睾丸特异性钙结合蛋白CBP86-IV相互作用蛋白的筛选与鉴定及其功能研究

Testis-specific calcium-binding protein CBP86-IV (TS-CBP86-IV) was one of the differentially expressed proteins which were identified in the total sperm protein of asthenozoospermia. Some proteins which belong to the same protein family with TS-CBP86-IV were already confirmed to regulate sperm motility in previous reports. Therefore, we considered that TS-CBP86-IV was related to sperm motility, but it is still unclear about the molecular mechanism. The functional proteomics is becoming the research focus with the purpose of confirming the unknown function of the interacted proteins with the target protein. According to the advanced experience of oversea country, the study of multiprotein complex of TS-CBP86-IV was carried out in vitro and in vivo independently. The coding gene of TS-CBP86-IV will be picked up and inserted into the yeast expression vector pGBKT7, and transformed into yeast strain AH109 for screening positive clones and further protein expression with auxotrophic culture medium. Cracking cells, protein purification, electrophoretic separation and mass spectrometic analysis will be followed for identification of interactional proteins with TS-CBP86-IV. Surface plasmon resonance (SPR) technology will be applied to confirm interactional proteins via Biacore system for vitro study. Base on the functional pathways of interactional proteins, the molecular mechanism of sperm motility regulated by TS-CBP86-IV will be discussed.

TS-CBP86-IV是我们前期研究弱精症蛋白质组所鉴定的差异表达蛋白质，已有研究发现该蛋白家族中的一些成员参与调控精子运动。因此，我们认为TS-CBP86-IV可能也与精子的运动调控有关，但确切的分子机制不清楚。当前，功能蛋白质组研究已成为新的热点，其目的是研究在接近生理条件下确定与已知蛋白相互作用的蛋白功能。为此，本研究分别从体内、体外两个方面对TS-CBP86-IV相互作用蛋白进行研究。首先提取TS-CBP86-IV蛋白质的编码基因，用克隆技术构建带有TAP标签的融合基因表达载体，插入酵母细胞表达载体pGBKT7中，转化AH109酵母菌株并获得表达，筛选阳性克隆进行细胞培养；然后裂解细胞，鉴定出与TS-CBP86-IV相互作的蛋白质。进一步采用Biacore方法进行体外实验，对所鉴定的蛋白质进行验证。然后，从相互作用的蛋白功能对TS-CBP86-IV调控精子运动的分子机制进行探讨。

功能蛋白质组学研究已成为当前新的热点，其目的是研究在接近生理条件下确定与已知蛋白相互作用的蛋白功能。睾丸特异性钙结合蛋白CBP86-IV（TS-CBP86-IV）是我们前期研究弱精症蛋白质组所鉴定的差异表达蛋白质，为了进一步对其功能研究，本项目采用TAP技术分别从大肠杆菌BL21和293T细胞中筛选和鉴定出了与TS-CBP86-IV可能有相互作用的蛋白24个。体外实验采用CO-IP和West Blot 技术在人精子中验证证实phosphoglycerate kinase2（PGK2）与TS-CBP86-IV有相互作用。PGK2是糖酵解的关键酶，特异性表达在精子细胞。表明TS-CBP86-IV和PGK相互作用蛋白在调控精子运动方面起重要作用。依据实验结果推断可能包括二个途径：一是TS-CBP86-IV与PGK2结合，促进糖酵解生成ATP，为精子的运动提供所需的能量；二是TS-CBP86-IV被PGK2激活，TS-CBP86-IV是一种钙调蛋白，被激活后调节精子膜内钙Ca2+浓度，Ca2+增加可促进精子运动。本项目所取得的成果为弱精症的诊断和治疗、以及男性避孕等研究奠定重要基础。