[Expression and activity determination of TNFR domain of osteoprotegerin in E.coli and corresponding antibody preparation].
Osteoprotegerin (OPG) plays an important role in the regulation of bone resorption and remodeling. The TNFR domain of OPG, which is involved in the inhibition of formation and activity of osteoclasts, was amplified by PCR and inserted into multiple cloning site of PET-28a. The recombinant plasmid was transferred into E.coli BL21 to express recombinant protein. It was found that expressed product existed in the form of inclusion body. The inclusion body was solubilized, renatured and purified by affinity chromatography. Polyclonal antibodies with high specificity were obtained from the serum of rabbit immunized with purified recombinant protein. Mice were used to determine the hypocalcemic effect of the recombinant protein. Results showed that the recombinant protein expressed in E.coli had the proper bioactivity.
Xiao
Hai-Long
HL
Hangzhou Institute of Calibration and Testing for Quality and Technical Supervision, Hangzhou 310004, China. xiao9988@126.com
Ren
Ai-Xia
AX
Zhang
Yao-Zhou
YZ
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002118:Calcium / Q000097:blood; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D053244:Osteoprotegerin / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011897:Random Allocation; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D014162:Transfection
D000906:Antibodies; D053244:Osteoprotegerin; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D002118:Calcium