转基因水稻中CAS9蛋白质的免疫印迹检测

发布时间：2019-06-11 16:28

【摘要】：【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。
[Abstract]:[objective] to prepare monoclonal antibody against CAS9 protein and establish an immunoblotting method for detection of CAS9 protein in transgenic rice to understand the expression characteristics of CAS9 protein in transgenic rice. [methods] using plasmid DNA with Cas9 as template, the fragment of Cas9 5 'end 810 bp was amplified by PCR and cloned into p ET30a vector, and the recombinant plasmid confirmed by restriction enzyme digestion and with correct sequence was transferred into the expression strain Condon Plus. The expression of CAS9 protein (N-terminal 270amino acid) was induced by IPTG. The purified recombinant protein was used as immunogen to immunize mice, and monoclonal antibodies were prepared. The specific and sensitive antibody cell lines were screened by immunoblotting. The Cas9, of transgenic rice was amplified by specific primers PCR and the CAS9 protein in transgenic rice was detected by immunoblotting. The recombinant CAS9 protein and the protein of rice seedling leaves with CAS9 were analyzed by parallel immunoblotting. The standard curve of CAS9 protein was drawn by Image J software, and then the CAS9 protein in rice leaves was quantitatively analyzed. The total protein of single grain rice was extracted, the detection limit of CAS9 protein was analyzed by immunoblotting after dilution, and the total protein of rice material at several stages was extracted, including shoot and underground part of seedling stage, stem, stem node, leaf sheath and leaf at tiller stage. The expression characteristics of CAS9 protein were detected and compared by immunoblotting after SDS-PAGE separation. [results] the expression characteristics of CAS9 protein were compared by cloning in vitro. The purified N-terminal fragment of recombinant CAS9 protein was induced and 42 positive hybridoma cell lines were obtained after immunization with mice. The # 12D2 cell line was identified as having good specificity and sensitivity for the detection of rice samples. Transgenic rice was detected by immunoblotting with this antibody. The detection limit of recombinant CAS9 protein by the established immunoblotting method was about 0.25 ng.. In the leaves of rice seedling stage, CAS9 protein accounted for about 0.00005% of the fresh weight, and the CAS9 protein in 8% of the single grain rice sample (about 2 mg) could be detected. The expression abundance of CAS9 protein in shoot was higher than that in underground part at seedling stage, and the expression level in stem and leaf at tiller stage was higher than that in root and leaf sheath. [conclusion] the monoclonal antibody against CAS9 with strong specificity and high sensitivity was obtained. The immunoblotting method for detection of CAS9 protein in transgenic rice revealed the expression characteristics of CAS9 protein in different parts of rice and showed its application potential in other plants.
【作者单位】：河北农业大学生命科学学院;北京华大蛋白质研发中心有限公司;
【基金】：高等学校博士学科点专项科研基金(20131302110006)
【分类号】：S511

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