抗P21激活的磷酸化蛋白激酶5多克隆抗体的制备..doc

抗P-21激活的磷酸化蛋白激酶5多克隆抗体的制备及其在牙胚细胞研究中的应用 摘要：目的??克隆PAK5-N 端基因并诱导其表达，进行多克隆抗体制备，为研究其在牙胚细胞中的生物学功能奠定基础。方法??根据人全长PAK5 cDNA序列，设计引物；利用PCR技术，以PAK5 全长cDNA为模板扩增PAK5-N端基因，将扩增产物克隆至pGEX-4T-1载体中，经EcoRI /XhoI 双酶切后进行重组质粒鉴定，DNA测序。以硫代半乳糖苷诱导其在大肠杆菌BL21中表达。利用GST融合蛋白纯化系统进行蛋白纯化，通过免疫家兔制备多克隆抗体，并在牙胚细胞中进行了初步研究。结果和结论??成功克隆了PAK5-N端基因，在E.coli中表达了PAK5-NT，并纯化了GST融合蛋白，制备了PAK5特异性抗体。Western blotting 表明PAK5 在牙胚细胞中过度表达，为其在口腔细胞中的生物学功能研究提供了依据。 关键词：PAK5；基因克隆；蛋白表达；多克隆抗体；牙胚细胞 中图分类号：R392.4??文献标识码：A??文章编号：1673-4254(2006)06-0730-04 Preparation of anti-P21-activated kinase 5 polyclonal antibody and its application in dental germ cellsAN Zheng-wen1,2； LIU Hong-wei1,3； JIA Zhi-min4； LI Zhao-feng1； Staffan Str觟mblad2； ZHANG Hong-quan2Department of Stomatology, Nanfang Hospital1, College of Pharmacy4, Southern Medical University, Guangzhou 510515，China; 2Karolinska Institute Department of Biosciences and Nutrition, Huddinge SE-141 57, Sweden; 3Affiliated Dental School of Tongji University, Shanghai 200072, ChinaAbstract: Objective To clone PAK5-N terminal sequence for expression in E.coli to prepare its polyclonal antibody, and examine the role of PAK5 in dental germ cells. Methods??Based on human PAK5 cDNA sequence, PCR primers were designed to amplify PAK5-N terminal sequence. The PCR product was cloned into the expression vector pGEX-4T-1 EcoRI/XhoI sites, and the recombinant plasmids were identified by agarose gel electrophoresis followed by DNA sequence analysis. The recombinant plasmids were transformed into E.coli BL21 and the expression of GST-fusion protein was induced by IPTG. Glutathione-Sepharose beads were used to purify GST-fusion PAK5-N-terminal fragment. Anti-PAK5 polyclonal antibody was obtained in immunizing rabbits with purified GST-PAK5 N-terminal fusion protein, and the antibodies were purified by protein A beads and used for detection of PAK5 expression in dental germ cells. Results and Conclusions We successfully cloned PAK5-N terminal gene fragment, and achieved protein expression, purif