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

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。

Preparation of anti-P21-activated kinase 5 polyclonal antibody and its application in dental germ cells

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 eDNA sequence, PCR primers were designed to amplify PAK5-N terminal sequence. The PCR product was cloned into the expression vector pGEX-4T-1 EcoRUXhoI 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 fragrnent. 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 Condusions We successfully cloned PAK5-N terminal gene fiagment, and achieved protein expression, purification and production of PAK5-NT polyclonal antibody. The results of Western blotting indicated that PAK5 can be highly expressed in the dental germ cells, suggesting that PAK5 may play an important role in biological function of dental germ cells.