Expression and identification of mutated osteoprotegerin in culture cells and larvae of silkworm.
The mutated osteoprotegerin (OPG-372) gene was inserted into the baculovirus transfer vector pBacPAK8, and the recombinant plasmid was co-transfected with linearized Bm-BacPAK6 virus DNA into BmN cells, then homologous recombination occurred inside the cells. The recombinant virus BmNPV-OPG-372 was screened and identified by Southern blotting. The recombinant human OPG-372 was expressed in cultured cells and the larvae of silkworm by inoculation of recombinant virus. The expression products were run on the SDS-PAGE and their immunoreactivities were determined by Western blotting. It was found that a 42 kD recombinant protein was expressed in BmN cells and a 46 kD one in larvae respectively. The bioactivities of the recombinant proteins were determined by hypocalcemic effect assay in the mice. The results showed that the recombinant proteins had a significant hypocalcemic effect on mice sera.
Xiao
Hai-Long
HL
Institute of Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, China.
Jin
Yong-Feng
YF
Zhang
Yao-Zhou
YZ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D012831:Bombyx / Q000235:genetics* / Q000378:metabolism* / Q000821:virology; D002118:Calcium / Q000097:blood; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism* / Q000506:poisoning*; D006801:Humans; D006996:Hypocalcemia / Q000097:blood / Q000139:chemically induced*; D007814:Larva / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation; D053244:Osteoprotegerin; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection / Q000379:methods
D006023:Glycoproteins; D053244:Osteoprotegerin; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; C506030:TNFRSF11B protein, human; C506031:Tnfrsf11b protein, mouse; D002118:Calcium