转基因水稻中CAS9蛋白质的免疫印迹检测  

Western Blot Detection of CAS9 Protein in Transgenic Rice

作　　者：郭亚璐[1] 马晓飞 史佳楠[1] 张柳 张剑硕 黄腾 武鹏程 康昊翔 耿广荟 陈浩 魏健[1] 窦世娟[1] 李莉云[1] 尹长城 刘国振[1] 

机构地区：[1]河北农业大学生命科学学院,河北保定071001 [2]北京华大蛋白质研发中心有限公司,北京101318

出　　处：《中国农业科学》2017年第19期3631-3639,共9页Scientia Agricultura Sinica

基　　金：高等学校博士学科点专项科研基金(20131302110006)

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�【Objective】The objective of this study is to generate monoclonal antibodies against CAS9 protein and establish immunological method for the detection of CAS9 protein in transgenic plants, and to understand the characters of the expression patterns of CAS9 protein in transgenic rice. 【Method】The 5′ fragment（810 bp） of Cas9 was amplified using plasmid DNA confers Cas9 as template. The amplican was cloned into expression vector p ET30 a. Restriction enzyme digestion identified recombinant plasmid were sequencing verified. The recombinant plasmid was transformed into E. coli Condon Plus strain. The induced CAS9 protein was purified and used as immunogene to generate monoclonal antibodies. Positive hybridoma cell lines were identified by western blot analysis. PCR amplifications were carried out to identify positive transgenic rice using specific primers of Cas9. Western blot was carried out to detect CAS9 protein in rice. The recombinant CAS9 protein and protein samples extracted from rice seedling were analyzed in parallel by Western blot, and standard curves were drawn based on Image J software extracted signals. CAS9 protein in rice tissues were analyzed quantitatively based on the standard curve. Total protein of single rice grain was extracted and the sensitivity of CAS9 protein detectable by Western blot was analyzed using diluted samples. The abundance of CAS9 protein in different tissues, including shoot and root at seedling stage, stem, node, sheath and leaf at tillering stage, were compared by Western blot. 【Result】The Cas9 was cloned and the plasmid was transformed into E. coli. Recombinant N-terminal portion of CAS9 protein was obtained and used as immunogen to inject mice. Forty-two positive hybridoma cell lines were obtained after immunization. Among them, cell line #12D2 showed higher specificity and sensitivity for the detection of CAS9 protein in rice tissues. Western blot analysis was carried out for the detection of transgenic rice via the antibody of #12D2-derived hybridoma cell

关 键 词：水稻 转基因植物 CAS9蛋白质 单克隆抗体 免疫印迹 

分 类 号：S511[农业科学—作物学]