肝片吸虫免疫显性抗原基因的筛选及鉴定

为筛选出特异的肝片吸虫诊断候选抗原,拓展诊断靶标,利用肝片吸虫阳性血清筛选肝片吸虫cDNA表达文库,获得肝片吸虫特异性抗原基因,采用RT-PCR技术扩增目的基因,连接表达载体pET-32a(+),构建重组质粒,转化入感受态细胞BL21(DE3)中,利用IPTG对重组蛋白进行诱导表达,通过SDS-PAGE及Western blot技术对蛋白表达情况进行鉴定。结果显示:经筛选获得了17个肝片吸虫免疫显性抗原基因,其中假定蛋白Fh010935为肝片吸虫特异性抗原基因;扩增得到的Fh010935核苷酸序列大小为144 bp,编码48个氨基酸,A+T含量为55.56%;Fh010935蛋白由1个α-螺旋,2个β-折叠和3个β-转角构成,具有3个抗原表位,推测该蛋白可能具有较好的抗原性;重组蛋白主要以包涵体形式表达,分子量大小约24 ku,可以被肝片吸虫感染阳性血清特异性识别,具有较好的反应原性。提示:假定蛋白Fh010935可作为肝片吸虫病诊断候选抗原,为疾病诊断制剂的开发提供前期基础。

Screening and identification of immunodominant antigen gene of Fasciola hepatica

In order to screen out the specific candidate antigen for diagnosis of Fasciola hepatica and to expand the diagnostic target of Fasciola hepatica, the cDNA expression library of Fasciola hepatica was screened using the Fasciola hepatica positive serum to obtain the specific antigen gene of Fasciola hepatica. The target gene was amplified by RT-PCR, and was ligated to the expression vector pET-32 a(+). A recombinant plasmid was constructed and transformed into the competent cell BL21(DE3). The recombinant protein was induced by IPTG, and was identified by SDS-PAGE and Western blot. The results showed that 17 immuno-dominant antigen genes of Fasciola hepatica were obtained, among which the hypothetical protein of Fh010935 was specific to Fasciola hepatica by sequence homology analysis. The size of the amplified nucleotide sequence was 144 bp, encoding 48 amino acids with an A+T content of 55.56%. The protein Fh010935 was mainly composed of one α-helix, two β-sheets, three β-bends, and three antigenic epitopes, and was predicted to possess good antigenicity. The recombinant protein was mainly expressed as an inclusion body and its molecular weight was about 24 ku;and it could be specifically recognized by the Fasciola hepatica infected sheep serum, indicating that it had good reactogenicity. All these results suggested that the hypothetical protein Fh010935 could be used as a candidate antigen for diagnosis of hepatic fascioliasis and it provided a preliminary basis for development of preparations for disease diagnosis.