利用昆虫杆状病毒表达系统表达人KCTD9蛋白

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

目的应用杆状病毒表达系统在昆虫细胞(sf9细胞)中表达人KCTD9蛋白,以进行其功能学研究。方法从pMD18-T-hKCTD9质粒中扩增hKCTD9全长基因片段,连接至pENTR/D-TOPO载体,将pENTR/D-TOPO-hKCTD9质粒与线形杆状病毒DNA在体外重组后,转染易感细胞系昆虫sf9细胞。采用免疫共聚焦和WesternBlot法分析融合蛋白的表达;在电子显微镜下观察病毒颗粒的形成。结果凝胶电泳分析hKCTD9基因存在于各载体中;激光免疫共聚焦显微镜证实感染重组杆状病毒的sf9细胞核内可见绿色荧光,提示目的基因表达正确;WesternBlot进一步显示hKCTD9蛋白大小约为55KD;透射电子显微镜观察发现被感染的sf9细胞核涨大、透亮,核内清晰可见大量的杆状病毒颗粒。结论 hKCTD9在昆虫杆状病毒表达系统中被成功表达,为进一步功能学研究奠定了基础。

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.