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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p H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p H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。
Objective: To detect the expression of human lysozyme protein 6 (LYZL6) in the male reproductive system to reveal its possible physiological function. Methods: The Pichia pastoris strain encoding LYZL6 gene was cultured in high density and purified by chitin affinity chromatography after induced by methanol. The bactericidal activity of recombinant LYZL6 was observed by double agar plate diffusion method. Rabbit anti-LYZL6 polyclonal antibody was prepared by using the purified recombinant protein as the immunogen, and the antibody titers were detected by ELISA. The distribution of LYZL6 protein in human tissues and semen was detected by Western blotting and the expression of LYZL6 in human testis was confirmed by immunohistochemistry. Results: The recombinant LYZL6 had a high bactericidal activity against M. lysosus at low p H. Antibodies prepared using recombinant LYZL6 as an immunogen had high titers, and Western blotting revealed that LYZL6 was expressed only in the testis and epididymis, with high testicular expression. LYZL6 was detected in human sperm protein extracts and no clear protein bands were detected in the seminal plasma. Immunohistochemistry showed that in the testicular tissue, LYZL6 was located in late stage spermatocytes and round spermatids. Conclusion: Under the condition of low p H, recombinant LYZL6 has a strong ability to dissolve the bacterial cell wall. LYZL6 is secreted by the testicular epithelium and then binds to the sperm, suggesting that LYZL6 may play a role in some aspects of sperm-egg fusion.