脂肪细胞膜抗血清对猪胴体品质和肉品质的影响

将36头5周龄的断奶上海大白猪随机分成6组.对照组C1和C2、皮下免疫试验组S1和S2、腹腔免疫试验组A1和A2,每组6头猪.试验周期为11周和17周.试验组A1和A2腹腔注射5.0 ml/kg BW山羊抗猪脂肪细胞膜抗血清,试验组S1和S2皮下注射5.0 ml/kg BW山羊抗猪脂肪细胞膜抗血清,对照组C1和C2以相同剂量注射正常山羊血清.11周末和17周末屠宰结果显示:①各试验组猪板油重和背膘厚都显著低于对照组(P＜0.05);11周末,与对照组相比,试验组S1和A1中猪眼肌面积均显著升高;17周末,腹腔免疫组猪眼肌面积显著高于对照组(P＜0.05).②11周末,腹腔免疫组猪半腱肌和里脊、皮下免疫组猪背最长肌和里脊中脂肪含量均明显低于对照组(P＜0.05);17周末,与对照组相比,腹腔免疫组猪半腱肌中脂肪含量显著下降(P＜0.05).③11周末,腹腔免疫组猪背最长肌和里脊中蛋白质含量显著高于对照组(P＜0.05);17周末,各试验组背最长肌和里脊中蛋白质含量与腹腔免疫组半腱肌中蛋白质含量均显著高于对照组(P＜0.05).表明,山羊抗猪脂肪细胞膜被动免疫具有较好改善猪胴体品质和肉品质的作用.     

Gene cloning of porcine adiponectin gene from adipose tissue and construction of its eukaryotic expression vector

To clone adiponectin (ADPN) gene from Shaziling porcine adipocyte and construct its eukaryotic expression vector, total RNA was extracted from subcutaneous fatty tissue. One pair of specific primers was designed by Primer 5.0 software according to the sequence of ADPN gene of porcine available in GenBank. The ADPN gene was amplified by PCR from cDNA and cloned into pMD18‐T vector to construct recombinant clonal vector pMD‐ADPN, sequenced and analysed. A recombinant expression plasmid pPICZaA‐ADPN was constructed by subcloning the cloned ADPN gene into the linearized pPICZaA vector. Then, the plasmid pPICZaA‐ADPN was expressed in Pichia pastoris (GS115) by electrotransformation. Western blot and Bradford analysis were used to determine the target protein induced by methanol. Results showed that the genome size of ADPN was 732 bp and encoded 244 amino acid, the nucleotide sequence of ADPN shared 100% identity with that of porcine available in GenBank. Western blot and Bradford analysis showed that the recombinant ADPN was expressed in GS115 correctly and has certain immune activity. The expression level of ADPN was 28.5 μg/ml. In conclusion, the recombinant ADPN could express in eukaryotic expression vector pPICZaA‐ADPN constructed in this study effectively.     

Adiponectin‐impaired adipocyte differentiation negatively regulates fat deposition in chicken

Adiponectin is a protein hormone secreted exclusively by adipocytes that plays an important role in the modulation of glucose and lipid metabolism. To investigate the effect of adiponectin on lipid metabolism in chicken, rosiglitazone (agonist of adiponectin) and dexamethasone (inhibitor of adiponectin) were used to treat 23‐day‐old broilers in vivo. To verify the functionality of adiponectin on fat deposition, chicken pre‐adipocytes were cultured in the medium containing 10 μg/ml adiponectin. Serum adiponectin and lipids and fat distribution were analysed. Oil Red O staining was used to determine lipid deposition in adipocytes. The expression levels of adiponectin, adiponectin receptors (AdipoR) and lipid metabolism–related genes in different tissues and pre‐adipocytes were measured using real‐time PCR, and the abundance of lipid metabolism–related proteins was measured by Western blot. Rosiglitazone increased serum adiponectin concentration and the expression levels of adiponectin and adiponectin receptor 1 (AdipoR1) in tissues and significantly decreased levels of serum lipids and fat deposition. Rosiglitazone significantly increased the expression levels of adipose triglyceride lipase (ATGL) and AdipoR1 and decreased the expression levels of fatty acid synthase (FAS). Dexamethasone had the converse effects compared with rosiglitazone. Oil red O staining results showed a marked decrease in fat deposition in cells treated with adiponectin. In adipocytes, adiponectin could decrease the expression levels of CCAAT/enhancer‐binding protein α (C/EBPα) and FAS and increased the expression levels of ATGL and AdipoR1. These results indicate that adiponectin has a remarkable effect on impairment of adipocyte differentiation, which contributes to the negative regulation of fat deposition in chicken.     

日粮中添加脂肪细胞膜蛋白抗体对猪脂肪沉积的影响

将160头体质量27 kg左右的杜×(长×梅)三元杂交商品猪随机分成试验组和对照组,每组设4个重复,每个重复20头,公母各半。在试验组基础日粮中添加75 mg.kg-1脂肪细胞膜蛋白抗体(简称OAAb),饲喂104 d后,从每组选出体质量相近的12头猪(公母各半)屠宰,记录内脏器官和脂肪组织质量,同时采集血样。结果显示:试验组平均日增质量、饲料报酬分别提高13.03%(P<0.01)和7.49%;试验组背膘厚、肾周脂肪指数、肠系膜脂肪指数和皮下脂肪指数分别下降24.14%(P<0.01)、27.27%(P<0.05)、20.42%(P<0.01)和29.11%(P<0.01),而肌内脂肪含量则不受影响;血清游离脂肪酸浓度在处理后明显升高(P<0.01),血清葡萄糖和甘油三酯浓度无明显差异;脾脏质量显著增加(P<0.05),但不影响肝脏和肾脏质量。提示:口服OAAb可抑制猪的脂肪沉积,改善猪的胴体品质。     

