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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (21): 174-182.doi: 10.11924/j.issn.1000-6850.2011-3598

所属专题： 生物技术

• 生物技术科学 • 上一篇    下一篇

病程相关蛋白-1a多克隆抗体的制备与应用

陈卓 刘开兴 刘家驹 王贞超 毕亮 李向阳 杨松 宋宝安   

  • 收稿日期:2011-12-02 修回日期:2011-12-20 出版日期:2012-07-25 发布日期:2012-07-25
  • 基金资助:

    国家重点基础研究发展计划（973 计划）“病毒及菌害调控的候选药物与分子靶标”;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”;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”;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”;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

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

  • Received:2011-12-02 Revised:2011-12-20 Online:2012-07-25 Published:2012-07-25

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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。试验表明：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 blot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Western blot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、96 h的烟草叶片总蛋白进行间接-ELISA，发现：BTH作用普通烟K-326 144 h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。

关键词: 理化性状, 理化性状

Abstract:

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 (KLH) by way of 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 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 specific band with molecular weight of 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 and 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 shown the PR-1a polyclonal antibody reached high sensitivity and specificity, and was used to the action mechanism of inducer of Systemic acquired resistance (SAR).