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目的以真核表达的人g2型溶菌酶(human goose-type lysozyme 2,LysG2)为免疫原制备抗LysG2的兔多克隆抗体,检测LysG2在睾丸组织中的定位,为阐明其生理功能提供线索。方法对LysG2毕赤酵母GS115转化菌株进行高密度培养,甲醇诱导目的蛋白表达并通过甲壳素亲和色谱纯化,以重组蛋白为免疫原制备兔抗LysG2多克隆抗体。通过ELISA检测抗体效价后,对人体各组织、精子及精液标本进行Western印迹分析,并对人睾丸组织进行免疫组化检测。结果以毕赤酵母表达LysG2为免疫原制备的抗体具有高效价,表达谱分析表明LysG2在睾丸中有表达,在精子蛋白提取物中没有检测到其存在,在精浆中检测到了LysG2。免疫组化检测结果显示LysG2在睾丸中主要定位于间质组织的Sertoli细胞。结论本研究成功以真核表达的LysG2制备了该蛋白多克隆抗体,LysG2在睾丸Sertoli细胞有表达,并分泌到精浆中,提示LysG2可能在男性生殖系统先天性免疫中发挥作用。
OBJECTIVE To prepare rabbit anti-LysG2 polyclonal antibody by using human goose-type lysozyme 2 (LysG2) as an immunogen to detect the localization of LysG2 in testis tissue, and to provide clues for clarifying its physiological function. Methods The LysG2 Pichia pastoris strain GS115 was cultured in high density. The target protein was induced by methanol and purified by chitin affinity chromatography. The recombinant protein was used as immunogen to prepare rabbit anti - LysG2 polyclonal antibody. After the antibody titers were detected by ELISA, Western blot analysis was performed on human tissues, sperm and semen samples, and immunohistochemistry was performed on human testicular tissues. Results Antibodies produced by using Pichia pastoris LysG2 as immunogen showed high titer. Expression profiling showed that LysG2 was expressed in testes, but not in sperm protein extracts. LysG2 was detected in seminal plasma. The results of immunohistochemistry showed that LysG2 mainly localized in interstitial Sertoli cells in testis. Conclusions This study successfully prepared polyclonal antibody against LysG2 expressed in E. coli. LysG2 was expressed in sertoli cells and secreted into seminal plasma, which suggested that LysG2 may play a role in innate immunity of male reproductive system.