核心蛋白聚糖在毕赤酵母中的表达及活性检测  

Expression of Decorin in Pichia pastoris and Activity of Expressed Product

作　　者：郝小夏[1] 王桂琴[1] 王艳红[1] 李凌霞[1] 郭松佳[1] 兰晶[1] 

机构地区：[1]山西医科大学微生物学与免疫学教研室,太原030011

出　　处：《中国生物制品学杂志》2008年第3期190-193,共4页Chinese Journal of Biologicals

基　　金：山西省自然科学基金资助项目(20011061)

摘　　要：目的利用巴氏毕赤酵母真核表达系统表达人核心蛋白聚糖DCN,并检测其抗肿瘤活性。方法利用DCN的特异引物,通过RT-PCR扩增人DCN基因,与载体pPIC9K连接,将序列正确的重组体扩增后,酶切线性化,通过醋酸锂法转化酵母菌HIS-/GS115,G418筛选阳性克隆,用含1%甲醇的BMMY培养基诱导表达,并观察纯化的表达产物对人肝母细胞瘤细胞(HepG2)增殖的影响。结果表达产物作用于HepG2细胞后,与对照组相比,细胞增殖数量及速度均显著降低。随着表达产物浓度的升高及作用时间的延长,抑制作用也增强,并表现出浓度和时间依赖性关系。细胞形态学观察表明DCN对HepG2生长有明显抑制作用。结论已成功构建了pPIC9K-DCN真核表达载体,并表达了有活性的蛋白产物。Objective To express decorin （DCN） by using Pichia pastoris eukaryotic expression system and determine the anti-tumor activity of expressed product.Methods Amplify human DCN gene by RT-PCR using a pair of specific primers and insert into vector pPICgK. The constructed recombinant plasmid was identified by sequencing, amplified and linearized by digestion with Bgl Ⅱ , then transformed to P. pastoris HIS/GSll5 by lithium acetate method.The positive clones were screened with G418 and inoculated to BMMY medium containing 1% methanol. Purify the expressed product by ammonium sulfate precipitation and observe its effect on proliferation of HepG2 cells. Results Both the count and proliferation rate of HepG2 cells with addition of expressed DCN decreased significantly as compared with those without addition of DCN. The inhibiting rate of proliferation of HepG2 cells increased with the increasing concertrafion of expressed DCN and the increasing time for incubation, indicating the concentration- and time-dependent inhibitory effect of DCN on proliferation of HepG2 cells. Morphological observation showed significant inhibiting effect of DCN on growth of HepG2 cells. Conclusion Eukaryotic expression vextor pPIC9K-DCN was successfully constructed,and the DCN with activity was expressed.

关 键 词：核心蛋白聚糖 巴氏毕赤酵母 表达 活性 

分 类 号：Q786[生物学—分子生物学]