日本血吸虫Sj16基因重组杆状病毒的构建和鉴定  

Construction and Identification of Recombinant Baculovirus with Schistosoma japonicum Sj16 Gene

作　　者：胡少敏[1,2] 王海[1] 阮志燕[1] 余新炳[1] 吴忠道[1] 

机构地区：[1]中山大学基础医学院寄生虫学教研室 [2]南华大学生命科学与技术学院生物技术实验中心,衡阳421001

出　　处：《热带医学杂志》2005年第1期8-11,60,共5页Journal of Tropical Medicine

基　　金：国家自然科学基金资助项目(No.30271165);广东省自然科学基金资助项目(No.031727)。

摘　　要：目的利用杆状病毒-昆虫细胞系统构建日本血吸虫(Schistosomajaponicum,Sj)Sj16基因重组杆状病毒。方法从日本血吸虫尾蚴提取总RNA,通过RT-PCR扩增出Sj16基因全编码区序列,将其克隆到载体pET30a(+)中,通过PCR将Sj16基因连同pET30a(+)多克隆位点下游的6×His·Tag一起扩增出来,插入donor载体pFastBacHTa中,构建重组杆状病毒donor载体pFastBacHTa-Sj16-His,转化大肠杆菌DH10Bac-GFP进行转座,提取重组Bacmid,用Lipofectin法转染昆虫细胞Sf9使其包装成有感染性的重组杆状病毒。结果构建了含日本血吸虫Sj16基因的重组Bacmid-GFP-Sj16-His,转染Sf9细胞后,获得了有感染力的重组杆状病毒。结论成功构建了日本血吸虫Sj16基因重组杆状病毒,为下一步重组Sj16蛋白表达和Sj16基因功能研究打下了基础。Objective To construct and identify recombinant baculovirus with Schistosoma japonicum Sj16 gene by baculovirus insect cell system. Methods The total RNA was extracted from cercaria of Schistosoma japonicum. The whole coding sequence of Sj16 gene of interest was synthesized by RT PCR and the cloned into pET30a(+) vector. A fusion fragment including Sj16 gene and the 6 ×His·Tag following the multiple cloning site was synthesized by PCR from the recombinant pET30a(+) Sj16. The fusion fragment Sj16 His and the empty plasmid pFastBacHTa were digested by EcoRⅠand SalI, ligated and transformed into competence E.coli TG1. Positive recombinants were identified by EcoR I/ Sal I digestion and sequence. The recombinant plasmid was extracted and transformed into competence E.coli DH10Bac GFP. The recombinant Bacmid GFP Sj16 His was constructed by transposion of pFastBacHTa Sj16 His and Bacmid GFP and was identified by PCR. Then, the Bacmid GFP Sj16 His DNA was extracted and transfected Sf9 insect cells. After the appearance of fluorescent green and growth changes of Sf9 cells about 3 days post transfection, the culture medium was collected as virus stocks. Results The recombinant Bacmid GFP Sj16 His has been constructed. After transfection, the infective recombinant virus was obtained. Conclusion Infective recombinant baculovirus with Sj16 gene of interest has been constructed successfully.

关 键 词：日本血吸虫 Sj16 杆状病毒 Bac—to-Bac杆状病毒表达载体系统 

分 类 号：R383.24[医药卫生—医学寄生虫学]