Prokaryotic expression of recombinant human p75NTR-Fc fusion protein and its effect on the neurite outgrowth of dorsal root ganglia neuron
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Prokaryotic expression of recombinant human p75NTR-Fc fusion protein and its effect on the neurite outgrowth of dorsal root ganglia neuron
Military Medical ResearchVol. 24, Issue 1, Pages: 1-9(2009)
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CLC:R741
Published:2009
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[1].Prokaryotic expression of recombinant human p75NTR-Fc fusion protein and its effect on the neurite outgrowth of dorsal root ganglia neuron[J].Journal of Medical Colleges of PLA,2009,24(01):1-9.
DOI:
[1].Prokaryotic expression of recombinant human p75NTR-Fc fusion protein and its effect on the neurite outgrowth of dorsal root ganglia neuron[J].Journal of Medical Colleges of PLA,2009,24(01):1-9.DOI:
Prokaryotic expression of recombinant human p75NTR-Fc fusion protein and its effect on the neurite outgrowth of dorsal root ganglia neuron
摘要
Abstract
Objective: To clone
express
and identify the extracellular domain gene of human p75 neurotrophin receptor with IgG-Fc (hp75NTR-Fc) in prokaryotic expression system
and investigate the effect of the recombinant protein on dorsal root ganglia (DRG) neuron neurites. Methods: The hp75NTR-Fc coding sequence was amplified from pcDNA-hp75NTR-Fc by polymerase chain reaction (PCR) and subcloned into vector pET30a (+)
in which hp75NTR-Fc expression was controlled under the T7 promoter. The recombinant vectors were amplified in E. coli DH5α and identified by PCR
enzyme digestion and sequencing
and then transformed into E. coli BL21 (DE3). The expression product was analyzed with SDS-PAGE and Western blot. Then after the recombinant protein purified with Protein A affinity chromatograph
and renaturated with dialysis
respectively
the effect of the recombinant protein on DRG neuron neuritis was further investigated. Results: The results of PCR
enzyme digestion
and sequencing demonstrated the success of inserting the hp75NTR-Fc fragment into vector pET30a (+). SDS-PAGE and Western blot showed a positive protein band with molecular weight about 50 kD in the expression product
which is accordant with the interest protein
and this band could be specifically recognized by rabbit anti-NGFRp75 antibody. The purified infusion protein following dialysis could promote neurite outgrowth of DRG neurons cultured with myelin-associated glycoprotein (MAG). Conclusion: The hp75NTR-Fc coding sequence was subcloned into the expression vector pET30a (+) correctly and expressed successfully in the prokaryotic expression system. The infusion protein could promote neurite outgrowth of DRG neurons cultured with MAG.
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