日本血吸虫新抗原基因的筛选、克隆、表达和免疫效果研究  

A New Antigen Gene of Schistosoma japonicum: Screening,Cloning, Expression and Immunization in Mice

作　　者：陈利玉[1] 易新元[1] 曾宪芳[1] 蔡春[1] L.McREYNOLDS 

机构地区：[1]中南大学湘雅医学院病原生物学系 [2]Molecular Parasitology Group,New England Biolabs,MA01915,USA

出　　处：《生物化学与生物物理进展》2005年第4期331-337,共7页Progress In Biochemistry and Biophysics

基　　金：TDR/WHO资助项目(980268);湖南省科技厅资助项目(00jzy2115).~~

摘　　要：为了寻找日本血吸虫(Schistosomajaponicum,Sj) 新的疫苗候选基因并进行免疫效果研究,用Sj雌虫抗原免疫家兔制备血清,对Sj成虫cDNA文库进行免疫筛选,将获得的新基因(命名为Sj-F1,GenBank登录号为AY261995) 克隆入原核表达载体pTWIN1和真核表达载体pcDNA3,经PCR、限制性酶切筛选和鉴定阳性重组子. 将pTWIN1/Sj-F1质粒转化大肠杆菌ER2566,在低温和低IPTG浓度下诱导表达可溶性重组融合蛋白(rSj-F1/intein2),并经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blot)分析鉴定. 将pcDNA3/Sj-F1质粒转化大肠杆菌ER2502,大量制备DNA疫苗. 用重组融合蛋白和DNA疫苗免疫小鼠,末次免疫后2周用Sj尾蚴进行攻击感染. 感染后42天剖杀冲虫,计算减虫率和减卵率. 感染前采血用ELISA法检测抗体. 免疫保护效果测定显示:重组蛋白疫苗以FCA作佐剂经皮下免疫和以壳聚糖作佐剂经粘膜免疫分别获得了28.07%、24.69%的减虫率和48.30%、46.38%的减卵率;DNA疫苗(pcDNA3/Sj-F1)单独免疫获得了18.47%的减虫率和35.06%的减卵率;用DNA疫苗启动免疫后用重组蛋白疫苗经皮下加强免疫,减虫率和减卵率分别提高到了40.42%和56.17%;用DNA疫苗启动免疫后用重组蛋白疫苗经黏膜加强免疫,减虫率和减卵率增高更明显,分别提高到了42.38%和62.In order to search for new effective Schistosoma japonicum vaccine candidate genes and study its immune protection against challenge infection in mice, S. japonicum adult worm cDNA library was screened with sera from rabbits immunized with S. japonicum female worm antigen. The novel gene (designated Sj-F1, GenBank accession number are AY261995) was cloned into the prokaryotic expression vector pTWIN1 and the eukaryotic expression vector pcDNA3 respectively. The positive recombinants were identified by PCR and restriction enzyme digestion. The plasmid pTWIN1/Sj-F1 was transformed into E.coli ER2566. The soluble recombinant fusion protein (rSj-F1/intein2) was expressed in E. coli under low IPTG concentration and low temperature, then analyzed by SDS-PAGE and Western blot. The plasmid pcDNA3/Sj-F1 was transformed into E.coli ER2502 for preparing DNA vaccine. Mice were immunized with rSj-F1 protein or/and pcDNA3/Sj-F1 DNA vaccine. Two weeks after the third vaccination, a challenge infection was carried out with S. japonicum cercariae. Worms and eggs collected from the livers of mice were counted 42 days after challenge infection. Levels of specific antibody were detected by ELISA before infection. Immunization experiment showed the recombinant Sj-F1 protein with FCA adjuvant or with chitosan adjuvant provided 28.07%, 24.69% worm reduction rates and 48.30%, 46.38% egg reduction rates in mice respectively. The naked pcDNA/Sj-F1 provided 18.47% worm reduction rate and 35.06% egg reduction rate. The worm and egg reduction rates were increased to 40.42%, 42.38% and 56.17%, 62.87% respectively when mice were immunized with the pcDNA/Sj-F1 at priming and with rSj-F1 by subcutaneously or intranasally at booster. The results suggest that both recombinant Sj-F1 protein and naked DNA can induced partial protection immunity against S. japonicum infection and the protection can be enhanced via DNA priming and rSj-F1 boosting.

关 键 词：日本血吸虫 免疫筛选 雌虫 Sj-F1 免疫 

分 类 号：R383[医药卫生—医学寄生虫学]