SMCY性别特异融合抗原的克隆表达及其抗体制备  

The application of the cloning and expression of SMCY gender-specific fusion antigens with the corresponding polyclonal antibodies

作　　者：安志远[1,2] 田露[2] 王建霞[3] 冯晓燕[3] 唐建平[4] 陈新[4] 陈荣华[4] 毕钢[4] 肖雄[2] 邱志军[2] 胡寅[2] 周文斌[5] 周怀谷[4] 

机构地区：[1]上海公安高等专科学校,上海200137 [2]上海市公安局浦东分局,上海200135 [3]军事医学科学院基础医学研究所,北京100850 [4]上海市公安局物证鉴定中心法医物证学现场应用技术公安部重点实验室上海市现场物证重点实验室,上海200083 [5]上海市公安局长宁分局,上海200335

出　　处：《中国法医学杂志》2017年第4期375-378,共4页Chinese Journal of Forensic Medicine

基　　金：上海市科学技术委员会科研计划项目(14JG0500400)

摘　　要：目的利用蛋白检测的快速性优势,研究不同性别在SMCY抗原氨基酸序列的差异,筛选出特异性氨基酸序列,并克隆表达性别特异融合抗原,制备相应抗体,建立一种快速鉴别法医物证性别的方法。方法通过对人SMCY和SMCX进行序列分析,发现了三段差异片段,采用搭桥PCR方法获得差异片段全长基因,连接入p ET-28a载体进行原核表达,用Ni柱纯化后的性别特异融合抗原免疫制备多克隆抗体,用ELISA法和western blot检测SMCY多抗与抗原的反应特异性,制作胶体金试纸条检测样本。结果筛选出具有性别特异性的氨基酸序列,获得SMCY性别特异融合抗原,成功制备出多克隆抗体及胶体金试纸条。结论获得SMCY性别特异融合抗原具有很好的抗原活性,制备的多克隆抗体可以与抗原特异性地结合,用于性别鉴定。Objective with the advantages of rapidity in detection protein, We selected the gender-specific amino acid sequence based on human SMCY and SMCX, cloned and expressed SMCY gender-specific fusion antigens. The rabbits were immunized with purified antigens to obtain the polyclonal antibodies. A new method was established for rapidly sex identification of forensic evidence samples by detecting SMCY antigens with the corresponding polyclonal antibodies. Methods We found three differential fragments by analyzing the sequence of human SMCY and SMCX. Then we cloned this three fragments and ligated as a new recombinant.This SMCY gender-specific fusion antigen gene was sub-cloned into p ET-28 a and expressed in Escherichia coli. The fusion antigen was purified by Ni-NTA column. The rabbits were immunized with purified antigen to produce the specific polyclonal antibodies.The reactivity of the polyclonal antibody was evaluated by ELISA and Western blotting. We developed a colloidal gold test strip for detecting the gender of human samples. Results We successfully selected gender-specific amino acid sequence, the SMCY gender-specific fusion antigen was expressed by prokaryotic expression and the polyclonal antibody was prepared by immunizing rabbit. The results of colloidal gold strip tests showed that there is a significant difference between male and female serums. ConclusionThe results showed that the SMCY gender-specific fusion antigen could be recognized by the polyclonal antibody.The colloidal gold strip tests made by SMCY gender-specific fusion antigens and the corresponding polyclonal antibodies could be used for rapidly determining the gender of forensic evidence samples.

关 键 词：法医物证学 性别鉴定 SMCY性别特异融合抗原 克隆和表达 多克隆抗体 

分 类 号：D919.4[医药卫生—法医学]