GGNBP1抗体制备及小鼠组织表达谱分析

体外实验研究表明配子生成素结合蛋白1(GGNBP1)可能与GGN1相互作用形成睾丸特异性复合物,在精子生成过程中发挥作用.从小鼠睾丸总RNA中反转录扩增Ggnbpl全长cDNA,构建表达质粒,在大肠杆菌中表达GGNBP1,经聚丙烯酰胺凝胶纯化后免疫新西兰白兔,制备兔多抗血清.镍离子金属螯合柱纯化表达的GGNBP1蛋白,与NHS活化基团交联,制备GGNBP1抗体亲和层析柱,纯化GGNBP1多抗.在293FT细胞中瞬时表达Myc-GGNBP1融合蛋白,用于Mvc单抗验证GGNBP1抗体特异性,结果证明获得了特异性的GGNBP1抗体.分别制备小鼠脑、心、肺、肝、脾、肾、肌肉、卵巢、睾丸和子宫组织匀浆,用GGNBP1抗体进行Western印迹分析,结果仅在睾丸组织匀浆中检测到GGNBP1特异性条带,证明GGNBP1是睾丸特异性表达蛋白.

Preparation of an Antibody Specific to GGNBP1 and Analysis of GGNBP1 Expression Profile in Mouse Tissues

A novel protein GGNBP1 interacts with GGN1 and forms a testis-specific complex, which could be involved in spermatogenesis. Ggnbp1 cDNA was amplified by RT-PCR from mouse testis total RNA, and an expression plasmid and expressed (His)6-tagged GGNBP1 was constructed in E.coli. Using separated the recombinant GGNBP1 by SDS-PAGE as antigen, rabbit anti-GGNBP1 polyclonal antibody was prepared. The (His)6-tagged GGNBP1 was purified by Ni2 -charged HiTrap Chelating HP column, then coupled to a NHS-activated HiTrap column as an antigen column to immunoaffinity-purify specific anti-GGNBP1 antibody from the polyclonal serum. 293FT cells were transfected with pcDNA3.1Myc/HisA/GGNBP1, and transiently expressed Myc-GGNBP1 fusion protein. Myc-GGNBP1 was analyzed by immunoblotting with anti-GGNBP1 antibody and anti-Myc monoclonal antibody respectively to confirm the anti-GGNBP1 antibody specificity. GGNBP1 expression profile was studied in mouse tissues by western blotting analysis with the anti-GGNBP1 antibody, shows that GGNBP1 expresses in mouse testis tissue only, and suggests that GGNBP1 is a testis-specific protein.