EDS1多肽抗体的制备和应用

Preparation and Application of Polyclonal Antibody with a Peptide EDS1

EDS1蛋白是植物寄主SA信号转导通路中具有重要调控功能的蛋白,为获得效价高和选择性强的EDS1抗体,根据NCBI GenBank中报道的EDS1蛋白一级结构信息,采用Blastn、Blastx和ExPASy等生物信息学软件进行序列同源性分析,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GC-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89.37%,目的多肽分子量为1978.33。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,采用间接-ELISA和Western blotting测定其效价和特异性,经间接-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70kD,与预测分子量相符,表明利用该方法制备的EDS1多肽抗体具有较高特异性和灵敏度。

EDS1 is an important protein with regulation function at SA signal transduction pathway at plant host.In order to acquire polyclonal antibody of high titer and specificity against enhanced disease susceptibility 1（EDS1） ,according to the primary structure of information about EDS1 in NCBI GenBank,3 polypeptides with sequence-specific was obtained by using bioinformatics software contained Blastn,Blastx and Expasy.One polypeptide was synthetized by fmoc solid phase synthesis methods,and determined theirs purity and molecular weight by using HPLC and GC-MS,purity value reaching at 89.37%and molecular weight being at 1978.33.The polypeptide was coupled to keyhole limpet hemocyanin（KLH） to form a complex of Pep-KLH by us 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 Indirect-ELISA and Western blotting.The results shown that anti-sera and polyclonal antibody reacted with Pep-KLH and detected a specific band of 70 kD,and the size was agreed with the predicted molecular mass.The EDS1 polyclonal antibody revealed high sensitivity and specificity.