人脂联素球状结构域基因的真核、原核表达及多克隆抗体的制备

发布时间：2019-06-18 19:14

【摘要】：研究目的利用杆状病毒表达系统表达出有生物学活性的人脂联素球状结构域(globular domain of adiponectin,gAd)蛋白,为研究gAd蛋白在糖尿病、心血管疾病、子痫等疾病中的作用机制奠定基础。构建gAd基因的原核表达载体,并经诱导表达、纯化gAd后,制备gAd多克隆抗体,为后续对人血清脂联素检测方法的建立奠定基础。研究方法 (1)第一部分:以人基因组为模板,PCR法扩增人gAd基因,将gAd基因与质粒pFastBacHTB连接,转化含有穿梭载体Bacmid的大肠杆菌DH10Bac。筛选转座成功的重组穿梭载体Bacmid-gAd,通过脂质体介导,转染昆虫细胞Sf9,经SDS-PAGE,免疫印迹法检测表达产物。 (2)第二部分:以人基因组为模板,用PCR方法扩增人gAd基因,构建原核表达载体PET-28a(+)-gAd,转化大肠杆菌BL21(DE3),IPTG诱导蛋白表达,经SDS-PAGE,免疫印迹法检测并鉴定表达产物,表达的目的蛋白用镍亲和层析柱纯化后免疫新西兰大白兔,制备多克隆抗体。研究结果 (1)利用真核杆状病毒表达系统后,重组杆状病毒感染的Sf9细胞形态变化明显,SDS-PAGE图谱,在相对分子质量15～25KD之间有一条新生蛋白带,免疫印迹法证实能与相应的抗His标签抗体结合。 (2)原核表达载体PET-28a(+)-gAd构建成功,并获得gAd重组蛋白,免疫印迹证实能与抗his标签检测抗体结合,重组蛋白经纯化后电泳条带单一,制备的多克隆抗体经间接ELISA法测得效价为1:32 000。免疫印迹证实,抗血清能特异性地识别gAd蛋白和胎盘组织中的脂联素。结论 (1)人gAd基因成功在昆虫细胞中表达,为后续进一步研究脂联素在糖尿病、心血管等疾病发病过程中的作用机制及治疗价值奠定了基础。 (2)成功构建原核表达载体PET-28a(+)-gAd,并表达、纯化重组蛋白gAd,制备的多克隆抗体特异性好,效价较高,为进一步制备针对gAd的单克隆抗体、进而建立检测脂联素的方法奠定了基础。
[Abstract]:Objective to express human adiponectin spherical domain (globular domain of adiponectin,gAd protein with biological activity by baculovirus expression system, and to lay a foundation for studying the mechanism of gAd protein in diabetes, cardiovascular disease, eclampsia and other diseases. The prokaryotic expression vector of gAd gene was constructed and expressed by induction. after purification of gAd, gAd polyclonal antibody was prepared, which laid a foundation for the subsequent establishment of human serum adiponectin detection method. Methods (1) the first part: using the human genome as template, the human gAd gene was amplified by PCR. The gAd gene was ligated with plasmid pFastBacHTB and transformed into E. coli DH10Bac. containing shuttle vector Bacmid. The recombinant shuttle vector Bacmid-gAd, which was successfully inserted into insect cells, was mediated by liposomes and transformed into insect cells. The expressed products were detected by SDS-PAGE, immunoblotting. (2) the second part: using the human genome as template, the human gAd gene was amplified by PCR, and the prokaryotic expression vector PET-28a ()-gAd, was constructed to transform the expression of E. coli BL21 (DE3), IPTG induced protein). The expression product was detected and identified by SDS-PAGE, immunoblotting. The expressed target protein was purified by nickel affinity chromatography and immunized with New Zealand white rabbit to prepare polyclonal antibody. Results (1) after using eukaryotic baculovirus expression system, the morphology of Sf9 cells infected with recombinant baculovirus changed obviously, SDS-PAGE map, and there was a new protein band between the relative molecular weight 15~25KD. Immunoblotting confirmed that it could bind to the corresponding anti-His label antibody. (2) the prokaryotic expression vector PET-28a ()-gAd was successfully constructed and the recombinant protein of gAd was obtained. The recombinant protein could bind to the antibody detected by anti-his label by immunoblotting. After purification, the electrophoresis band of the recombinant protein was single, and the titer of the prepared polyclonal antibody was 1:32 000 by indirect ELISA method. Western imprinting confirmed that antiserum could specifically recognize gAd protein and adiponectin in placental tissue. Conclusion (1) Human gAd gene is successfully expressed in insect cells, which lays a foundation for further study on the mechanism and therapeutic value of adiponectin in the pathogenesis of diabetes, cardiovascular and other diseases. (2) the prokaryotic expression vector PET-28a ()-gAd, was successfully constructed and expressed. The polyclonal antibody prepared by purified recombinant protein gAd, had good specificity and high titer, which laid a foundation for the further preparation of monoclonal antibody against gAd and the establishment of a method for the detection of adiponectin.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

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