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捻转血矛线虫新基因Hcher-1的克隆与特性分析

DOI: 10.7685/j.issn.1000-2030.2012.06.015, PP. 89-96

Keywords: 捻转血矛线虫,Hcher-1基因,免疫原性,差异表达,实时定量PCR

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Abstract:

根据Caruorhabditiselegans、C.briggsae和Brugiamalayi的her-1基因的保守序列设计引物,从捻转血矛线虫cDNA中扩增出597bp的DNA片段。以此序列为基础,通过5’RACE和3’RACE技术分别获得该基因的5’端和3’端未知序列。将这些序列拼接后获得捻转血矛线虫Hcher-1基因的全长cDNA,该基因全长1091bp,包含一个960bp的最大开放阅读框(ORF)、67bp5’非翻译区和64bp3’非翻译区。根据该基因的ORF设计1对特异性引物,以捻转血矛线虫的总RNA为模板,采用RT-PCR技术,扩增出960bp的DNA片段,序列测定结果与推导的ORF序列一致。将该ORF亚克隆到pET-28a(+)载体中,转化大肠杆菌Rosseta,用IPTG进行诱导表达,SDS-PAGE电泳结果分析发现融合蛋白在上清液和包涵体中均有表达。以该重组蛋白免疫大鼠制备血清作为一抗,Westernblot方法分析天然HcHER-1蛋白,结果发现在全虫抗原的相对分子质量为35×103处出现特异性条带,与其理论大小一致。用荧光定量PCR技术对该基因在捻转血矛线虫的虫卵、第3期幼虫、雌虫和雄虫等不同发育阶段和不同性别虫体内的表达情况进行了定量分析,结果显示Hcher-1在雄性成虫中表达量最高,虫卵和第3期幼虫其次,在雌性成虫中最低,仅为雄虫表达量的1/7~1/8。

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