利用精子特异性抗体SPAG8分离混合细胞体系中的精子  

Separation of sperms from cell mixtures by sperm-specific antibody SPAG8

作　　者：李学博[1,2] 王清山[2] 宁淑华[3] 王业全[2] 封宇[2] 缪元颖[2] 李红卫[2] 

机构地区：[1]山东省高校证据鉴识重点实验室(山东政法学院),济南250014 [2]重庆市公安局物证鉴定中心,重庆400021 [3]重庆市公安局江北分局法医科,重庆400020

出　　处：《第三军医大学学报》2014年第12期1322-1326,共5页Journal of Third Military Medical University

基　　金：山东省高校证据鉴识重点实验室(山东政法学院)开放课题(2014-01);重庆市公安局科技攻关项目(2012-05)~~

摘　　要：目的利用精子特异性抗体SPAG8(Sperm associated antigen 8)结合免疫磁珠技术分离混合细胞系中的精子,建立混合斑中精子细胞分离的新方法。方法制备含有精子103、104、105/mL与阴道上皮细胞104/mL的3种混合悬液各10例,各样本制作混悬液及模拟混合斑各2份,分别以生物素标记的SPAG8单克隆抗体孵育特异结合混合悬液及混合斑中的精子,然后用亲和素包被的免疫磁珠通过生物素-亲和素复合体捕获分离精子细胞,提取精子DNA进行STR复合扩增和DNA分型检测,同时以差异裂解法检测混悬液及混合斑中的精子进行方法比较。结果免疫磁珠-亲和素-生物素-抗体复合物可分离混合细胞体系中的精子,当混合悬液内的精子数量达到103/mL时,90%的样本可得到13个STR基因座的单一男性分型,精子数量达到104/mL时,90%的样本均可得到16个STR基因座的单一男性分型;含有104/mL以上数量精子的混悬液制作的混合斑,单一男性13个STR基因座的正确分型率达80%以上,混悬液及混合斑的免疫磁珠法有效STR检出率高于传统的差异裂解法。结论本方法简便快捷,可作为混合斑法医学检验的有效补充手段。Objective To establish a new method for the separation of sperms from the mixture of sperms and vaginal epithelial cells by applying a sperm-specific antibodiy against sperm-associated antigen 8 （SPAG8） and immunomagnetic bead sorting. Methods Cell mixture suspensions containing vaginal epithelial cells （104/mL） and sperms at 3 different concentrations （103/mL, 104/mL and 105/mL separately） were prepared, 10 samples for each suspension. Biotin-labeled anti-SPAG8 monoclonal antibody and avidin-coated immunomagnetic beads were incubated with the mixed cell systems and the sperm cells were connected with the magnetic beads by the antibody-biotin-avidin system. The DNA of sperm cells was extracted and amplified by PCR. PCR products were separated and analyzed. Differential lysis was also conducted for the mixed samples to evaluate the effectiveness of immunomagnetic bead sorting. Results Sperm cells were separated by the immunomagnetic beads from the mixtures successfully. The antibody-biotin-avidin-magnetic bead system was stable for the separation. The sperm DNA was extracted and the DNA typing of 13 STR loci and 16 STR loci was acquired in 90% suspension samples when the sperm cell number was larger than 103/mL and 104/mL, respectively. DNA typing of 13 STR loci was acquired in 80% mixed stain samples when the sperm cells number was larger than 104/mL. The detection rate of immunomagnetic bead sorting for mixed samples was higher than that of the differential lysis method. Conclusion Immunomagnetic bead sorting is a simple and quick method for the separation of sperms from sperm and vaginal epithelial cell mixture, and can be utilized as a supplementary means in forensic practice.

关 键 词：SPAG8 免疫磁珠 混合斑 精子 DNA 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学] R392.11[医药卫生—基础医学]