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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。
Objective: To detect the localization of LYZL4 in testis by using the prokaryotic recombinant human Lysozyme 4 (LYZL4) as immunogen to provide the basis for elucidating the physiological function of LYZL4. Methods: The LYZL4 gene was cloned into prokaryotic expression vector pET32a and IPTG was used to induce the expression of recombinant LYZL4 (r LYZL4). The rLYZL4 was purified by Ni-NTA resin and chitin affinity chromatography, respectively, and the bactericidal activity was tested by double-layer agar plate diffusion method. The rabbit anti-rLYZL4 polyclonal antibody was prepared by using Ni-NTA resin purified product as the immunogen. The antibody titers were determined by ELISA. After detecting the antibody specificity by Western blotting, the distribution of LYZL4 protein in various tissues of the human body and its distribution in sperm and seminal plasma Exist, immunohistochemistry confirmed the localization of LYZL4 in human testis tissue. Results: Prokaryotic expression system can effectively express rLYZL4, which has no killing activity against Micrococcus lysolyticus and Escherichia coli. The prepared rabbit anti-rLYZL4 polyclonal antibody has high potency and specificity. LYZL4 can be detected in the testis, epididymis and human sperm protein extracts. In the seminiferous tubules, LYZL4 is located in the round sperm cells and long Shaped sperm cells on the acrosome. CONCLUSION: Polyclonal antibody of rLYZL4 prokaryotic expression was successfully constructed in this study. LYZL4 was expressed in the testis and epididymis and localized on the acrosome, suggesting that it may be related to the acrosome structure or function.