【摘要】 为了表达猪带绦虫的缺氧诱导因子-1(HIF-1)并制备其特异性抗体,根据本课题组对猪带绦虫全基因组测序获得的HIF-1基因序列设计了1对特异性引物,以提取的猪带绦虫的总RNA为模板,通过RTPCR获得猪带绦虫HIF-1基因。将该基因定向克隆至原核表达载体pET-30a(+)中,将阳性质粒转化大肠杆菌BL21(DE3)感受态细胞,并用IPTG诱导表达,对表达产物进行SDS-PAGE分析。结果显示,获得了约为17.0ku的表达产物,与目的蛋白的大小相符。原核表达的蛋白纯化后免疫新西兰白兔,制备多克隆抗体,ELISA和Western-blot结果表明,制备的多克隆抗体有较高的抗体效价(1∶51 200)和抗原特异性。该基因又被定向克隆至真核表达载体pcDNA3.1中进行了真核表达。结果成功构建了猪带绦虫HIF-1因子的原核表达系统和真核表达系统,并制备出其多克隆抗体,为进一步研究其功能奠定了基础。更多还原

【Abstract】 To express the hypoxia inducible factor-1(HIF-1)gene of Taenia soliumagainst HIF-1protein,apair of primer was designed according to the unpublished HIF-1gene sequenced in our laboratory.Total RNA was extracted fromT.solium,and then the HIF-1gene was amplified by RT-PCR and cloned into pET-30a(+)vector.The positive plasmid was transformed into competent Escherichia coli BL21(DE3),and then expressed under the induction of IPTG.A recombinant protein with 17.0ku of molecular weight was confirmed by SDS-PAGE.New Zealand White rabbits were immunized with purified HIF-1protein to obtain the polyclonal antibodies against this protein.Western-blot analysis showed that the antibodies had good specificity.ELISA analysis showed that the antibodies had high titer(1∶51 200).Then,the HIF-1gene was cloned into pcDNA3.1vector,and the recombinant vector pcDNA3.1-HIF-1 was expressed.The result showed that the HIF-1gene of T.solium was successfully cloned and expressed in the prokaryotic and eukaryotic expression systems.The polyclonal antibodies against HIF-1protein were prepared.This study provided a basis for studying the functions of HIF-1.更多还原