人抑瘤素M在大肠杆菌中的表达、纯化及其对胆固醇代谢关键基因表达的影响  

Prokaryotic expression and purification of human oncostatin M and its effects on the expression level of key players in cholesterol metabolism

作　　者：杜郁[1] 贾晓健[1] 袁芳[1] 王丽[1] 王丽非[1] 洪斌[1] 

机构地区：[1]中国医学科学院医药生物技术研究所卫生部抗生素生物工程重点实验室,北京100050

出　　处：《中国医药生物技术》2015年第3期203-210,共8页Chinese Medicinal Biotechnology

基　　金："十二五"国家科技支撑计划(2012BAK25B01);协和青年科研基金(3332013088)

摘　　要：目的构建人抑瘤素M(OSM)的原核表达载体,通过表达及纯化获得重组蛋白,为深入研究OSM的功能及机制奠定基础。方法利用PCR技术,从质粒pOTB7-h OSM中扩增出人OSM蛋白编码序列,与原核表达载体pET-16b连接,构建表达质粒pET16b-OSM。在BL21(DE3)工程菌中表达目的蛋白,Western blot法检测OSM蛋白的表达。为提高OSM的可溶性表达,采用共表达分子伴侣的方法提高其上清产物的表达量。将放大发酵的产物处理后,经镍亲和层析法纯化,获得OSM成熟蛋白,SDS-PAGE电泳分析蛋白纯度。MTT法检测OSM对A375细胞的抑制活性,实时荧光定量PCR试验分析OSM对Hep G2细胞中胆固醇代谢相关基因(LDLR、ABCA1、Apo A-I和SR-BI)表达的影响。结果成功构建pET16b-OSM原核表达质粒,酶切鉴定和测序分析与预期结果完全一致。在E.coli BL21(DE3)中实现了目的蛋白的表达,通过共表达分子伴侣质粒pTf16,进一步提高了可溶性表达的水平。放大发酵产物经镍亲和层析纯化获得OSM成熟肽,电泳检测纯度大于98%。MTT法检测所制备的OSM蛋白抑制A375细胞生长的EC50为315ng/ml。mRNA水平检测证实OSM能剂量依赖地上调胆固醇代谢相关的低密度脂蛋白受体和ABCA1基因的表达。结论成功构建了OSM的原核表达载体,获得了具有生物学活性的目的蛋白,证实了其对胆固醇代谢关键基因转录的影响,为下一步功能及机制研究奠定基础。Objective To construct a new human oncostatin M （OSM） prokaryotic expression vector, and obtain recombinant protein by expression and purification, which is expected to provide basis for the functional mechanism study. Methods The gene encoding mature human OSM protein was amplified from plasmid pOTB7-OSM by PCR and cloned into pET-16b. The fusion protein was expressed in E. coli BL21（DE3） and identified by Western blot. In order to increase the yield of soluble fractions, molecular chaperones were co-expressed along with the OSM. The protein in the cellular lysate supernatant was purified by nickel affinity chromatography after fermentation. The purified HIS-OSM was identified by SDS-PAGE. The biological activity was measured in a growth inhibition assay using A375 cells by MTT test. The functional activity of the HIS-OSM on expression of genes involved in cholesterol metabolism in HepG2 cells was analyzed in vitro by real time PCR assay. 〈 Results The prokaryotic expression plasmid pET16b-OSM was successfully constructed. The results identified by enzyme digestion and nucleotide sequencing corresponded exactly to the expected. The HIS-OSM protein expressed in E. coli BL21（DE3） and identified as OSM by Western blot, and the soluble form of the product was increased by co-expression of chaperone plasmid pTf16. SDS-PAGE analysis showed that HIS-OSM with purity 〉 98% was harvested by nickel affinity purification. Biological activity tests showed that HIS-OSM inhibits A375 human melanoma cell growth and the EC50 was 315 ng/ml. The mRNA levels of ABCA1 and LDLR were increased by HIS-OSM in a dose-dependent manner in HepG2 cells. Conclusion The prokaryotic expression vector of human OSM protein is successfully constructed. The function of OSM protein on gene transcription related to cholesterol metabolism is identified in vitro. This study establishes a foundation of further research and utilization of OSM.

关 键 词：抑瘤素M 原核表达 分子伴侣 胆固醇代谢 

分 类 号：R392[医药卫生—免疫学]