摘要
弓形虫棒状体蛋白32(Rop32)是由弓形虫棒状体在感染细胞中分泌于宿主细胞中的磷酸激酶,但其功能尚不清楚。为筛选与Rop32相互作用的宿主蛋白,本研究利用构建的p GBKT7-Rop32诱饵质粒转化酵母菌株Y2H,通过表型从人包皮成纤维细胞文库中筛选与Rop32相互作用的宿主蛋白。结果表明p GBKT7-Rop32诱饵质粒无自激活和细胞毒性作用。通过酵母双杂交,共筛选到12个与Rop32具有相互作用的宿主细胞蛋白。将筛选的阳性克隆进行序列分析结果表明,筛选到的蛋白包括1个参与蛋白转运相关蛋白、1个凋亡相关蛋白、1个细胞骨架蛋白、1个参与能量转化的磷酸转移酶以及8个其它蛋白。利用免疫共沉淀试验进一步验证了Rop32和SEC63蛋白之间的特异性相互作用。Rop32与参与蛋白转运的SEC63的相互作用提示Rop32可能在弓形虫对营养物质和代谢物的运输过程中发挥作用。
Rhoptry protein 32 （Rop32） of Toxoplasma gondii is secreted from rhoptry into the parasite infected cells, but the function of Rop32 is still unknown. To identify the host proteins which interacted with Rop32, a partial Rop32 gene of T.gondii was cloned into the pGBKT7 plasmid to construct the recombinant plasmid of pGBKT7-Rop32, which was transform into the yeast to express as a bait for Rop32-interacting proteins screening from a cDNA library constructed with human foreskin fibroblast through a phenotypic screening method. The results showed that the pGBKT7-Rop32 had no self-activation and cytotoxicity, and 12 proteins interacted with Rop32 were obtained all together. Sequence analysis indicated that positive clones of the sequences encoded a transportation protein, an apoptosis-related protein, a cytoskeletal protein, a phosphotransferase involved in energy conversion and 8 other proteins. In addition, the specific interaction between Rop32 and SEC63 was further validated using Co-IP. The interaction between Rop32 and SEC63 might suggest that Rop32 plays a role in protein transportation into parasitophorous vacuolar membrane of T.gondii.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2016年第6期471-474,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
兽医生物技术国家重点实验室自主课题(SKLVBP2015012)
