Exp ression of hKCTD9 protein in insect-baculovirus expression system in vitro

Objective To construct human KCTD9 protein in sf9 expression system and harvest the protein for further functional study. Methods To express hKCTD9 in insect cells,the full length hKCTD9 gene was obtained from pMD18-T-hKCTD9 plasmid. The gene was then inserted into pENTR/D-TOPO plasmid. After homologous recombination of the plasmid with BaculoDirectTM linear DNA,the recombinant baculovirus DNA containing the hKCTD9 gene fragment was obtained. Sf9 cells were transfected with baculovirus DNA via Cellfectin and cultured for 3 days. After 3 rounds amplification of recombinant baculovirus,the expression of hKCTD9 in sf9 cells was detected and identified by the laser confocal microscopy and Western blot respectively. And finally the baculovirus particle were catched by transmission electron microscopy(TEM). Results PCR confirmed the identity of recombinant plasmid pENTR/D -TOPO -hKCTD9 and the recombined baculovirus containing hKCTD9 fusion gene. The laser confocal microscopy found that the hKCTD9 fusion proteins were expressed efficiently in infected sf9 cells and localized in the nuclei of sf9 cells. The expression hKCTD9 fusion protein was 55KD as shown by Western blot,other than 43KD in gene bank. TEM photograph showed that numerous recombinant baculocirus located in nuclei and cytoplasm in sf9 cells. Conclusion Human KCTD9 protein is expressed successfully using baculovirus expression system,which provides the basic tool for its further functional study.