摘要
目的:研究以非复制型重组腺病毒表达人轮状病毒NSP4基因作为新型轮状病毒基因疫苗的可行性．方法:用AdEasy系统构建表达NSP4蛋白的重组腺病毒,并通过形态学观察、PCR、RT- PCR、Southern blot和Western blot等对重组腺病毒进行鉴定．通过滴鼻途径免疫♀小鼠,对免疫后母鼠生产的乳鼠(4日龄)用SA11株轮状病毒灌胃攻击,观察腹泻的产生情况并进行腹泻评级．结果:获得了重组腺病毒rvAdEasyNSP4． Southern blot表明,在重组腺病毒中确有特异性的NSP4基因整合,Western blot证明,目的蛋白得到表达．初次免疫小鼠后血清IgG抗体的滴度可达1:1 000,但阳转率仅为28．5％;再次免疫后,血清抗体阳转率达到了85．7％;第3次免疫后,血清抗体阳转率达到100%．血清IgA 阳转率可以达到71．4％．实验组乳鼠攻毒后腹泻的百分率和腹泻评分(反应腹泻严重程度) 均较对照组低．结论:腺病毒载体可有效表达轮状病毒非结构蛋白NSP4并可有效诱导小鼠免疫反应,这些结果的取得为新型轮状病毒疫苗的研制提供了依据．
AIM： To investigate the efficacy of passive protection induced by the recombinant adenovirus bearing human rotavirus NSP4 gene and to explore the clues for novel rotavirus vaccine development. METHODS： Human rotavirus NSP4 protein was expressed in a recombinant adenovirus. AdEasy system was recruited to facilitate the preparation and the NSP4 gene was inserted into the early 1 region of the vector by homologous recombination in E.coli. The expression of NSP4 was confirmed by Western blotting. The resultant recombinant adenovirus rvAdEasyNSP4 was induced to mice by intranasal （i.n.） immunization. Specific IgG and IgA antibodies were detected in the murine serum. Murine pups born to the rvAdEasyNSP4 immunized dams were challenged with simian rotavirus SA11 strain orally 4 d after birth, and the induced diarrhea was graded. RESULTS： The recombinant adenovirus rvAdEasyNSP4 showed typical morphology under electron microscope. The transcription of NSP4 specific mRNA by rvAdEasyNSP4 was confirmed with RT-PCR in infected 293 cells, and the expression of the NSP4 protein was verified by Western blotting. After immunization for three times, the positive rates of serum IgG antibody were 28.5%, 85.7% and 100%, respectively, while after the first immunization, the titer of serum IgG reached 1 ： 1 000. After immunization, the positive rate of IgA antibody reached the level of 71.4%. Moderate protection was achieved after simian rotavirus SA11 strain challenged in murine pups. CONCLUSION： NSP4 protein of human rotavirus strain can be expressed by a recombinant adenovirus vector and trigger effective immune response in mice, laying a solid foundation for the development of novel rotavirus genetic engineering vaccine.
出处
《世界华人消化杂志》
CAS
北大核心
2006年第9期858-862,共5页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No 30000145
30370066国家863计划资助项目
No.2003AA215071
