人源类溶菌酶蛋白4的多克隆抗体制备及其表达分析

发布时间：2018-09-05 15:30

【摘要】：目的:以原核表达的人源类溶菌酶蛋白4(LYZL4)为免疫原制备多克隆抗体,检测LYZL4在睾丸组织中的定位,为阐明其生理功能提供依据。方法:将LYZL4编码基因克隆于原核表达载体pET32a,IPTG诱导重组LYZL4(r LYZL4)表达。分别使用Ni-NTA树脂和甲壳素亲和层析纯化rLYZL4,采用双层琼脂平板扩散法检测杀菌活性。以Ni-NTA树脂纯化产物为免疫原制备兔抗rLYZL4多克隆抗体,通过ELISA测定抗体效价,Western印迹检测抗体特异性后,检测LYZL4蛋白在人体各组织的分布及其在精子和精浆中存在情况,免疫组化确认LYZL4在人睾丸组织中的定位。结果:原核表达系统可有效表达rLYZL4,其对溶壁微球菌及大肠杆菌无杀灭活性。制备的兔抗rLYZL4多克隆抗体具有很高的效价和特异性,在睾丸、附睾及人精子蛋白提取物中可检测到LYZL4存在,在生精小管中,LYZL4定位于圆形精子细胞及长形精子细胞的顶体上。结论:本研究成功以原核表达的rLYZL4制备了该蛋白的多克隆抗体,确认LYZL4在睾丸和附睾有表达,并定位于精子顶体上,提示其可能与顶体结构或功能相关。
[Abstract]:Aim: to prepare polyclonal antibodies against human lysozyme protein 4 (LYZL4) expressed in prokaryotic cells and to detect the localization of LYZL4 in testis and provide evidence for elucidating its physiological function. Methods: LYZL4 encoding gene was cloned into prokaryotic expression vector pET32a,IPTG to induce recombinant LYZL4 (r LYZL4) expression. RLYZL4, was purified by Ni-NTA resin and chitin affinity chromatography respectively. The bactericidal activity was detected by double layer Agar plate diffusion method. Rabbit anti rLYZL4 polyclonal antibody was prepared by using purified Ni-NTA resin as immunogen. The specificity of the antibody was detected by ELISA assay. The distribution of LYZL4 protein in human tissues and its presence in spermatozoa and seminal plasma were detected. Immunohistochemistry confirmed the localization of LYZL4 in human testis. Results: prokaryotic expression system could express rLYZL4, effectively. It had no killing activity against micrococcus and Escherichia coli. The rabbit polyclonal antibody against rLYZL4 has high titer and specificity. LYZL4 can be detected in testis, epididymis and human sperm protein extract, and LYZL4 is located on acrosome of round spermatocytes and long sperm cells in seminiferous tubules. Conclusion: the polyclonal antibody of rLYZL4 expressed in prokaryotic cells was successfully prepared in this study. It was confirmed that LYZL4 was expressed in testis and epididymis and located on acrosome of spermatozoa, suggesting that it might be related to the structure or function of acrosome.
【作者单位】：上海健康医学院基础医学院;上海健康医学院药学院;上海纽约大学文理学部;
【基金】：上海市自然科学基金(15ZR1421800) 上海市教育委员会科研创新项目(15ZZ116) 上海市青年科技英才扬帆计划(15YF1408600)~~
【分类号】：R392.11

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