蓝氏贾第鞭毛虫a-4和a-11贾第素的细胞定位和功能研究

Giardia lamblia，a primitive unicellular eukaryote, similar to higher eukaryotes, has highly developed, fine structured cytosleletal system which is closely related to parasitic pathogenicity. The multi-gene α-giardin family, a Giardia-specific cytoskeletal protein, has 21 members. The subcellular location and biological function of α-4 and α-11 giardins, two members of α-giardin family, are still unknown. In this study, the subcellular location of α-4 and α-11 giardins will be detected by bioinformatics，immunoelectron microscopy and immunofluorescence methods. To identify biological function of the two giardins, vector-mediated hammerhead ribozymes will be designed and constructed. After the expression of α-4 and α-11 giardins are inhibited by ribozymes respectively, a series experiments will be performed to detect changes in biological characteristics of Giardia lamblia trophozoites. The α-4 and α-11 giardins interacting phospholipid components will be identified by phospholipid binding assay. The yeast two-hybrid assay and co-immunoprecipitation(or His pull down) will be applied to identify α-4 and α-11 giardins interacting protein components. When the expression of α-4 and α-11 giardin are inhibited respectively, the expression level changes of otherα-giardin family members will be detected by real-time PCR. Systemically analyze the biological function and function mechanism based on the above experimental results. The research results will provide valuable reference materials for intensive study of α-giardins.

蓝氏贾第鞭毛虫是一种原始的单细胞真核生物，但却有着与高等真核生物相似的高度发达的、结构精细的、与虫体致病力密切相关的细胞骨架结构。其中，α-贾第素是贾第虫特有的骨架蛋白成分，其21个家族成员中的α-4和α-11贾第素至今尚无亚细胞定位及功能研究的成功报道。本研究拟采用生物信息学、免疫电镜、免疫荧光等技术对α-4、α-11贾第素进行亚细胞定位；通过构建锤头状核酶载体抑制α-4、α-11贾第素的表达，采用多种实验检测两种贾第素表达受抑制后贾第虫滋养体生物学性状的改变；通过膜磷脂结合实验鉴定与两种贾第素作用的磷脂成分，酵母双杂交和免疫共沉淀（或His pull down）等技术手段鉴定作用的蛋白，实时定量PCR检测α-4、α-11贾第素表达受抑后其它贾第素表达水平的改变，综合以上实验结果系统分析两种贾第素的生物学功能和作用机制。研究结果将为贾第素的深入研究提供有价值的实验资料。