摘要
目的克隆、表达降钙素原(PCT),制备相应的多克隆抗体。方法根据GenBank上PCT的基因序列,设计10条用于基因拼接的DNA引物,利用PCR技术扩增PCT基因并构建pGEX-KG-PCT重组质粒,在大肠杆菌中表达融合蛋白PCT-GST,然后用融合蛋白免疫新西兰大白兔制备多克隆抗体,Western blot法进行生物活性鉴定,并利用琼脂免疫扩散实验检测了多克隆抗体的特异性和效价。结果成功获得PCT全长基因,重组质粒pGEX-KG-PCT在大肠杆菌中成功表达,SDS-PAGE电泳显示蛋白相对分子质量(Mr)大小为36 000左右。利用纯化的蛋白同时制备了GST和PCT-GST多克隆抗体,琼脂免疫扩散实验显示多克隆抗体可以特异性识别PCT,效价为1∶4。结论成功克隆、表达并纯化出PCT蛋白,制备了相应的多克隆抗体。
Objective To clone, express the procalcitonin （PCT） and prepare its polyclonal antibody. Methods We designed 10 DNA primers according to the encoding sequence of human PCT gene from GenBank, amplified PCT gene by PCR and constructed the recombinant vector pGEX-KG-PCT. Then the fusion protein, PCT-GST, was expressed in E. coil and purified by affinity chromatography. The corresponding polyclonal antibody was produced in the New Zealand white rab- bits immunized with the fusion protein, and its biological activity was detected by Western blotting. The specificity and titer of the polyclonal antibody was identified by agar gel immunodiffusion test. Results We successfully obtained the full-length PCT gene and expressed the fusion PCT-GST in E. coli. SDS-PAGE showed that relative molecular mass （ Mr） was about 36 000. Agar gel immunodiffusion test revealed that the prepared polyclonal antibody could specially recognized PCT and its titer was 1：4. Conclusion The PCT is successfully cloned, expressed and purified. The high specific polyclonal antibody of PCT is prepared.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2013年第5期504-506,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(30801014)
关键词
降钙素原
克隆表达
多克隆抗体
procalcitonin
clone and express
polyclonal antibody
