小鼠睾丸膜联蛋白A1的克隆、表达及定位

目的:克隆、表达小鼠睾丸膜联蛋白A1,并研究其在睾丸中的定位。方法:利用RT-PCR技术从小鼠睾丸组织中扩增膜联蛋白A1的cDNA序列,将该基因插入GST融合表达载体pGEX-5T。以亲和层析法纯化蛋白免疫家兔制备多抗,并做睾丸切片免疫组织化学检测。结果:重组质粒测序结果表明,插入片段与小鼠膜联蛋白A1的序列完全一致。K802重组菌高效表达出相对分子质量为63 000的融合蛋白,表达量占菌体总蛋白的30%。多抗效价达1∶102 400,可特异识别重组蛋白。免疫组织化学显示膜联蛋白A1主要分布于精原细胞核、精子细胞顶体帽及精子胞质残余体内。结论:成功克隆、表达了小鼠膜联蛋白A1基因;膜联蛋白A1在睾丸生精细胞中的不同分布预示其可能与生精过程有关。

Cloning, expression of mouse Annexin A1 and its localization in testis

Objective: To clone and express mouse annexin A1 gene and explore its localization in the testis.Methods: The encoding sequence of mouse annexin A1 gene was amplified from mouse testis tissue by RT-PCR and was inserted into the GST fusion expression vector pGEX-5T.The products were purified and used to immunize rabbit to obtain polyclonal antibody,which was then used to study localization of annexin A1 in testis by immunohistochemistry.Results: The sequence of the inserted segment was coincident with the published encoding sequence of mouse annexin A1.The fusion protein had a molecular weight of 63()000 and the yield was 30 percent in total proteins.The polyclonal antibody specifically recognized annexin A1 protein by immunoblotting.Annexin A1 mainly distributed in nuclei of spermatogonia,acrosomal cap of spermatid and residual body of sperm.Conclusion: Annexin A1 cDNA was successfully cloned and expressed.Differential localization of annexin A1 in spermatogenic cells suggests that it may play a role in spermatogenesis.