一种新的混合斑精子分离方法  

New Method for Sperm Separation from Mixed Stain Samples

作　　者：伍贤军[1,2] 赵兵[2] 缪璇 钱程[1] 王冰瑜 李晓萌[2] 杨百全[1] 

机构地区：[1]吉林省公安厅物证鉴定中心,长春130051 [2]东北师范大学生命科学学院分子表观遗传学教育部重点实验室,长春130024

出　　处：《高等学校化学学报》2015年第5期886-892,共7页Chemical Journal of Chinese Universities

基　　金：国家公安部重点研究计划项目(批准号:2011ZDYJJNWZ007)资助~~

摘　　要：制备了精子表面膜抗原受精素β蛋白（Fertilinβ）的特异性抗体,通过抗原-抗体反应和抗体固相偶联技术,将抗体连接到琼脂糖球珠上,建立了混合斑精子分离纯化的新方法.首先,通过聚合酶链式反应（PCR）扩增编码人受精素β蛋白第341~373位氨基酸的基因序列,构建PGEX-4T-1/Fertilinβ原核表达质粒;其次,诱导表达GST-Fertilinβ融合蛋白,制备受精素β蛋白多克隆抗体,免疫荧光检测结果表明,受精素β蛋白定位于精子的头后部,并且阴道上皮细胞没有受精素β蛋白的表达;最后,将受精素β抗体与ProteinA琼脂糖球珠连接构建固相抗体系统,将精子吸附于表面,可从混合斑（精子与阴道上皮细胞混合液）中分离纯化精子.本文为性犯罪案件侦破提供了新的方法.In sex crimes cases,mixed stain samples（a mixture of sperm and vaginal epithelial cells） are common and important biological evidence.Identification of sperm sources is the key of cases detected.The lack of effective technique of sperm separation from mixed stain samples is the bottleneck of sperm test.In the present study,the group chose sperm surface antigen,Fertilin β,as antigen to prepare specific antibodies,and further coupled the antibody to a solid phase of agarose beads,in order to establish a new sperm separation system.First,antigen Fertilin β recombinant plasmid was constructed.A c DNA for Fertilin β 341—373aa was amplified by polymerase chain reaction（PCR）,and was cloned into a prokaryotic expression plasmid,p GEX-4T-1 vector.Second,GST-Fertilin β（341—373aa） fusion protein was induced and purified.Against this antigen,the Fertilin β antibodies were obtained from serum of rabbit immunized.ELISA and Western blot analysis demonstrated the specificity of the antibody against Fertilin β antigen.Then,the specific recognition of the antibody to sperm was identified by the immunofluorescence assay,in which Fertilin β protein in the sperm head can detected by the antibody,whereas in the vaginal epithelial cells showed negative detection.Finally,the sperm detection system using the solid phase of Fertilin β antibody was established,in which the Fertilin βantibody was coupled to Protein A / G agarose beads and was able to separate sperm from the mixture.In conclusion,the Fertilin β antibody was successfully prepared,which was able to bind to the rear of the sperm head.Furthermore,the Fertilin β solid phase antibody system was generated,which could bind to the sperm surface,so as to isolate the sperms from the mixed stain samples.This sperm separation method from mixed stain samples provided a new detection for sexual offenses.

关 键 词：混合斑 受精素β蛋白 多克隆抗体 

分 类 号：O657[理学—分析化学]