摘要
【目的】FAM3A(family with sequence similarity 3,memberA)为新近发现的细胞因子FAM3的家族成员之一,本研究检测FAM3A的相互作用蛋白。【方法】克隆FAM3A并连接到pGB载体,转化酵母Y190宿主菌,加入含主要信号传导细胞因子人cDNA文库,按标准程序进行酵母双杂交,以X-Gal显色评估活性。提取扩增阳性克隆,连接pACT2载体,测序、检索基因库确定阳性克隆基因。克隆阳性基因并与FAM3A质粒共转染酵母进一步确定相互作用。【结果】酵母双杂交检测到一个与FAM3A相互作用的阳性克隆,提取扩增后测序分析此相互作用蛋白为一睾丸特异性丝氨酸/苏氨酸激酶(TSSK4),一种与精子的发育、成熟、及生育功能有关的蛋白质.共转染pGB-FAM3A质粒与pACT2-TSSK4质粒入Y190酵母证实上述相互作用。【结论】FAM3A可能通过与TSSK4的相互作用参与精子功能与生育能力的调节。
[Objective] FAM3A （family with sequence similarity 3, member A） is a member belong to the newly discovered FAM3 family. We conducted this study to search for interaction proteins with FAM3A. [Method ] FAM3A DNA was PCR-amplified and cloned to plasmid pGB and transferred to Y190 yeast. A yeast two-hybrid screen in Y190 yeast strain of a human cDNA library （contains most of the cytokins and signaling pathways） was performed. A standard transformation procedure was used. Beta-galactosidase activity of clones was estimated using X-Gal. Positive clones were amplified and linked to pACT2 plasmid. The interaction was confirmed by cotransformation of FAM3A and the TSSK4 into Y190 yeast. [ Result ] A potential binding protein was identified. Isolation and sequence analysis of the interacting spectrum clone revealed that FAM3A was binding to a testis-specific serine/threonine kinases （TSSK4）, a member of the TSSK family that might have a role in sperm differentiation and/or subsequent sperm functions during maturation, capacitation, and fertilization. The re-introduction of the constructs into Y190 yeast has shown that cotransformation of FAM3A and TSSK4 into Y190 yeast activated expression of the lacZ reporter genes in Y190 yeast, confirming the specificity of their interaction with FAM3A. [Conclusion] FAM3A may be a potentional factor involving in the sperm functions and fertilization through interaction with TSSK4.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2008年第5期521-525,共5页
Journal of Sun Yat-Sen University:Medical Sciences
基金
国家自然科学基金(30570887)
教育部回国人员科研启动基金(141008)
中山大学回国人员科研启动基金(132023)
