Construction of Prokaryotic Expression Vector for pbv220/NT4–ADNF–9

Abstract

ObjectiveTo construct a prokaryotic expression vector bearing fusion gene NT4–ADNF–9 for future studies on genetic therapies for sensorineural deafness. MethodsDouble strand ADNF–9 cDNA was synthesized using asymmetrical primer/ templates and ligated to the 3’ terminal of signal and leader peptides of neurotrophin 4 (NT4). The fusion gene NT4–ADNF–9, was subcloned into prokaryotic expression vector pBV220, and named pBV220/ NT4–ADNF–9. DNA sequence of the fusion gene was analyzed. The fusion protein was isolated by SDS–PAGE and its bioactivity was evaluated using primary culture of day 8 chicken embryonic DRGcells. ResultsThe correct sequence of fusion gene NT4–ADNF–9 was successfully subcloned into the pBV220 vector. The expressed ADNF–9 protein showed its effects in promoting cell survival and neurite growth. ConclusionProkaryotic expression vector pBV220/NT4–ADNF–9 was constructed successfully and the expressed fusion protein demonstrated satisfactory bioactivity.