猪传染性胃肠炎病毒S基因核酸疫苗免疫小鼠体液免疫功能变化的研究

本实验对TGEV S基因核酸疫苗免疫后体液免疫功能变化进行了研究,同时比较了两种核酸疫苗单独免疫小鼠后体液免疫应答的变化,证明重组核酸疫苗诱导小鼠产生体液免疫应答。     

扩增片段长度多态性（AFLP）在动物遗传育种中的应用进展

AFLP是一种新的分子遗传标记技术，它结合了RFLP和PCR技术的优点，具有RFLP的稳定性和PCR的高效性，被称为“最有威力的分子遗传标记”或“下一代分子标记”。相对于国外而言，我国在这方面的研究还比较少。本文综述了扩增片段长度多态性(AFLP)这种遗传标记的原理、技术特点、技术发展及其在动物遗传多样性、种质鉴定、遗传图谱构建、基因定位和标记辅助育种等研究中的应用。     

猪繁殖与呼吸综合征病毒GP5、M蛋白基因的克隆及其基因疫苗构建

参照GenBank中猪繁殖与呼吸综合征病毒(PRRSV)美洲型代表株VR2332 GP5和M蛋白基因序列,设计并合成2对引物,用RT-PCR方法分别扩增出PRRSV野毒株GP5和M蛋白基因603,525bp片段,并将其分别克隆到pMD18-T载体。测序正确后,将GP5和M蛋白基因分别克隆到真核表达载体pEGFP-C1上,成功构建基因疫苗表达载体pEGP5-C1和pEM-C1。小鼠免疫试验证实,这些基因疫苗质粒可以诱导小鼠产生特异性抗体,并在二免后1周开始检测到特异性淋巴细胞增殖反应。     

Expression and Bioinformatic Analysis of ADIPOQ Gene in Hanper Sheep

The aim of this study was to investigate the expression and biological characteristics of adiponectin (ADIPOQ) gene in longissimus dorsi muscle of Hanper sheep at different months of age.The ADIPOQ gene mRNA expression was detected in longissimus dorsi muscle of Hanper sheep at 3 different growth periods (1,7 and 13 months old) by Real-time quantitative PCR.The bioinformatics method was used to analyze the nucleotide and amino acid sequences of ADIPOQ gene,protein characteristics and network interaction protein.The results showed that ADIPOQ gene was expressed in longissimus dorsi muscle of Hanper sheep,which showed an increasing trend with the growth of the months age without significant difference (P>0.05).There was higher homology of ADIPOQ gene with Capra hircus (98.60%,99.00%) and Bos taurus (98.60%,87.00%) in nucleotide and amino acid sequences.The results of physical and chemical properties analysis showed that the molecular formula of ADIPOQ protein was C₁₁₇₂H₁₇₇₆N₃₁₄O₃₄₉S₅,the molecular mass was 26.00 ku,and the theoretical isoelectric point (pI) was 5.88,which suggested that the protein was acidic.The average value of ADIPOQ hydrophilic protein (GRAVY) was －0.403,which indicated that the protein was hydrophilic and didn’t belong to transmembrane protein.In addition,the signal peptide sequence was Met¹-Ala¹⁷,and the cleavage site was between Ala¹⁷ and Arg¹⁸.ADIPOQ existed collagen structure domain and C1Q conserved structure domain at position 45－101 and 101－237,respectively.The secondary structure mainly composed of random coil (66.95%).The construction of protein interaction network indicated that ADIPOQ might interact with ADIPOR1,ADIPOR2,CDH13 and LEP.The results provided a theoretical basis for further exploring the molecular biological functions of ADIPOQ gene in the process of muscle development in Hanper sheep.     

鸡褪黑激素受体基因在开产前后不同组织中的表达差异

为了从分子水平上了解鸡褪黑激素受体（MTNR）基因在开产前后不同组织中的表达差异,以β-actin基因为内标基因,运用实时荧光定量PCR方法,比较了MTNR1A、MTNR1B和MTNR1C这3个受体基因在心脏、肝脏、输卵管、卵巢、脑和视交叉各组织及在鸡开产前后表达量的差异。结果表明,MTNR1A只在开产后各组织中的表达量存在差异,其中以卵巢组织表达量最高,心脏中表达量最低。MTNR1B和MTNR1C的表达量在开产前或开产后均表现出组织间的显著差异;在开产前后的各组织中,MTNR1B的表达量均以视交叉为最高,卵巢为最低;而MTNR1C的表达量在开产前以心脏中最高,开产后在视交叉中最高,在卵巢组织中的表达量开产前后均为最低。同一组织开产后与开产前比较,MTNR1C基因在心脏、输卵管和脑组织中的表达量呈显著下调趋势。因此,3个受体基因在鸡的不同组织和生理时期存在表达差异。     

