Jagadish N, Rana R, Selvi R, Mishra D, Shankar S, Mohapatra B, Suri A
Genes and Proteins Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Mol Reprod Dev. 2005 May;71(1):58-66. doi: 10.1002/mrd.20245.
The present study was conducted to isolate macaque proteomic homologue of human SPAG9 (EMBL nomenclature human sperm associated antigen 9: hSPAG9; Shankar et al., 1998: Biochem Biophys Res Commun 243:561-565) in order to find out whether the macaque can provide a suitable model for examining its immunocontraception effects. Macaque SPAG9 was cloned and sequenced from the macaque testis cDNA library. The macaque cDNA contained open reading frame encoding 712 amino acids. A 84.9% and 94% homology between macaque and human SPAG9 was found at protein and DNA levels. Northern analysis and RNA in situ hybridization experiments revealed testis- and stage-specific expressions of macaque SPAG9 mRNA, mainly confined to round spermatid suggesting haploid germ cell expression. Anti-human SPAG9 antibodies recognized native SPAG9 in macaque sperm extract in Western blotting and the acrosomal compartment region of macaque sperm in indirect immunofluorescence. Flow cytometry analysis further revealed surface localization of macaque SPAG9 in live macaque sperm. The amino acid sequence data for nonhuman primate SPAG9 suggest that antibodies generated by vaccinating macaque with hSPAG9 will recognize nonhuman primate SPAG9, supporting the testing of SPAG9 contraceptive vaccine based on hSPAG9 in the nonhuman primate model.
本研究旨在分离人类SPAG9（EMBL命名法：人类精子相关抗原9；hSPAG9；Shankar等人，1998年：《生物化学与生物物理研究通讯》243：561 - 565）的猕猴蛋白质组同源物，以确定猕猴是否能为研究其免疫避孕效果提供合适的模型。从猕猴睾丸cDNA文库中克隆并测序了猕猴SPAG9。猕猴cDNA包含编码712个氨基酸的开放阅读框。在蛋白质和DNA水平上，发现猕猴与人类SPAG9之间的同源性分别为84.9%和94%。Northern分析和RNA原位杂交实验揭示了猕猴SPAG9 mRNA的睾丸特异性和阶段特异性表达，主要局限于圆形精子细胞，提示单倍体生殖细胞表达。抗人类SPAG9抗体在蛋白质印迹中识别猕猴精子提取物中的天然SPAG9，并在间接免疫荧光中识别猕猴精子的顶体区。流式细胞术分析进一步揭示了猕猴SPAG9在活猕猴精子中的表面定位。非人灵长类动物SPAG9的氨基酸序列数据表明，用hSPAG9免疫猕猴产生的抗体将识别非人灵长类动物SPAG9，支持在非人灵长类动物模型中基于hSPAG9测试SPAG9避孕疫苗。
