An Zheng-wen, Liu Hong-wei, Jia Zhi-min, Li Zhao-feng, Strömblad Staffan, Zhang Hong-quan
Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2006 Jun;26(6):730-3.
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.
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.
We successfully cloned PAK5-N terminal gene fragment, 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.
克隆PAK5的N端序列，在大肠杆菌中表达以制备其多克隆抗体，并研究PAK5在牙胚细胞中的作用。
根据人PAK5 cDNA序列设计PCR引物，扩增PAK5的N端序列。将PCR产物克隆到表达载体pGEX-4T-1的EcoRI/XhoI位点，通过琼脂糖凝胶电泳鉴定重组质粒，随后进行DNA序列分析。将重组质粒转化到大肠杆菌BL21中，用IPTG诱导GST融合蛋白表达。用谷胱甘肽-琼脂糖珠纯化GST融合的PAK5-N端片段。用纯化的GST-PAK5 N端融合蛋白免疫兔子获得抗PAK5多克隆抗体，并用蛋白A珠纯化抗体，用于检测PAK5在牙胚细胞中的表达。
成功克隆了PAK5的N端基因片段，实现了蛋白表达、纯化及PAK5-NT多克隆抗体的制备。Western印迹结果表明，PAK5在牙胚细胞中高表达，提示PAK5可能在牙胚细胞的生物学功能中发挥重要作用。
