mechanism of AGO2 protein in insect RNAi pathway.