细粒棘球绦虫成虫表膜抗原特异性基因的筛选及克隆

IMMUNOSCREENING AND CLONING OF TEGUMENTAL SURFACE ANTIGEN OF ECHANOCOCCUS GRANULOSUS ADULT WORM

为筛选细粒棘球绦虫（Echinococcus granulosus,Eg）表膜抗原基因或具有诊断性的特异性基因,提取Eg成虫表膜抗原,经ELISA、Western blot对该抗原的免疫学特性进行初步研究。以Eg成虫表膜抗原高免鼠血清为探针,筛选Eg成虫cDNA文库,将阳性噬菌斑的PCR产物和pGEM-T载体连接,转染到DH5α,对得到的重组子进行测序,并进行同源性分析。ELISA检测显示,表膜抗原免疫鼠诱导产生了特异性抗体,Western blot鉴定该抗体能识别Eg成虫表膜抗原、原头蚴、Eg发育不成熟、Eg发育成熟抗原。筛选出6个阳性克隆,DNA片段大小在1~2kb之间。对阳性克隆进行同源性分析,结果与EgP-29mRNA同源性为99%,与Eg14-3-3蛋白mRNA同源性80%-99%。筛选Eg成虫cDNA文库所获得的基因,证明了Eg虫体表膜抗原的存在,有望成为犬细粒棘球绦虫的诊断抗原以及犬抗细粒棘球绦虫的候选基因。

Tegumental surface antigen of canine Echinoccocus granulosus（Eg） was extracted from adult worms and used for immunoscreening and cloning of novel genes that encode proteins for vaccine or immunodiagnostic reagents.The immunological activities of the Eg tegumental surface antigen were tested in ELISA and Western blot.The cDNA library was screened by using murine sera against Eg tegumental surface antigen and inserts of positive clones were specifically amplified by PCR.The PCR products were cloned into pGEM-T-easy vector and ligation of framgments was transformed into DH5α cells.Recombined DNA products were sequenced and analyzed for homology.Murine antiserum was specific for Eg tegumental surface antigen as demonstrated in ELISA.This antiserum also reacted in Western blot with antigens from protoscolexes,immature adults and mature adults.Six positive clones were obtained from Eg cDNA and the size of the insert ranged from 1 kb to 2 kb.Sequence analysis showed 99% homology to EgP-29 mRNA and 80%~99% homology to Eg14-3-3 protein mRNA.The present study has demonstrated the existence of Eg tegumental surface antigen,which might be useful for development of vaccine candidate or diagnostic tool for dog infection.