日本血吸虫SjPP基因重组杆状病毒的构建和在昆虫细胞中的表达

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。

Construction and Expression of Recombinant Baculovirus with Schistosoma japonicum SjPP Gene

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 DH10Bac 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.