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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。
OBJECTIVE: To prokaryotic express the recombinant fusion protein of leptin receptor (OBR) near the transmembrane region and to study the effect of SD on the fat deposition in SD rats. Methods A pair of primers specific to BamHⅠand HindⅢ was designed and used to amplify the extracellular extracellular transmembrane region of OBR (1705 ~ 2364 bp). After digested with restriction enzyme BamHⅠ and HindⅢ, The OBR recombinant plasmid was constructed and expressed in E.coli BL21 (DE3). SDS-PAGE and western blotting were used to identify the expression products. Subsequently, OBR recombinant fusion protein was prepared and immunized SD rats to observe the effects of OBR on body weight, feed intake, body length, Lee’s index, abdominal fat percentage, hepatic fat deposition and subcutaneous fat deposition . Results SDS-PAGE electrophoresis analysis and Western blotting results showed that the IPTG-induced expression of fusion protein of about 27 600 molecular weight in OBR recombinant E. coli cells. Body mass, body length, Lee’s index, abdominal fat rate and liver fat deposition were not significantly different from those of the control group after immunization of OBR recombinant fusion protein in extracellular and transmembrane regions of the OB rats, while the intake of OBR was significantly increased. The subcutaneous adipocytes Volume reduction. Conclusion The recombinant fusion protein of leptin receptor of Tibetan miniature piglets was successfully expressed in vitro and immunized SD rats can promote feed intake and inhibit subcutaneous fat deposition.