抗脂肪细胞特异膜蛋白单克隆抗体对猪内分泌代谢的影响*

 通过动物试验测定抗脂肪细胞特异膜蛋白单克隆抗体对猪内分泌代谢的影响。结果表明：给15kg仔猪阶段腹腔注射0.1,0.5和1.0mg/kg体重抗体后，血浆Ins含量不同程度降低，注射1.0mg/kg体重抗体Ins含量与对照组差异显著（P＜0.05）；血浆T₃,T₄,GH,IGF-I含量不同程度提高，注射1.0mg/kg体重抗体这些指标的含量与对照组差异显著（P＜0.05）；给60kg肥育猪腹腔注射抗体后，血浆Ins,T₃,T₄,GH,IGF-I的变化趋势与仔猪相同，但变化幅度不如仔猪明显。表明抗脂肪细胞特异膜蛋白单克隆抗体可降低血浆Ins含量，提高血浆T₃,T₄,GH,IGF-I水平，对猪内分泌代谢有明显调节作用。     

鸭脂肪细胞中circ-FBLN2鉴定与功能预测

Circular RNA(circRNA)是一类在转录过程中形成的首尾相接的环状非编码RNA,其在多种生物学过程以及疾病发生过程中发挥重要作用。基于前期鸭胚原代脂肪细胞分化过程中circRNA测序结果,本研究选择由FBLN2编码的circ-FBLN2进行验证分析。通过PCR扩增和焦磷酸测序技术获得接合序列,采用Rnase R消化实验进一步验证了circ-FBLN2的存在,采用核质分离技术得出circ-FBLN2主要存在于细胞质中,且其在脂肪细胞诱导分化后显著下调,在樱桃谷鸭各组织中的表达具有一定的差异,主要在脂肪和腿肌中表达。生物信息学分析结果显示,circ-FBLN2与预测到的靶标基因主要参与MAPK,Toll样受体,以及细胞因子与受体互作通路等信号通路,揭示其可能通过对靶基因的调控作用间接调控鸭脂肪细胞分化过程。     

ADCY2基因表达对延边黄牛脂肪前体细胞成脂分化的影响

本试验旨在研究环腺苷酸（3',5'-cyclic adenosine monophosphote,cAMP）环化酶合成酶2（adenylyl cyclase 2,ADCY2）基因对延边黄牛脂肪前体细胞成脂分化的影响。选取3日龄的延边黄牛腹股沟皮下脂肪组织进行脂肪前体细胞的分离、培养和诱导分化;设计ADCY2基因的干扰片段（siADCY2-1、2、3）,构建pEX4过表达载体;分别收集正常诱导（对照组）、干扰和过表达0、5和10 d的脂肪细胞。用实时荧光定量PCR检测过氧化物酶体增殖激活物受体（PPARγ）、CCAAT/增强子结合蛋白（C/EBPα）和ADCY2在细胞分化过程中mRNA的转录水平,并用Western blotting法检测其蛋白的表达;用油红O染色检测脂滴含量的变化;用甘油三酯试剂盒检测甘油三酯含量。试验结果表明,在成脂分化过程中,与未分化相比,ADCY2基因在分化的第5和10天表达量均极显著上升（P<0.01）,而分化第10天与第5天相比水平极显著下降（P<0.01）;siADCY2-1干扰效率最高,过表达ADCY2基因使其mRNA水平升高;与对照组相比,过表达组细胞内ADCY2基因mRNA水平提高了约4 000 000倍,脂肪细胞内脂滴含量和甘油三酯的含量极显著提高（P<0.01）,成脂关键基因C/EBPα和PPARγ表达极显著上调（P<0.01）,而RNA干扰组细胞内ADCY2基因mRNA水平极显著降低（P<0.01）,脂肪细胞内脂滴含量和甘油三酯的含量极显著下降,成脂关键基因C/EBPα和PPARγ表达极显著下调（P<0.01）。因此,ADCY2基因过表达能显著增加成熟脂肪细胞的脂滴含量及甘油三酯含量,促进成脂关键基因C/EBPα和PPARγ的表达,说明ADCY2基因对脂肪细胞分化具有正向调控作用。     

Generation and Identification of Monoclonal Antibody Against Porcine Adipocyte Plasma Membrane Proteins

Production of monoclonal antibody against porcine adipocyte plasma membrane proteins to explore a new way of controlling body fat deposition and improving carcass quality is discussed in this article. Membrane proteins of pig adipocyte plasma membrane proteins were extracted with the help of sucrose density gradient centrifugation, and two kinds of proteins were obtained. The monoclonal antibody （designated 3B2 and 3F3） of IgG1 and IgG2b subclass against adipocyte membrane proteins were produced by immunization, with adipocyte membrane proteins as an antigen, and its titer was 1：105 detected by enzyme-linked immunoadsorbent assay （ELISA）. The cell strains were identified by analyzing the number of chromosomes, the heat stability, the acid and alkali, the types and subtypes of immnoglobulin, and its peculiarities and affinities. Through identification, the chromosome number of hybridoma cell strains was from 80 to 100 and the strains formed good hybridomas colonies. The strains＇ affinity constants were 4.63 × 10^9 and 3.75 × 10^9 （mol L^-1）-1, respectively. At the same time, the McAb secreted was stable to environmental factors, such as, temperature, acid, alkali and so on. The monoclonal antibodies had been obtained and their specificity to porcine adipocyte plasma membrane proteins had been identified.