病程相关蛋白-1a多克隆抗体的制备与应用  

The Preparation and Application of Polyclonal Antibody with a Peptide PR-1a

作　　者：陈卓[1,2] 刘开兴[1,2] 刘家驹[1,2] 王贞超[1,2] 毕亮[1,2] 李向阳[1,2] 杨松[1,2] 宋宝安[1,2] 

机构地区：[1]贵州大学绿色农药与农业生物工程国家重点实验室培育基地,贵阳550025 [2]贵州大学精细化工研究开发中心,贵阳550025

出　　处：《中国农学通报》2012年第21期174-182,共9页Chinese Agricultural Science Bulletin

基　　金：国家重点基础研究发展"973"计划"病毒及菌害调控的候选药物与分子靶标"(2010CB-126105);农业科技成果转化资金项目"防治稻飞虱及其传媒病毒药剂中试与转化"(2009GB2F200330);贵州省教育厅自然科学研究项目重点项目"新型抗植物病毒药物的化学生物学研究"[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"新型抗植物病毒药物-病毒星与靶标分子的化学生物学研究"[黔省专合字(2009)104号];贵州省科技厅农业攻关项目"新型氨基膦酸酯类抗病毒剂与harpin binding protein的作用机制研究"[黔科合NY字[2011]3052号]

摘　　要：为获得效价高和选择性强的PR-1a抗体。根据NCBI GenBank中普通烟PR-1a一级结构信息,采用Blastn、Blastx和Expasy等软件进行序列同源性分析,获得1段序列特异性多肽,采用9-氟甲氧羰基固相合成法获得目的多肽,采用HPLC和LC-MS测定目的多肽的浓度和分子量。结果表明:目的多肽纯度达93.92%、分子量为2088.17;采用碳化二亚胺法制备获得Pep-KLH,并通过免疫新西兰大白兔获得多克隆抗体,采用间接-ELISA和Western blotting测定其抗体效价和特异性,结果表明:抗血清在1:32000条件下,抗血清的吸光值(OD)约为1.0;经Westernblotting试验表明:抗血清和多克隆抗体可特异性识别烟草叶片组织内的PR-1a;同时,试验采用系统性获得性的免疫激活剂-BTH作用普通烟K-326,对作用后的0、6、12、24、48、76、144、96h的烟草叶片总蛋白进行间接-ELISA,发现:BTH作用普通烟K-326144h后,PR-1a蛋白表达呈上调趋势;同时采用半定量RT-PCR试验也同样证实了BTH可诱导PR-1a基因表达上调,其表达上调趋势与间接-ELISA的研究结果相似。表明采用该方法制备的PR-1a多肽抗体具有较高的特异性和灵敏度,可用于免疫诱导剂等方面的研究。In order to acquire polyclonal antibody of high titer and specificity against PR-1a, 1 polypeptide with sequence-specific were obtained by Blastn, Blastx and Expasy software based on the primary structure of information about PR-1a of tobacco in NCBI GenBank. The polypeptide was synthetized by fmoc solid phase synthesis methods, and its purity and molecular weight were also determined by HPLC and LC-MS, respectively. The purity value and molecular weight reached at 93.2% and 2088.17, respectively. The polypeptide was coupled with keyhole limpet hemocyanin immunizing rabbit with Pep-KLH emulsified by Complete Adjuvant （IFA）, and polyclonal antibody was purified by （KLH） by way of EDC. Anti-sera were acquired by Freund＇s Adjuvant （CFA） and Incomplete Freund＇ s affinity chromatography. The titer and specificity of anti-sera and polyclonal antibody were determined by indirect-ELISA and Western blotting with the OD value reaching at 1.0 at the dilution of 1：32000 against anti-sera. The results from Western blotting showed that,anti-sera and polyclonal antibody could detected the specitic band with molecular weight ol 18 kD in Nicotiana tabacum K-326 leaves, the results was accord with molecular weight which was the predicted. Benzothiadiazole （BTH）, a class of inducers of systemic was sprayed on leaf of Nicotiana tabacum K-326 at the 500 μg/mL, and then the total protein from leaf was extracted at the interval of 0, 6, 12, 24, 48, 96, 144, 192 h. The results indicated that, PR-1a was up-regulated at the 48 h by indirect-ELISA assays. Meanwhile, RT-PCR of semi-quantity was also used to verify the expression trends about PR-1a, the results indicated to become a similar expression trends with that of indirect-ELISA. The results showed the PR-la polyclonal antibody reached high sensitivity and specificity, and was used to the action mechanism of inducer of systemic acquired resistance （SAR）.

关 键 词：病程相关蛋白-1a 序列分析 多肽合成 多克隆抗体 

分 类 号：S1[农业科学—农业基础科学]