猪内源氨产生机制及其营养调控措施

猪内源氨产生过量对动物危害甚大,能引发多种炎症、抑制生长性能、降低免疫力、造成贫血和组织缺氧、丧失食欲,同时还能产生严重的环境污染。全面了解猪内源氨产生机制,并采取相应的营养调控减排措施从根本上减少氨的排放,对动物健康有着重要的意义。本文首先阐述了饲粮蛋白质降解产氨机制、肠道微生物产氨机制和谷氨酰胺的脱酰氨基作用产氨机制;然后从氨在肝脏和血液中循环过程解释其在体内的代谢过程;最后综述了目前国内外有关猪内源氨的营养调控措施,包括降低饲粮蛋白质水平、添加植物提取物和益生菌,以期为减少环境中氨的浓度及提高猪的生长性能提供理论依据。     

肌肉糖原代谢调控对猪肉品质的影响

肌肉糖原酵解潜能对肌肉品质有比较重要的影响,国内外学者对此做了大量的研究工作,本文从糖酵解潜能与肌肉品质的关系、影响糖原代谢的因素及通过糖酵解途径提高肌肉品质等方面进行综述,为探讨糖原代谢规律及未来实现优质风味猪肉的选育与营养调控提供重要理论依据。同时也为人类运动生理学及糖原代谢相关疾病的理论研究具有借鉴意义。     

Prokaryotic Expression and Immunoreactivity Analysis of VP2 Protein of Bluetongue Virus Serotype 1

The study was aimed to test the immunoreactivity of the VP2 protein of bluetongue virus serotype 1 (BTV-1) in vitro. Based on the published BTV-1 L2 gene of Y863 strain, specific cloning PCR primers were designed and synthesized. The L2 gene was amplified through RT-PCR method and then was purified and cloned into the expressing vector pEASY-Blunt E1. The cloned recombinant plasmids were identified. The positive recombinant L2 plasmid was cloned into BL21(DE3) competent cells to express VP2 protein. The acquired purified recombinant BTV-1 VP2 protein was analyzed through the methods of Western blotting, ELISA and blocking ELISA. The results showed that:BTV-1 VP2 protein was expressed as the inclusion bodies in the pEASY-Blunt E1 vector; 160 and 200 mmol/L glyoxaline were the best condition to wash down the expressed protein. The molecular weight of this purified recombinant protein with N-terminal His-tag was about 105 ku. Through the results of Western blotting, ELISA and blocking ELISA, it had been proved that this recombinant protein could combine with BTV-1 specific antibody and this combination could be blocked by BTV-1 virus. The study showed that the recombinant BTV-1 VP2 protein, expressed through the prokaryotic expression vector pEASY-Blunt E1, possessed good immunoreactivity and this study had established foundation for locating the serotypic epitopes of the BTV-1 VP2 protein.     

Analysis of TLR1,TLR2,TLR4 and TLR5 Expression in Different Tissues of Jinding Duck

The aim of this study was to explore the expression profiles of Toll-like receptor (TLR) in different tissues,and to lay the foundation of TLRs research for ducks.Ten male Jinding ducks of 300 days of age were slaughtered,and the blood sample and 14 different kinds of tissues (spleen, liver, testise, lung,hypothalamus,pituitary gland,skin,leg muscle,heart,kidney, chest muscle,caecum,small intestine and thymus) were collected. The Primer Premier 5.0 software was used to design specific primers and the quantitative Real-time PCR method was used to detect the relative expression levels of TLR1,TLR2,TLR4 and TLR5 mRNA. The results showed that there were only one specific peak on the melting curve of each gene amplification products,indicating that the specificity of the primers were strong. The amplification efficiency of the target genes was 101.4% to 105.0% and the correlation coefficient (R²) was 0.98 to 1.000. The four kinds of TLRs expressed in all of the 14 tissues and blood with varying abundance in different tissues for each of the 4 kinds of TLRs.The expression levels of TLR1 was lowest in hypothalamus,and highest in chest muscle. The expression levels of TLR2 was lowest in small intestine,and highest in lung. The lowest expressed tissues for TLR4 and TLR5 were testis, and the highest expressed tissues were skin.The study indicated that TLRs were widely expressed in various tissues of duck,which laid the foundation for further study on the role mechanism of duck TLRs in the process of pathogen infection.     

刚地弓形虫丝裂原活化蛋白激酶1基因克隆、表达及抗原性分析

克隆、表达刚地弓形虫(Toxoplasma gondii)丝裂原活化蛋白激酶1(TgMAPK1)基因片段,并分析其抗原性。提取弓形虫GT1株速殖子总RNA,逆转录合成cDNA。RT-PCR扩增TgMAPK1基因。扩增产物经双酶切后连接入pET28a(+)载体,重组质粒转化大肠埃希菌(Escherichia Coli)DH5α,阳性菌落经PCR和双酶切鉴定,并测序。将测序正确的重组质粒pET28a(+)-TgMAPK1转化至E.coli BL21并加入异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结合考马斯亮蓝染色检测表达产物。以鼠抗弓形虫血清为一抗,蛋白质印迹(Western blotting)分析重组蛋白的抗原性。RT-PCR扩增产物约为1 599bp。菌落PCR、双酶切和测序结果显示,重组质粒pET28a(+)-TgMAPK1构建成功。SDS-PAGE结果显示,经IPTG诱导获得相对分子质量约58 000的包涵体重组蛋白。Western blotting分析证实其能被鼠抗弓形虫血清识别。刚地弓形虫GT1株TgMAPK1基因片段可在原核表达系统中表达,且该重组蛋白具有抗原性。     