miR-127和miR-136在转基因克隆绵羊胎盘中的表达分析及其靶基因的鉴定

克隆动物胎盘发育缺陷是造成动物克隆效率低下的一个重要原因。目前认为克隆胎盘发育异常通常是由于一些基因表达的异常所致,与表观遗传修饰有关。microRNA是一种重要的表观遗传修饰方式,对动物胚胎发育和胎盘的形成有着重要的调控作用。为研究miR-127和miR-136在克隆动物胎盘中的表达情况及其与克隆动物发育缺陷的关系,本实验运用荧光定量PCR分析了死亡克隆绵羊胎盘和同期普通绵羊胎盘组织中miR-127和miR-136的相对表达量,并鉴定了miR-127和miR-136的靶基因及靶基因在胎盘中的表达情况。结果显示,miR-127和miR-136在克隆绵羊胎盘中的表达量分别增加了3.1和2.8倍。EGFP荧光敲除实验证实,胎盘发育相关基因Rtl1是miR-127和miR-136的靶基因,同时定量PCR分析发现Rtl1基因在克隆绵羊胎盘中的表达量降低了3/5。结果说明,miR-127和miR-136在克隆胎盘中的异常表达可导致Rtl1基因的低表达,这很可能是导致克隆动物死亡的一个重要原因。     

马鹿Zfx/Zfy基因的克隆及序列分析

根据GenBank中公布的牛Zfx/Zfy基因的mRNA序列,设计特异性引物,对马鹿Zfx/Zfy基因进行扩增,并对该序列进行分析。结果表明,克隆得到马鹿Zfx基因序列长为2 115 bp,GenBank登录号为KP257294.1,编码696个氨基酸;Zfy基因序列长为2 406 bp,GenBank登录号为KU041539,编码801个氨基酸。依据Zfx/Zfy基因氨基酸序列构建进化树,分析表明,马鹿Zfx基因与人的亲缘关系较近,与聚为一类的野牛、藏羚羊、虎、家猫、家牛、家犬、绵羊的亲缘关系稍远;Zfy基因与家牛、野牛、藏羚羊、绵羊的亲缘关系较近,与其他动物及人的亲缘关系较远。本研究首次克隆了马鹿Zfx/Zfy基因,为研究该基因蛋白质的结构和功能奠定了基础。     

Effects of forage type,body condition and single‐nucleotide polymorphisms in the bovine cytochrome P450 regulatory region on cow productivity*

Single‐nucleotide polymorphisms (SNP) in the coding sequence of cytochrome p450 (CYP3A28) have been associated with milk yield and composition, and calving traits in cows. In this study, we aimed to determine whether (i) the CYP3A28 regulatory region was polymorphic and (ii) SNP genotype, forage type, body condition and their interactions affect cow productivity. Primers for CYP3A28 promoter were designed to amplify a 483‐bp segment by PCR. Amplicon sequences revealed seven SNP (T‐318C, T‐113A, C‐189T, T‐78G, A6G, G17A and T21C) in Brahman (38 cows), Brahman x Angus reciprocal crosses (47 cows) and crossbreds (98 cows). Angus cows (n = 41) appeared to be fixed at those SNP locations. Genotype and forage {endophyte‐infected tall fescue [KY+; Lolium arundinaceum (Schreb.) S. J. Darbyshire] vs. bermudagrass [Cynodon dactylon (L.) Pers.]} effects on lifetime (8‐years) calving rate, and calf weaning weights and heights were determined in Herd 1 (126 cows); genotype and BC (low vs. moderate) effects on calving date and calving percent were determined in Herd 2 (98 cows). Four SNP (T‐318C, T‐113A, A06G and T21C) appeared to be related to cattle productivity, CC cows at T‐318C having a lower (p < 0.05) lifetime calving rate than TC or TT cows (65%, 85% and 81% respectively). Cows that grazed KY+ and were TT at T‐318C produced calves that tended (p < 0.07) to weigh less than their contemporaries. Moreover, calves of TT cows were shorter (p < 0.05) at weaning than calves of CC or TC cows. In Herd 2, moderate‐BC cows that were TT or AA at T‐318C, T‐113A, T‐78G, A6G and T21C had greater (p < 0.05) calving rates (74–80%) than heterozygous cows (46–60%), and low‐BC cows that were AA at G17A calved at least 6 days earlier (p < 0.05) than heterozygous cows. Our findings suggest that SNP in the CYP3A28 regulatory region of Brahman‐influenced cows are associated with cattle productivity.     

荧光定量PCR技术及其在动物营养中的应用

实时荧光定量PCR技术是一种通过检测PCR荧光信号的变化进行核酸定量的技术。目前,该技术已经应用于病原微生物、基因表达、基因组变异和多态性检测等许多方面。本文综述了定量PCR技术的基本原理及其在动物营养中的应用。