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【目的】制备抗CAS9蛋白质的单克隆抗体,建立转基因水稻中CAS9蛋白质的免疫印迹检测方法,了解CAS9蛋白质在转基因水稻中的表达特征。【方法】以带Cas9的质粒DNA为模板,PCR扩增Cas9 5′端810 bp片段后克隆到p ET30a载体中,将酶切验证且序列正确的重组质粒转入表达菌Condon Plus中,经IPTG诱导表达CAS9蛋白质(N端270氨基酸),用纯化的重组蛋白质作为免疫原免疫小鼠,制备单克隆抗体,用免疫印迹分析筛选特异性和灵敏度高的抗体细胞株。用特异性引物PCR扩增转基因水稻的Cas9,用免疫印迹检测转基因水稻中的CAS9蛋白质。将重组CAS9蛋白质和带有CAS9的水稻苗期叶片蛋白质进行平行免疫印迹分析,用Image J软件采集信号绘制CAS9蛋白质的标准曲线,进而对水稻叶片中的CAS9蛋白质进行定量分析。提取单粒稻米的总蛋白质,稀释后用免疫印迹分析CAS9蛋白质的检测下限,提取多个时期、部位的水稻材料的总蛋白质,包括苗期的地上部和地下部,分蘖期的茎、茎节、叶鞘、叶片等,用SDS-PAGE分离后免疫印迹检测比较CAS9蛋白质的表达特征。【结果】通过体外克隆、诱导表达获得了纯化的重组CAS9蛋白质N端片段,免疫小鼠后得到42株阳性杂交瘤细胞株,经筛选鉴定到#12D2细胞株对水稻样品的检测具有较好的特异性和灵敏度。用该抗体通过免疫印迹检测了转基因水稻,所建立的免疫印迹方法对重组CAS9蛋白质的检测下限约为0.25 ng。在水稻苗期叶片中,CAS9蛋白质约占鲜重的0.00005%,可检出单粒稻米8%样品(约2 mg)中的CAS9蛋白质。在苗期地上部CAS9蛋白质的表达丰度高于地下部,分蘖期茎和叶片中表达量较高,根和叶鞘表达量较低。【结论】获得特异性强、灵敏度高的抗CAS9单克隆抗体,建立了检测转基因水稻中CAS9蛋白质的免疫印记方法,揭示了CAS9蛋白质在水稻不同部位的表达特征,并展示了在其他植物中的应用潜力。
【Objective】 To prepare a monoclonal antibody against CAS9 protein and establish a western blot assay for the CAS9 protein in transgenic rice to understand the expression characteristics of CAS9 protein in transgenic rice. 【Method】 The 810 bp fragment of 5 ’end of Cas9 was amplified by PCR using the plasmid DNA with Cas9 as a template. The fragment was cloned into p ET30a vector. The recombinant plasmids verified by restriction endonucleases and the correct sequence were transferred into Condon Plus and expressed by IPTG The CAS9 protein (N-terminal 270 amino acids) was induced to express and the purified recombinant protein was used as an immunogen to immunize mice to prepare monoclonal antibodies. The specific and sensitive antibody cell lines were screened by immunoblotting. The specific primers were used to amplify Cas9 of transgenic rice and the CAS9 protein in transgenic rice was detected by Western blotting. Western blot analysis of recombinant CAS9 protein and rice leaf protein with CAS9 was carried out. The standard curve of CAS9 protein was acquired by Image J software and the CAS9 protein in rice leaves was quantitatively analyzed. The total protein of single grain of rice was extracted, and the lower limit of detection of CAS9 protein was analyzed by Western blotting. The total protein of rice material at different time and place was extracted, including shoot and shoot at seedling stage, , Leaf sheaths, leaves and so on. SDS-PAGE was used to detect the expression characteristics of CAS9 protein by Western blot. 【Result】 N-terminal fragment of purified recombinant CAS9 protein was obtained by in vitro cloning and induced expression. After being immunized, 42 positive hybridoma cell lines were obtained. Screening showed that the # 12D2 cell line had good detection on rice samples Specificity and sensitivity. Transgenic rice was detected by immunoblotting using this antibody. The lower limit of detection of recombinant CAS9 protein by the established Western blot was about 0.25 ng. CAS9 protein accounted for about 0.00005% of the fresh weight at the seedling stage of rice, and CAS9 protein in 8% of single grain rice (about 2 mg) was detected. At the seedling stage, the expression level of CAS9 protein was higher than that in the lower part. The expression level of CAS9 protein was higher in stems and leaves at the tillering stage, and lower in the roots and sheaths. 【Conclusion】 We obtained the anti-CAS9 monoclonal antibody with high specificity and sensitivity and established the western blot method to detect the CAS9 protein in transgenic rice, revealing the expression characteristics of CAS9 protein in different parts of rice and showed that in other plants Application potential.