长链非编码RNA及其在畜禽生长调控中的研究进展

长链非编码RNA(long non-coding RNA,lncRNA)是一类长度大于200个核苷酸、没有编码蛋白能力的RNA,通常被认为是一类异构体RNA。近年来,越来越多的研究表明,lncRNA在许多重要的生物作用及人类疾病发展中起关键作用。lncRNA作为调节因子参与基因表达调控的各个层次,在表观遗传、转录调控及转录后调控等方面有着广泛功能。研究结果已表明,lncRNA表达水平的紊乱与人类各种癌症及其他疾病有很大的关系。作为基因调控网络的调控因子,lncRNA被越来越多的研究者所关注。相较于人类医学,lncRNA在畜禽上的研究尚处于起步阶段。作者对lncRNA的特点、分类、作用机制、研究方法及其在畜禽生长调控方面的研究进展进行了综述,并对其在畜禽养殖中的应用进行了展望,以期为lncRNA在畜禽生长调控方面开展深入研究提供理论基础。     

猪传染性胃肠炎病毒重组N蛋白间接ELISA抗体检测方法的建立

为建立一种检测猪传染性胃肠炎病毒(TGEV)血清抗体的间接ELISA方法,将TGEV的N基因片段克隆到p ET-28a载体中,转化至大肠杆菌BL21感受态细胞中进行诱导表达,并对表达的蛋白进行Western-blot鉴定。以纯化的重组N蛋白作为包被抗原,最终建立了检测TGEV抗体的间接ELISA方法。该ELISA方法与猪流行性腹泻病毒(PEDV)、猪瘟病毒(CSFV)、猪繁殖与呼吸系统综合征病毒(PRRSV)、口蹄疫病毒(FMDV)、猪圆环病毒2型(PCV2)等5种病毒阳性血清不发生交叉反应,表明建立的ELISA方法具有良好的特异性。本研究可为猪传染性胃肠炎的流行病学调查、诊断与防控奠定基础。     

Cloning,Expression of Clostridium perfringens α-toxin Gene and Preparation of its Antisera

One pair of primers had been designed and synthesized based on the α-toxin gene of Clostridium perfringens.The complete α-toxin gene fragment was amplified by polymerase chain reaction (PCR), and then was cloned into pGEM-T Easy vector to construct pGEM-T-α.Digested with EcoRⅠ and Hind Ⅲ, a fragment of 1125 bp was cloned into the expression plasmid vector pET-28a(+).The recombinant plasmid was transformed into the BL21(DE3)plys and induced by 1.0 mmol/L IPTG at 37 ℃.The expression product was found to be 46.1 ku as expected one identified by SDS-PAGE, and confirmed by Western blotting with Clostridium perfringens type A antisera, indicating similar reactivity with native α-toxin.Recombinant α-toxin protein was simultaneously found in culture supernatant, postsonic supertanant and inclusion bodies, most protein was expressed in inclusion bodies, which indicated recombinant α-toxin protein was expressed in the extracellular, periplasm and cytoplasm.Recombinant α-toxin protein in postsonic supertanant could not make mice die, indicating its non-toxicity.Toxin-antitoxin neutralization test showed that antisera of recombinant α-toxin protein were specific to α-toxin.Upon immunization of rabbit with the recombinant α-toxin protein, antisera with high antibody titer neutralizing 100 MLD toxin per 1 mL were prepared.     

Expression,Purification and Activity Evaluation of Mb1230 Gene of Mycobacterium bovis

To explore the value of Mb1230 protein of Mycobacterium bovis in the diagnosis of bovine tuberculosis,we obtained the Mb1230 gene by PCR and constructed recombinant plasmid pET-22b-Mb1230.Recombinant Mb1230 protein was obtained by IPTG induction and purified by affinity chromatography.The activity of the recombinant protein was evaluated by TST test,IGRA test and indirect ELISA.The size of the recombinant protein matched with the theoretical value proved by SDS-PAGE;Western blotting result showed that the recombinant protein could react with mouse anti-His antibody,and had specific band;The results of TST test,IGRA test and indirect ELISA test also showed the recombinant protein had antigenic activity.The results indicated the recombinant protein Mb1230 had good B cell and T cell activity,so,it had the potential application in the diagnosis of bovine tuberculosis.     

Study on Developmental Expression of ApoCⅡ Gene mRNA in Liver Tissues of Pigs

The aim of this study was to investigate the developmental patterns of ApoCⅡ gene mRNA in liver in Mashen and Large White pigs, and study the relationship between the expression level of ApoCⅡ and the lipid metabolism in pigs.The mRNA relative expressions of ApoCⅡ gene in liver at seven stages of 1,30,60,90,120,150,and 180-day old in Mashen and Large White pigs were determined by quantitative Real-time PCR.The results showed that the developmental trend of ApoCⅡ mRNA expression in liver between Mashen and Large White pigs was different.The ApoCⅡ mRNA abundance was decreased from birth to 60-day old,then increased at 90-day old,and decreased again after that in Mashen pig.However,the relative expression amount in Large White pig was gradually decreased from birth to 150-day old and increased again at 180-day old.Except for the ApoCⅡ mRNA expression amount at 1-day old,the differences of the expression amount at other stages in Mashen and Large White pigs were significant or extremely significant (P<0.05 or P<0.01).The ApoCⅡ mRNA expression in liver was affected by age and breed,and could play an important role in lipid metabolism in pigs.