Expression of a Novo Human Osteoprotegerin-Mycobacterial Heat Shock 70 Fusion Protein with Baculovirus-Insect Expression System
- VernacularTitle: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
- Author:
Shu LIU
;
Quan-Geng ZHANG
;
Yue ZHANG
;
Jing MA
;
Zhen-Long LIU
;
Wen-Ming ZHAO
;
- Publication Type:Journal Article
- Keywords:
Human osteoprotegerin Heat shock protein70 BaculoDirectTM baculovirus-expression system Insect cell Gene expression
- From:
China Biotechnology
2006;0(05):-
- CountryChina
- Language:Chinese
-
Abstract:
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
