摘要
根据NCBI GenBank中报道的NPR1一级结构信息,采用Blastn、Blastx、ExPASy和Protean等软件进行序列同源性和抗原性指数分析,获得三段序列特异性较高的多肽,并从中优选一段序列特异性多肽,采用9-氟甲氧羰基固相合成法获得序列特异性最好的多肽,采用HPLC和LC-MS测定合成多肽的浓度和分子量,试验表明目的多肽纯度达88%、目的多肽分子量为1.92234 kD。采用碳化二亚胺法将多肽与KLH进行偶联获得免疫原Pep-KLH,并将其免疫新西兰大白兔以获得抗血清和多克隆抗体,采用ELISA和Western blotting测定其效价和特异性,经ELISA检测表明抗血清和多克隆抗体可与Pep发生特异性免疫反应,经Western blotting试验表明抗血清和多克隆抗体可识别烟草叶片特异性条带,其相对分子量为65 kD,与预测分子量相符,表明利用该方法制备的NPR1多肽抗体具有较高特异性和灵敏度。
According to the primary structure of information about NPR1 in NCBI GenBank,3 polypeptides with sequence-specific were obtained using Blastn and Blastx software.One polypeptide was synthetized by fmoc solid phase synthesis methods,and determined theirs purity and molecular weight using HPLC and LC-MS with purity value reaching at 88% and molecular weight being at 1.92234 kD.The polypeptide was coupled to keyhole limpet hemocyanin（KLH） to form a complex of Pep-KLH by EDC.Anti-sera were acquired by immunizing rabbit with Pep-KLH emulsified by complete freund＇s adjuvant（CFA） and incomplete freund＇s adjuvant（IFA）,and polyclonal antibody was purified by affinity chromatography.The titer and specificity of anti-sera and polyclonal antibody were determined by ELISA and Western blotting.The results showed that anti-sera and polyclonal antibody reacted with Pep-KLH and detected a specific band of 65 kD,and the size was agreed with the predicted molecular mass.The NPR1 polyclonal antibody revealed high sensitivity and specificity.
出处
《生物技术通报》
CAS
CSCD
北大核心
2012年第1期145-150,共6页
Biotechnology Bulletin
基金
贵州省教育厅自然科学研究项目重点项目[黔科教(2009)0132号]
贵州省优秀科技教育人才省长专项资金项目[黔省专合字(2009)104号]
农业科技成果转化资金项目(2009GB2F200330)
科技人员服务企业行动项目(2009GJF20047)
贵州省科技厅农业攻关项目(黔科合NY字[2011]3052号)
关键词
非表达型病程相关蛋白
序列分析
多肽
合成
多克隆抗体
Nonexpressor of pathogenesis-related genes 1（NPR1） Sequence analysis Polypeptide synthesizing Polyclonal antibody
