南方医科大学学报 ›› 2013, Vol. 33 ›› Issue (06): 832-.

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免疫瘦素受体胞外近跨膜区重组融合蛋白对SD大鼠脂肪沉积的影响

刘闻,吴丽红,徐名衬,郭日红,顾为望,施振旦,袁进   

  • 出版日期:2013-06-20 发布日期:2013-06-20

Effects of immunization with recombinant fusion protein of extracellular neartransmembrane domain of Tibet minipig leptin receptor on fat deposition in SD rats

  • Online:2013-06-20 Published:2013-06-20

摘要: 目的原核表达瘦素受体(OBR)近跨膜区重组融合蛋白并进行SD大鼠免疫实验,观察其对SD大鼠脂肪沉积的影响。方
法设计1对带有BamHⅠ和HindⅢ酶切位点特异性引物,扩增OBR胞外近跨膜区基因片段(1705~2364 bp),经酶切后连接到
表达载体pRSETA的BamHⅠ和HindⅢ两酶切位点之间,构建OBR胞外近跨膜区重组表达质粒并在大肠杆菌E.coli BL21
(DE3)中表达,SDS-PAGE电泳分析和western blotting鉴定表达产物。随后制备OBR胞外近跨膜区重组融合蛋白并免疫SD大
鼠,观察其对SD大鼠体质量、采食量、体长、Lee’s指数、腹脂率、肝脏脂肪沉积、皮下脂肪沉积的影响。结果SDS-PAGE电泳分
析和Western blotting鉴定结果显示,在IPTG诱导下OBR胞外近跨膜区重组表达菌表达了相对分子质量27 600左右的融合蛋
白。对SD大鼠免疫OBR胞外近跨膜区重组融合蛋白后,体质量、体长、Lee’s指数、腹脂率、肝脏脂肪沉积与对照组无明显差异,
而采食量显著增加、皮下脂肪细胞体积减小。结论成功表达西藏小型猪瘦素受体胞外近跨膜区重组融合蛋白,免疫SD大鼠可
促进采食和抑制皮下脂肪沉积。

Abstract: Objective To investigate the effect of immunization with prokaryotically expressed recombinant fusion protein of
extracellular near-transmembrane domain of Tibet minipig leptin receptor (OBR) on fat deposition in SD rats. Methods A pair
of specific primers containing BamHI and HindIII restriction enzyme sites was designed to amplify the extracellular
near-transmembrane domain (1705-2364 bp) of Tibet minipig OBR gene. After digestion, the amplified fragment was inserted
into the plasmid pRSETA between BamHI and HindIII sites. The recombinant plasmid was transformed and expressed in E.coli
BL21(DE3) and the product was analyzed by SDS-PAGE and Western blotting. SD rats were immunized with the fusion
protein, and the changes in body weight, feed intake, body length, Lee’s index, percentage of abdominal fat, liver fat
deposition and subcutaneous fat deposition were assessed. Results The recombinant fusion protein obtained (about 27.6 kD)
was expressed in E.coli induced by IPTG and identified by SDS-PAGE and Western blotting. The rats immunized with the
fusion protein showed no significant changes in body weight, body length, Lee’s index, percentage of abdominal fat or liver fat
deposition as compared with the control rats. Nevertheless, the immunization caused significantly increased feed intake and
significantly decreased volume of subcutaneous fat cells. Conclusions Immunization with the fusion protein of extracellular
near-transmembrane domain of Tibet minipig OBR can promote feed intake and suppress subcutaneous fat deposition in SD
rats.