摘要
目的 :为了便于追踪瘦素 (Leptin)在体内的去向 ,对其进行体内定位 ,设计合成Leptin荧光分子蛋白探针。方法 :用PCR技术将LeptincDNA与绿色荧光蛋白cDNA构建成融合基因LG ,然后将其克隆进原核表达载体pET3c中 ,构建成原核表达工程菌BL2 1(DE3) /pET3c LG。用Western blot杂交检测重组蛋白的免疫原性 ,用荧光显微镜观察融合蛋白及其诱导菌休的发光活性。结果 :DNA测序结果证实设计合成的融合基因与预期一致 ;构建的工程菌BL2 1(DE3) /pET3c LG获得了表达 ,融合蛋白的表达量占菌体总蛋白的 4 0 %以上 ;Western blot杂交检测表明重组蛋白具有Leptin的免疫原性 ;经IPTG诱导后的菌体及蛋白粗提液在荧光显微镜下可观察到其能发射出强烈的绿色荧光。结论 :融合基因在大肠杆菌中实现了高效表达 ,表达的融合蛋白具Leptin的免疫原性和GFP的发光特性。为利用绿色荧光蛋白标记Leptin在动物体内的药物治疗的研究提供了一种新的途径。
AIM:To track the trace of Leptin in vivo ,designing constructing the Leptin fluoresent protein probe.METHOD:With polymerase chain reaction(PCR),constructing the LG fusion gene,which the Leptin cDNA was connected with 5′end of the cDNA of green fluoresent protein gene(GFP).The LG fusion gene was cloned into pET3c expression vector.The recombinant plasmid was transformed into BL21(DE3) for expressing the aim protein.Using the Western bloting to analyze the immunogenic of the Leptin,and using the fluorescence microscope to observe the green fluorescence of the fusion protein and the cell after induceing.RESULT:It was confirmed that the DNA sequence of the synthetic fusion gene was identical with that of the designed gene by DNA sequencing.The recombinant was expressed in BL21(DE3) successfully.The expression level of the recombinant LG in BL21(DE3) was above 40% of the total protein.Western blot analysis shows that the recombinant protein has the immunocompetence of leptin.Fusion protein and the cell after induceing can emit the green fluorescence.CONCLUSION:Recombinant LG gets overexpression in E.coli BL21(DE3) successfully.The fusion protein of expressed has the immunocompetence of Leptin and the normal function of the GFP.
出处
《中国药科大学学报》
CAS
CSCD
北大核心
2004年第3期276-279,共4页
Journal of China Pharmaceutical University
基金
广东省生物技术与生物药物重点实验室基金资助项目 (No .2 0 0 2B60 119)~~