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瘦素与绿色荧光蛋白融合基因的合成及其高效表达

Fusion of Leptin Gene with Green Fluoresent Protein Gene and Its Overexpression in E.coli Cell
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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)~~
关键词 瘦素 绿色荧光蛋白(GFP) 融合基因 高效表达 Leptin Green Fluoresent Protein (GFP) Fusion Gene Overexpression
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  • 1[1]Ahima R S et al. 1996. Role of leptin in the neuroendocrine response to fasting[J]. Nature, 382:250~252.
  • 2[2]Bjorbaek C, Uotani S,da Silva B, Flier J S. 1997. Divergent signaling capacities of the long and short isoforms of the leptin receptor [ J ]. J Biol. Chem., 272: 32686~32695.
  • 3[3]Borstein S R et al. 1997. Evidence for a novel peripheral action of leptin as a metabolic signal to the adrenal gland. Leptin inhibits cortisol release directly[J]. Diabetes, 46: 1235~1238.
  • 4[4]Campfield L A et al. 1995. Recombinant mouse OB protein: Evidence for a peripheral signal linking adiposity and central neural networks [ J ]. Science ( Wash DC), 269: 546 ~549.
  • 5[5]Carro J F et al. 1997. Regulation of in vivo growth hormone secretion by leptin[J ]. Endocrinology, 138:2203~2206.
  • 6[6]Chehab F F et al. 1997. Early inset of reproductive function in normal female mice treated with leptin[J]. Science, 275:88~90.
  • 7[7]Cohen S L et al. 1996. Characterization of endogenous human leptin[J ]. Nature, 382:589.
  • 8[8]Coleman D L. 1978. Obese and Diabetes: two mutant genes causing diabetes- obesity syndromes in mice[J].Diabetologia,14: 141~148.
  • 9[9]Coleman D L. 1973. Effects of parabiosis of obese with diabetes and normal mice [J ]. Diabetologia, 9:294~298.
  • 10[10]Cusin I et al. 1995. The ob gene and insulin A relationship leading to clues to the understanding of obesity[J ]. Diabetes, 44: 1467~1470.

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