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  

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

作　　者：刘闻[1,2] 吴丽红[1,2] 徐名衬[1] 郭日红[3] 顾为望[1,2] 施振旦[3] 袁进[1] 

机构地区：[1]南方医科大学实验动物中心暨比较医学研究所,广东广州510515 [2]东莞松山湖明珠实验动物科技有限公司,广东东莞523808 [3]华南农业大学动物科学学院,广东广州510642

出　　处：《南方医科大学学报》2013年第6期832-837,共6页Journal of Southern Medical University

基　　金：国家973计划(2011CBA01006);863项目(2011AA100304-1);广东省科技计划项目(2009B060300009,2010A011200003,2010A030400001,2011B060300028,2010B060500001);广州市科技计划(2010U1-E021);广州市重大科技专项(2010U1-E00811-3)~~

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Hindlll酶切位点特异性引物，扩增OBR胞外近跨膜区基因片段（1705～2364bp），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27600左右的融合蛋白。对SD大鼠免疫0BR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。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.

关 键 词：瘦素受体 原核表达 重组融合蛋白 脂肪沉积 

分 类 号：R-332[医药卫生]