摘要
目的构建人热休克蛋白40(HSP40)的2种同源物HDJ-1和HDJ-2基因真核表达载体,观察其在人成神经细胞瘤细胞株(SK-N-SH)中的表达。方法从流产胚胎脑组织中抽提总RNA,RT-PCR扩增HDJ-1和HDJ-2cDNA,经限制性内切酶双酶切后,克隆至真核表达载体pcDNA3.1(+)和pEGFP-N1质粒中。HDJ-1和HDJ-2基因测序结果与基因库登录序列比对,序列正确的重组质粒用脂质体转染SK-N-SH细胞,Western blot和荧光显微镜观察基因表达情况。结果HDJ-1和HDJ-2基因测序结果与基因库登录序列比对显示完全一致。Western blot证实pcDNA3.1(+)/HDJ-1转染SK-N-SH细胞24h后有HDJ-1的过表达,至少持续72h;pEGFP-N1/HDJ-1和pEGFP-N1/HDJ-2转染的细胞有HDJ-1/GFP和HDJ-2/GFP融合蛋白的表达,至少持续72h。荧光显微镜观察到pEGFP-N1/HDJ-1和pEGFP-N1/HDJ-2转染的细胞中有绿色荧光蛋白的表达。结论本实验成功构建pcDNA3.1(+)/HDJ-1、pEGFP-N1/HDJ-1和pEGFP-N1/HDJ-2真核表达质粒,并在SK-N-SH细胞中表达。
Objective To construct the eukaryotic expression vectors of two human heat shock protein 40 （HSP 40） gene homologues named HDJ-1 and HDJ-2, and to detect their expression in human neuroblastoma SK-N-SH cell lines. Methods The total RNA was extracted from the aborted fetal brain. The full-length cDNA encoding HDJ-1 and HDJ-2 genes was obtained using the RT-PCR method and subcloned to eukaryotic expression vector pcDNA3.1 （ ＋ ） and pEGFP-N1 by restriction enzymes. The sequence of HDJ-1 and HDJ-2 genes was confirmed by blasting to Genbank. The recombinant plasmids were transfected into SK-N-SH cells by lipofectamin method. The expression of HDJ-1 and HDJ-2 genes in SK-N-SH cells was assayed by Western blot and fluorescence microscopy. Results The sequence of HDJ-1 and HDJ-2 genes was corrected by blasting to Genbank. Western Blot showed that HDJ-1 gene was overexpressed for at least 72 h, which was detected 24 h after pcDNA3.1 （ ＋ ）/HDJ-1 plasmids were transfected into SK-N-SH cells. The HDJ-1/GFP and HDJ-2/GFP fusion proteins were expressed in the cells transfected with pEGFP-N1/HDJ-1 and pEGFP-N1/HDJ-2 plasmids and lasted 72 h at least. Green fluorescence was observed by fluorescence microscopy on the cells transfected with pEGFP-N1/HDJ-1 and pEGFP-N1/HDJ-2. Conclusion The eukaryotic expression plasmids pcDNA3.1 （ ＋ ）/HDJ-1, pEGFP-N1/HDJ-1 and pEGFP-N1/HDJ-2 were successfully constructed and expressed in the dopaminergic neurons SK-N-SH cell lines.
出处
《上海交通大学学报（医学版）》
CAS
CSCD
北大核心
2006年第1期54-58,共5页
Journal of Shanghai Jiao tong University:Medical Science
基金
国家重点基础研究发展规划("973"项目)(G1999054008)
国家自然科学基金(39970263
30171025)
上海市卫生系统百名跨世纪优秀学科带头人培养计划(97BR001)资助项目
