Huang Peng, Yang Zhi-Fang, Xu Yi-Xin, Bao Jian-Ying, Zhang Ning, Cao Xiao-E, Li Wen-Shu
School of Basic Medicine, Shanghai University of Medical and Health Sciences, Shanghai 201318, China.
School of Pharmacy, Shanghai University of Medical and Health Sciences, Shanghai 200237, China.
Zhonghua Nan Ke Xue. 2017 Jan;23(1):3-10.
To prepare a polyclonal antibody against human lysozyme-like protein 4 (LYZL4) expressed in the prokaryotic system and identify the distribution of LYZL4 in the testis.
The full-length cDNA of LYZL4 was cloned into the pET32a plasmid and the expression of the recombinant LYZL4 (rLYZL4) was induced by IPTG. The rLYZL4 was purified by Ni-NTA and chitin affinity chromatography respectively and its bactericidal activity was observed by bilayer agar plate diffusion assay. The purified rLYZL4 was used as an immunogen to generate the polyclonal antibody, followed by examination of the antibody titer by ELISA and its specificity by Western blot. The distribution of LYZL4 in human tissue, sperm and seminal plasma was identified and its subcellular localization in the testis was determined by immunohistochemistry.
rLYZL4 was expressed efficiently in the prokaryotic system and exhibited no bacteriolytic activity against M. lysodeikticus and E. coli. The anti-rLYZL4 polyclonal antibody could bind the recombinant protein with a high sensitivity and specificity. LYZL4 was identified in the testis, epididymis and sperm protein extracts and localized in the acrosomal region of round and elongating spermatids.
An anti-rLYZL4 polyclonal antibody was successfully prepared using the prokaryotic expression system. LYZL4 was detected in the acrosomal region of round and elongating spermatids, suggesting an association with the structure and function of the acrosome.
制备针对在原核系统中表达的人溶菌酶样蛋白4（LYZL4）的多克隆抗体，并鉴定LYZL4在睾丸中的分布。
将LYZL4的全长cDNA克隆到pET32a质粒中，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导重组LYZL4（rLYZL4）的表达。分别通过镍-亚氨基二乙酸（Ni-NTA）和几丁质亲和层析纯化rLYZL4，并通过双层琼脂平板扩散试验观察其杀菌活性。将纯化的rLYZL4用作免疫原制备多克隆抗体，随后通过酶联免疫吸附测定（ELISA）检测抗体效价，并通过蛋白质印迹法检测其特异性。鉴定LYZL4在人体组织、精子和精浆中的分布，并通过免疫组织化学确定其在睾丸中的亚细胞定位。
rLYZL4在原核系统中高效表达，对溶壁微球菌和大肠杆菌无溶菌活性。抗rLYZL4多克隆抗体能以高灵敏度和特异性结合重组蛋白。在睾丸、附睾和精子蛋白提取物中鉴定出LYZL4，其定位于圆形和伸长型精子细胞的顶体区域。
利用原核表达系统成功制备了抗rLYZL4多克隆抗体。在圆形和伸长型精子细胞的顶体区域检测到LYZL4，提示其与顶体的结构和功能有关。
