The establishment of recombinant PAEP eukaryotic plasmid and the application in forensic medicine
目的 构建Glycodelin基因（PAEP）重组质粒，并对其在真核细胞中的表达及法医学应用进行研究。方法 提取人胎盘组织总RNA，应用 RT-PCR技术，特异性扩增PAEP基因，并将其插入到真核质粒pCMV-HA中，构建重组质粒pCMV-HA-PAEP。脂质体介导法将重组质粒转染至HEK293细胞中，免疫印迹检测蛋白表达，免疫荧光检测Glycodelin与精细胞的结合。结果 成功构建了PAEP真核表达质粒pCMV-HA-PAEP，该重组质粒可在真核细胞HEK293中高效表达，Glycodelin可对精细胞进行识别与结合。结论 构建的PAEP重组质粒在真核细胞中高表达，表达产物与精细胞的特异结合为深入研究PAEP基因在疾病中的分子机制及法医学应用奠定基础。
LI Fengrui1,， PANG Hao2,*
（ 1、School of Forensic Medicine, China Medical University, Shenyang 110001； 2、 Department of Forensic Medicine, Baotou Medical College, Baotou 014060； ）
Abstract： To establish eukaryotic expression plasmid for PAEP and to study its expression and forensic application. Method Total RNA was extracted from the placenta of human and PAEP was amplified by RT-PCR. The obtained PCR products were subcloned into plasmid pCMV-HA. HEK293 cells were transfected with purified pCMV-HA-PAEP plasmid by lipofectamine 2000, the expressed product in the cells was detected by immunoblotting technique. Overexpressed glycodelin combined with spermatozoa was analyzed by immunofluorescent method. Results A recombinant PAEP expression plasmid pCMV-HA-PAEP was successfully constructed and efficiently expressed in HEK293 cells. Spermatozoa might be recognized and bound by overexpressed glycodelin. Conclusion The recombinant pCMV-HA-PAEP was constructed and expressed successfully in eukaryotic HEK293. Overexpressed glycodelin is useful in further study on the PAEP and application in forensic medicine.
Keywords： forensic genetics; PAEP; cloning; eukaryotic expression; spermatozoa