【作者】 张国栋；

【导师】 杨在清；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 脂联素是由脂肪组织特异表达的细胞因子，分泌后进入血液循环，调节体内能量代谢。脂联素球状区在体内独立存在，与脂联素功能相似。虽然由脂肪组织特异表达，但是当动物脂肪过度沉积时脂联素表达及血液脂联素水平反而下降。另外，2型糖尿病、一些心血管疾病患者中的血液脂联素水平很低，而增加体内脂联素有助于这些疾病的改善。研究证实脂联素能增强胰岛素敏感性并有一定的抗炎症功能。 本文以猪为研究对象，旨在建立猪体内脂联素表达量的检测方法。首先用RT-PCR扩增脂联素编码区和球状区，构建pGEX-Adiponectin和pGEX-gAdiponectin表达载体，转化到BL21(DE3)中进行表达，得到猪脂联素GST融合蛋白及脂联素球状区GST融合蛋白。然后用重组蛋白免疫新西兰白兔而产生兔抗猪抗血清。其次建立了半定量PCR方法研究瘦肉型和脂肪型猪脂联素表达差异。半定量PCR要求PCR扩增在指数期进行，所以先要摸索指数期循环数，发现在25～29个循环脂联素和β-actin均处于指数期。以27为最终循环数进行半定量PCR，结果显示瘦肉型猪的表达量高于脂肪型。第三建立了竞争性PCR方法对长白猪脂联素mRNA进行绝对定量。用限制性内切酶Ava Ⅰ处理脂联素编码区，回收N端267bp和C端372bp片段，连接，然后进行PCR扩增，从而得到竞争性模板。对竞争性模板进行稀释，与猪脂联素cDNA进行竞争性PCR反应，通过分析PCR产物计算出长白猪脂肪组织中脂联素mRNA的含量。最后应用辐射杂种细胞系技术将猪脂联素定位在13号染色体。更多还原

【Abstract】 Adiponectin is a cytokine produced exclusively in adipocyte. After released from fat cells, it circulates along with blood cycle and regulates energy metabolism in vivo . Globular domain of adiponectin which present in plasma has similar function to full length adiponectin. Though adipose-specific, expression level and plasma concentrations of adiponectin decrease when animal has exess adipose tissue. In addition, plasma concentrations of adiponectin is significantly lower in type 2 diabetes and some cardiovascular disease patients than healthy one. Syndrome of these disease will be improved if plasma concentrations of adiponectin elevated. It was approved that adiponectin can increase insulin sensitivity and has anti-inflammatory effect.In this study, we try to establish assays to understand expression profile of pig adiponectin in vivo. First, coding sequence and globular domain of pig adiponectin were amplified with RT-PCR, and recombinant expression vector pGEX-Adiponectin and pGEX-gAdiponectin were constructed. Recombinant vector were induced in BL21(DE3) and fusion protein adiponectin-GST and globular adiponectin-GST were produced. Antiserum were raised against the recombinant proteins after immunized them to New Zealand rabbits. Second, a semi-quantitative PCR method was employed to measure adiponectin gene expession level of pig in vivo. Semi-quantitative PCR should be carried out at exponential phase. Cycles between 25 and 29 were appropriate for both adiponectin and 3 -actin. Then Semi-quantitative PCR was performed with 27 cycles and the result was that adiponectin mRNAs in lean pigs were higher than obese one. Next we established competitive PCR assay for quantification of pig adiponectin mRNA. Coding sequence of pig adiponectin was digested with restriction endonuclease Aval, 267bp fragment of N terminus and 372bp fragment ofC terminus were purified and ligated. PCR was perfomed with the ligation product as template and competitive template was produced.After series dilution of the competitive template, PCR was performed using different concentration of them and pig adiponectin mRNA as template. The PCR product was analyzed and pig adiponectin mRNA was calculated. Finally, we located pig adiponectin on chromosome 13 using Radiation hybrid panel.更多还原