人脂联素基因在昆虫杆状病毒表达系统中的表达

Expression of human adiponectin gene in the baculovirus-insect cell expression system

目的利用杆状病毒表达系统探讨人脂联素(ADPN)基因在Sf9昆虫细胞中的高效表达。方法利用PCR方法扩增人脂联素基因,与pFastBac1质粒连接,转化入含有穿梭载体bacmid的大肠杆菌DH10Bac中,筛选发生转座作用的重组穿梭载体Bacmid-ADPN并转染Sf 9昆虫细胞,使其表达重组杆状病毒,经SDS-PAGE、Western blot检测表达产物。结果重组杆状病毒感染的Sf 9细胞形态变化明显,能够表达出与脂联素多克隆抗体相结合的蛋白,蛋白分子质量约为30 ku。结论人脂联素基因在真核细胞中成功得到表达,为进一步研究其生物学活性和作用机制奠定基础。

Objective To investigate the expression of recombinant human adiponectin （ADPN） using baculovirus expression system in Sf9 insect cell line. Methods PCR method was used to amplify human ADPN gene, and then the gene was inserted into plasmid pFastBacl. Recombinant plasmid was transformed into E. coli DH10Bac contai- ning a shuttle vector, Bacmid. Recombinant shuttle vector Bacmid-ADPN was obtained by site-specific transposition and transfect Sf9 cells. To identify the expressed product by SDS-PAGE,Western blot. Results Sf9 cells infected by the recombinant baculovirus was observed for morphological changes. The Mw of recombinant protein was about 30 ku and Western blot proved that expressed protein could combine with ADPN polyclonal antibody. Conclusion Human ADPN gene was successfully expressed in Sf9 cells, and thus provided the material for studying its bioactivity and mechanism of action.