人骨保护素-热休克蛋白70融合蛋白在昆虫细胞中的表达和活性分析

目的:利用BaculoDirect杆状病毒表达系统融合表达人OPG功能片段p22-194和分枝杆菌HSP70 p111-125基因，并鉴定重组蛋白及其生物学活性。方法:将编码人OPG功能片段和分枝杆菌HSP70功能片段基因克隆至杆状病毒转座载体，将重组转座载体与BaculoDirectTM Linear DNA进行LR重组连接反应，构建出重组杆状病毒DNA，转染Sf9昆虫细胞，获得重组病毒。在Sf9细胞中进行表达，并对表达产物进行SDS-PAGE电泳、Western blotting分析，用Ni柱纯化。采用破骨细胞生成抑制试验和抑炎试验鉴定表达产物的生物学活性。结果:重组病毒在感染昆虫细胞后48h开始出现一相对分子质量为28 kDa大小的特异条带，感染后72～96 h蛋白量达到高峰。破骨细胞生成抑制实验及抑炎试验结果显示，重组蛋白能明显抑制破骨细胞的生长和分化，同时亦具有抑制炎症反应的作用。结论:利用杆状病毒表达系统在昆虫细胞中成功表达OPG-HSP70融合蛋白，该融合蛋白具有抑制破骨细胞生成和抑制炎症反应生物学活性。

Expression of a Novo Human Osteoprotegerin-Mycobacterial Heat Shock 70 Fusion Protein with Baculovirus-Insect Expression System

Objective:Osteoprotegerin (OPG) is a key molecule negatively regulating osteoclast differentiation and activation; and the conserved mycobacterial heat shock 70 (HSP70) peptide p111-125 has also been found to inhibit inflammation reactions in chronic arthritis. BaculoDirectTM baculovirus expression system was selected to express recombinant OPG-HSP70 in insect cells.Methods:The human functional fragment (p22-194) of OPG and functional fragment (p111-125) of mycobacterial HSP70 gene were cloned into the transfer vector pENTRTM/SD/D-TOPO. The recombinant plasmid was performed an LR reaction with the BaculoDirectTM Linear DNA to generate recombinant baculovirus DNA. The cultured Sf9 insect cells were directly transferred with the recombinant baculovirus DNA, and the pure recombinant baculovirus was obtained. Then recombinant baculovirus was infected Sf9 insect cells again to express the OPGHSP70 gene.Results:The target protein was detected at the time of 48h post infection, reached at highest yield at the time of 72h post-infection. A 28kDa protein immunostaining band was detected by Western blotting from lysate of those cells.And the purified protein was obtained by using Ni-NTA system. Functional stuies on the fusion protein showed it significantly reduce osteoclast cell number［(3.10±0.640) cells under each microscope field in treatment group by comparing to (10.70±0.817)cells in the control group］ in the osteoclast inhibition test，and reduce the inflammation reaction in a delayed type hypersensitivity (DTH) mice model (P<0.01).Conclusion:OPGHSP70 fusion protein was successfully expressed by using eukaryotic protein expression system (baculovirus-expression system). Results from functional studies on it suggested that the novo fusion protein may be a potential therapeutic agent for Rheumatoid Arthritis.