摘要
对雌激素受体α基因的氨基酸序列进行抗原性分析,筛选合适的基因片段进行克隆,经RT-PCR扩增得到大小为945bp的片段,经测序鉴定后,将该片段定向亚克隆于pMAL-c2x表达载体中,转化至TB1感受态细胞中进行诱导表达。经1mmol/L IPTG诱导4h观察到有新的重组蛋白获得了表达,采用SDS-PAGE和Western blot试验进行检测,结果表明表达的重组蛋白的分子质量约为78.1ku,表达产物占菌体总蛋白的42.3%,且具有免疫学活性。进一步纯化和蛋白酶切后,得到单一的雌激素受体α蛋白。以该蛋白免疫小鼠制备抗体,在免疫第7周时达到最大滴度1.158±0.232。将制备的多克隆抗体进行黄鳝性腺免疫组化分析,结果表明其能特异性地与卵巢中的激素受体α反应,在细胞膜上形成褐色沉淀。
To study the function of the estrogen receptor a （ERα） gene, the recombinant pMAL- ERa was constructed and expressed in TB1 E. coli. Partial cDNA encoding ERα was amplified from total RNA of swamp eel （Monopterus albus） ovary by reverse transcription-polymerase chain reaction （RT- PCR）. The analysis of the sequence data indicated that the cDNA fragment was 945 bp in length, enco- ding 315 amino acid residues. The amplified cDNA fragment was cloned into the prokaryotic expression vector, pMAL-c2x and the recombinant plasmid was then transformed into E. coli TB1. ERa-MBP fusion protein was obtained after the IPTG addition into the growth media. SDS-PAGE analysis showed that the ERα-MBP was expressed after induction by IPTG for 4 h. A protein band of 78. 1 ku appeared on SDS-PAGE gel and was identified by Western blot. The production of the recombinant protein was about 42.3% of total bacteria protein. After purification and cleavage of the fusion protein, purified ERa protein was obtained. Then the purified protein was used to immunize ICR mice to produce anti- ERa anti- body. This purified protein could significantly elicit specific antibody response in immunized mice compared with the control groups,and the titers against ERa reached the peak （1. 158±0. 232） at the 7th week. The immunohistochemical analysis showed that the polyclonal antibody can induce specific reaction with ERa receptor in ovary and produced brown precipitation on the ovary cell membrane.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2014年第4期99-105,共7页
Journal of Huazhong Agricultural University
基金
公益性行业科研专项(201003076)
国家自然科学基金项目(31200918)
中央级公益性科研院所基本科研业务费专项(2013JBFM02
2011JBFA12)
江苏省自然科学基金项目(BK2011184)
江苏省科技支撑计划(BE2013315)
江苏省水产三项工程项目(PJ2011-51)
关键词
黄鳝
雌激素受体
原核表达
抗血清
免疫组化分析
swamp eel
estrogen receptor α
prokaryotic expression
polyclonal antibody
immuno-histochemical analysis
