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  

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

作　　者：韩金红[1,2] 韩金芬[3] 王国栋[1] 包巍[1] 范文艳[1] 李和[2] 

机构地区：[1]新乡医学院基础医学院,河南新乡453003 [2]华中科技大学同济医学院组织学和胚胎学教研室,湖北武汉430030 [3]新乡医学院第三附属医院儿科,河南新乡453003

出　　处：《新乡医学院学报》2011年第4期401-405,共5页Journal of Xinxiang Medical University

基　　金：国家杰出青年科学基金项目(编号:30225024);新乡医学院基金项目(编号:2007YJA46)

摘　　要：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。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 antigens to raise polyclonal antibodies specific for HAP1A and HAP1B,respectively,in rabbits and guinea pigs.The specificity and titers were identified by using enzyme linked immunosorbent assay（ELISA）,immunoblotting and immunohistochemistry ABC,respectively.Results The identification of the recombinant plasmid pGEX-4T-2-HAP1N by restriction enzyme analysis and DNA sequencing showed that inserted DNA sequence was HAP1N.Assaying of relative molecular mass by SDS-PAGE showed that the produced three fusion proteins above were coincident with that of purpose.There were five kinds of antibodies：rabbit against HAP1N（TJ1NRB）,rabbit against HAP1BC（TJ1BcRB）,guinea pig against HAP1N（TJ1NGP）,guinea pig against HAP1AC（TJ1AcGP） and guinea pig against HAP1BC（TJ1BcGP）.The results revealed that the above five antibodies were high specificity antibodies of HAP1A and HAP1B,whose valences exceeded 110 000.The ELISA showed the antigenicity of three fusion proteins above.The immunoblotting showed TJ1NGP/ TJ1NRB could integrate to HAP1A and HAP1B,and the specificity was similar to EM78（a gift from overseas）,which was the antibody against the middle sequence polypeptide of HAP1 in rabbit or guinea pig.TJ1AcGP could specifically integrate to HAP1A.TJ1BcRB and TJ1BcGP could specifically integrate to HAP1B.The immunohistochemistry showed that the HAP1 of neurons could be marked by the above five antibodies,and the neuron distribution in rat brain marked by TJ1NRB,TJ1NGP,TJ1BcRB or TJ1BcGP was identica

关 键 词：亨廷顿蛋白相关蛋白1 氨基末端 羧基末端 抗体制备 酶联免疫吸附试验 免疫印迹 免疫组织化学 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]