筛选和鉴定抗卵巢癌活性六肽的靶点蛋白  

Screening the receptor of anticancer hexapeptide in human ovarian carcinoma cells

作　　者：李小勤[1] 唐宜桂[1] 刘琛[1] 雷婷[1] 秦宜德[1] 

机构地区：[1]安徽医科大学生物化学与分子生物学教研室,合肥230032

出　　处：《安徽医科大学学报》2014年第7期879-882,共4页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:30872992)

摘　　要：目的采用pull-down技术筛选乳源抗癌六肽(PGPIPN)在人卵巢癌细胞株(SKOV3)上的靶点蛋白(受体)并进行鉴定。方法以PGPIPN的序列为参照,设计出PGPIPN基因,以BamHⅠ/XhoⅠ为酶切位点,将PGPIPN基因构建到表达质粒载体pGEX-4T-1中,转化到E.coli BL21中,用诱导剂异丙基-β-D-硫代吡喃半乳糖苷(IPTG)低温诱导表达的谷胱甘肽S转移酶(GST)标签融合蛋白作为诱饵蛋白;从SKOV3中提取的总蛋白作为捕获蛋白,运用GST pulldown技术,筛选出靶点蛋白质,运用SDS-PAGE电泳进行初步鉴定,并用异硫氰酸荧光素(FITC)标记的PGPIPN孵育鉴定。结果 SDS-PAGE电泳中有两条条带,一条为诱饵蛋白,一条为目的条带,在免疫荧光显微镜下观察到荧光PGPIPN结合到该条带上。结论筛选和鉴定了PGPIPN作用于卵巢癌细胞上的靶蛋白,为研究其抗癌的作用机制及其信号转导通路奠定了基础。Objective To screen and identify the receptor of anticancer hexapeptide derived in bata casein in human ovarian cancer cell lines （SKOV3） by pull-down. Methods According to its sequence, PGPIPN gene was designed and synthetized with the restriction sites of BamHⅠ/XhoⅠ, then was linked with the expression plasmid vector, pGEX-4T-1. This expression plasmid vector was transformed into E. coli, BL21. The GST-tagged fusion protein as the bait protein was expressed in BL21 by IPTG induced at low temperature. The total proteins were extracted from SKOV3 as the capture protein. By GST pull-down, the target protein was captured and screened from the above extracted proteins. By SDS-PAGE, the target protein was identified with PGPIPN labelled fluorescein isothiocyanate （ FITC） . Results The two protein bands were seen on SDS-PAGE gel, one being the bait protein and another being the target protein which was identified by immunofluorescence microscopy with the fluorescence labelled peptides. Conclusion The receptor of anticancer hexapeptide derived in bata casein is screened in human ovarian cancer cell lines （SKOV3） by pull-down. This study will lay the foundation for understanding the anticancer mechanism and transduction pathways of PGPIPN.

关 键 词：乳源抗癌六肽 GST pull-down 诱饵蛋白 捕获蛋白 靶点蛋白 

分 类 号：R392.11[医药卫生—免疫学] R394.2[医药卫生—基础医学]