抗亨廷顿蛋白相关蛋白1氨基末端和羧基末端多肽抗体的制备

目的探讨抗亨廷顿蛋白相关蛋白1(HAP1)异构体——HAP1A和HAP1B的氨基末端和羧基末端多肽抗体的制备。方法对大鼠脑总RNA进行逆转录多聚酶链反应(RT-PCR)获取HAP1氨基末端多肽(HAP1N)基因片段,并插入载体构建HAP1N原核表达质粒。提取原核表达的谷胱甘肽转硫酶(GST)-HAP1N末端、GST-HAP1AC末端和GST-HAP1BC末端的融合蛋白,免疫豚鼠和兔分别制备抗HAP1N、HAP1A和HAP1B抗体,采用酶联免疫吸附试验(ELISA)、免疫印迹法和免疫组织化学技术对其特异性和效价进行鉴定和检测。结果构建的HAP1N原核表达质粒经酶切和DNA测序鉴定显示,目的基因插入部位正确,长度符合设计要求,为大鼠HAP1氨基末端片段基因。电泳结果显示,提取的3种融合蛋白相对分子质量大小与设计表达的目的蛋白相吻合。获得了豚鼠或兔分别抗3种融合蛋白的抗体,能与提取的融合蛋白特异性结合。豚鼠/兔抗HAP1N(TJ1NGP/TJ1NRB)可同时识别下丘脑提取组织的HAP1A和HAP1B,其特异性与国外的豚鼠/兔抗HAP1中间段序列抗体EM78相同,豚鼠抗HAP1AC(TJ1AcGP)特异性识别HAP1A,豚鼠/兔抗HAP1BC(TJ1BcGP或TJ1BcRB)特异性识别HAP1B;TJ1NRB、TJ1BcRB、TJ1NGP、TJ1AcGP和TJ1BcGP 5种抗体均能有效标记大鼠脑组织的HAP1,TJ1NRB、TJ1BcRB、TJ1NGP和TJ1BcGP免疫反应阳性物的分布与EM78完全一致,TJ1AcGP免疫反应阳性物主要以颗粒状形式定位在Stigmoid小体上,弥散分布产物极少;3种检测结果显示5种抗体的效价均达1 10 000以上。结论针对HAP1A和/或HAP1B氨基末端和羧基末端多肽抗体特异性识别HAP1A和/或HAP1B,可用于HAP1的研究;针对HAP1A/HAP1B氨基末端多肽抗体和HAP1B羧基末端多肽抗体可替代针对HAP1中间段多肽抗体。

Preparation of antibodies against amino terminal and carboxyl terminal poly peptide of huntingtin-associated protein 1

Objective To prepare polyclonal antibodies against N-terminal and C-terminal polypeptide of two isoforms of huntingtin-associated protein 1(HAP1),HAP1A and HAP1B.Methods The recombinant plasmid pGEX-4T-2-HAP1N of the prokaryotic expression,which was combined with N-terminal sequence of HAP1(1-381 bp) and plasmid pGEX-4T-2 was constructed.Three recombinant proteins,the fusion protein of GST with N-terminal and C-terminal of HAP1A and that of HAP1B,GST-HAP1N,GST-HAP1A,GST-HAP1B were produced and used as antig...