【摘要】 采用Ｓｊ未成熟虫卵抗原超免疫兔血清（ＲＡＳｊＩＥＡ）对Ｓｊ成虫ｃＤＮＡ文库进行筛选，并对可能具有抗日本血吸虫生殖／卵胚发育潜能的抗原分子进行克隆、表达。常规法免疫筛选Ｓｊ成虫文库，获得单个阳性克隆，ＰＣＲ扩增后琼脂糖电泳测定基因大小，ＤＮＡ测序，基因库搜索，找出同源基因。共鉴定出６个不同的基因克隆。选择其中的铁蛋白（ＳｊＦｅｒ）基因亚克隆于ｐＧＭＣ载体。重组蛋白以ＧＭＣＳＦ（粒细胞巨噬细胞集落刺激因子）－ＳｊＦｅｒ融合蛋白的形式在细菌中高效表达。表达产物仅溶于８Ｍ尿素溶液中，分子量为４０ｋＤ。更多还原

【Abstract】 Our previous work showed that immunization of mice with Schistosoma japonicum (Sj) immature eggs induced significant immunity against fecundity and embryonation of the parasite. The Sj adult cDNA library was screened by sera from rabbits against Sj immature egg antigen (RASjIEA). The genes encoding molecules which may induce immunity against fecundity/embryonation were chosen for further cloning and expression. First of all, RASjIEA was absorbed with E.coli lysate to remove cross reactive antibodies. The cDNA library was then immunoscreened using the routine method. The resulted positive plaques were rescreened till individual clones were confirmed. Phagemids were obtained using in vivo excision. The positive clones were amplified using PCR. The sizes of the genes were determined by agarose gel electrophoresis. After DNA sequencing of the genes cloned, Gene bank was searched and six different genes were identified from a total of 102 positive clones. One of six identified genes, Sj ferritin (SjFer) was chosen to subclone into pGMC vector. According to DNA sequences of Sj Fer and MCS (multiple cloning site) of the vector, forward primer (Fer/GMC1) and reverse primer (Fer/GMC2) were designed and used to amplify Sj Fer by PCR. The Sj Fer cDNA and expression vector pGMC were digested with BamHI and XhoI. The digested cDNA and pGMC were ligased by T4 DNA ligase to construct a recombinant which was then used to transform E.coli strain ER2566. The fusion protein GMCSF Sj Ferritin was expressed in insoluble form, the inclusion body. Pellets were harvested and resolved in Tris HCl buffer containing 8M urea. GMCSF Sj Ferritin was purified by affinity chromatography using Ni NTA resin. The molecular weight was determined by SDS PAGE. This study first reports the gene encoding S.japonicum ferritin as a new candidate for schistosome vaccine.更多还原