摘要
目的 构建日本血吸虫中国大陆株SjF1原核表达重组质粒,并进行免疫保护效果测定。方法 用聚合酶链反应(PCR)法将SjF1基因从已剪切阳性克隆中扩增出来,克隆入原核表达载体pTWIN1上intein2的N端,经PCR和限制性酶切筛选阳性重组子。将阳性重组子转化大肠杆菌,在低温和低IPTG浓度下诱导表达可溶性重组融合蛋白。经鉴定后分别以FCA作佐剂经皮下免疫和以壳聚糖作佐剂经黏膜免疫昆明鼠,观察诱导产生的减虫和减卵效果。感染前采血和唾液用ELISA法检测抗体。结果 成功克隆并表达了rSjF1。rSjF1以FCA作佐剂经皮下免疫获得了29.92％的减虫率和51.08％的减卵率;rSjF1以壳聚糖作佐剂经滴鼻免疫获得了25.74％的减虫率和44.04％的减卵率。结论 获得了rSjF1,该蛋白以FCA作佐剂经皮下免疫和以壳聚糖作佐剂经黏膜免疫均能诱导小鼠产生部分抗血吸虫感染的保护力。
To clone and express the novel gene SjF1 of Schistosoma japonicum and to study its immune protective effect of immunization, the SjF1 gene amplified by PCR from excised positive clones of bacteria was subcloned into the N-terminal of intein 2 of prokaryotic expression vector pTWIN1.The positive recombinant was identified by PCR and restriction enzyme digestion;and then was transformed into E. colt .Under low concentration of IPTG and low temperature, the soluble recombinant fusion protein rSjF1-intein2 was expressed in E.coli, and analyzed by SDS-PAGE and Western blotting.Mice were immunized subcutaneously or in-tranasally with rSjF1 plus FCA or chitosan as adjuvant. 42 days after the challenge infection with cercariae of S. japonicum, worms and eggs were collected and countered. Levels of the specific antibodies were determined by ELISA before infection. The results showed that SjF1 gene was successfully subcloned into vector pTWIN1 and expressed in E coli. In mice vaccinated subcutaneously with rSjF1 plus FCA, the worm reduction rate was 29.92% and the egg reduction rate was 51. 08% . While in mice vaccinated in-tranasally with rSj F1 and chitosan adjuvant, the worm reduction rate was 25.74 % and the egg reduction rate was 44.04 % . It concludes that the rSjF1 gene subcloned and expressed can induce partial protection against S. japonicum infection after immunization mice with rSjF1 and FCA or chitosan adjuvant subcutaneously or intranasally.
出处
《中国人兽共患病杂志》
CSCD
北大核心
2004年第6期467-470,共4页
Chinese Journal of Zoonoses
基金
TDR/WHO资助项日(No.980268)
