摘要
目的在昆虫细胞中表达获得可溶性的日本血吸虫SjPP重组蛋白。方法提取日本血吸虫成虫总RNA,通过RT-PCR扩增出SjPP基因的编码区序列,将其定向克隆到供体质粒pFastBac HT-B中,构建重组杆状病毒供体质粒pFastBac-SjPP,转化入大肠埃希菌DH10Bac进行转座,提取重组杆粒Bacmid-SjPP,用阳离子脂质体法转染粉纹夜蛾(TN5B1-4)细胞。利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)分析重组蛋白表达情况。结果构建了含日本血吸虫SjPP基因的重组杆粒Bacmid-SjPP,转染TN5B1-4细胞后,获得有感染力的重组杆状病毒,并在TN5B1-4细胞中成功表达SjPP蛋白。该重组蛋白可被抗SjPP蛋白的兔血清识别。结论成功构建SjPP基因重组杆状病毒,并在TN5B1-4细胞中表达,该蛋白具有良好的抗原性。
Objective To express the soluble recombinant Schistosoma japonicum SjPP proteins in TN5B1-4 cells. Methods The total RNA was extracted from adult worms of Schistosoma japonicum. The whole coding sequence of SjPP gene was synthesized by RT-PCR and cloned into donor plasmid. The recombinant donor pFastBac-SjPP was transformed into E.coli DHIOBac forming Bacmid-SjPP which was transfected into insect cell with cational lipofectin. The fusion protein SjPP was analyzed with SDS-PAGE and Western blotting. Results The infective recombinant baculovirus Bacmid-SjPP was obtained and SjPP protein was expressed in insect cells. Conclusion The recombinant protein SjPP has been expressed in insect TN5B1-4 cells with proper antigenicity.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2008年第2期124-127,共4页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家“863”计划(No.2006AA10A207)
国家科技支撑计划(No.2006-BAD06A09)~~
