Objective: To study the expression of human lysozyme-like protein 6 (LYZL6) in the male reproductive system and its physiological role.
Methods: The recombinant P. pastoris strain was cultured and induced with methanol to express LYZL6, followed by purification using chitin affinity chromatography. The bactericidal activity of the recombinant LYZL6 was observed by bilayer agar plate diffusion assay, and then the recombinant protein was used as an immunogen to generate polyclonal antibodies, whose specificity was examined by ELISA. The distribution of LYZL6 in the human tissue and semen was identified by Western blotting and the subcellular localization in the testis was investigated by immunohistochemistry.
Results: At pH 5.6, recombinant LYZL6 exhibited a high bacteriolytic activity against M. lysodeikticus. ELISA analysis showed that the anti-LYZL6 polyclonal antibodies could bind the recombinant protein with a high specificity. Western blot manifested the expression of LYZL6 in the testis and epididymis, higher in the former than in the latter. LYZL6 was also detected in the sperm protein extract, while protein bands were not observed in the seminal plasma. Immunodetection with a specific antiserum localized the LYZL6 protein in the late spermatocytes and round spermatids.
Conclusions: LYZL6 has a higher bacteriolytic activity under low pH condition and is bound to spermatozoa after secreted in the testicular epithelia, suggesting that LYZL6 could act as a potential hydrolase for carbohydrates in zona pellucida penetration.
目的：检测人源类溶菌酶蛋白6（LYZL6）在男性生殖系统中的表达情况，以揭示其可能的生理功能。方法：将整合LYZL6编码基因的毕赤酵母菌株进行高密度培养，甲醇诱导表达后甲壳素亲和色谱纯化。采用双层琼脂平板扩散法观察重组LYZL6的杀菌活性。以纯化的重组蛋白为免疫原制备兔抗LYZL6多克隆抗体，ELISA检测抗体效价。通过Western印迹检测LYZL6蛋白在人各组织及精液中的分布，并采用免疫组化法确认LYZL6在人睾丸中的表达情况。结果： 在低pH条件下重组LYZL6对溶壁微球菌杀菌活性较高。以重组LYZL6为免疫原制备的抗体具有高效价，Western印迹检测表明LYZL6仅在睾丸和附睾中表达，且睾丸表达水平较高。在人精子蛋白提取物中检测到了LYZL6，精浆中并未检测到明显蛋白条带。免疫组化检测显示在睾丸组织中，LYZL6定位于晚期精母细胞及圆形精子细胞。 结论： 在低pH条件下，重组LYZL6具有较强的溶解细菌细胞壁的能力；LYZL6被睾丸上皮分泌后结合于精子上，提示LYZL6有可能在精卵融合过程的某个环节发挥作用。.
Keywords: Pichia pastoris; bacteriolytic activity; eukarytoic expression; human lysozyme-like protein 6; polyclonal antibody; spermatozoa; testis.