利用中芯一号SNP芯片检测隆林猪全基因组拷贝数变异

[目的] 检测隆林猪的全基因组拷贝数变异。[方法] 采集33头隆林猪的耳组织样本,通过酚-氯仿法提取DNA后,使用猪中芯一号50K SNP芯片进行基因分型,得到的原始数据通过Genomestudio软件和Linux系统进行处理,使用CNVPartition和PennCNV软件分别检测拷贝数变异（copy number variation,CNV）,并利用Bedtools软件将CNV合并为拷贝数变异区域（copy number variation region,CNVR）,使用Biomart对CNVR进行基因定位,利用David网站对定位到的基因进行GO和KEGG富集分析,使用猪QTL数据库对共同CNVR进行QTL注释。[结果] CNVPartition软件共检测到260个CNVs,合并为47个CNVRs,其中缺失型40个、获得型5个、混合型2个,共定位到84个基因,显著富集到13条信号通路;PennCNV软件共检测到96个CNVs,合并为15个CNVRs,其中缺失型9个、获得型1个、混合型5个,共定位到8个基因,显著富集到8条信号通路;2个软件检测结果定位到的基因主要富集在嗅觉相关通路和G-蛋白偶联相关通路中,其中INPP5B、NEURL1和GAPDHS基因显著富集到精子活力通路;2个软件获得了3个共同CNVRs,其中缺失型、获得型和混合型均为1个,共定位到8个基因,显著富集到涉及嗅觉感官知觉的化学刺激检测通路、嗅觉受体活性通路、嗅觉转导通路、G-蛋白偶联受体活性通路、G-蛋白偶联受体信号通路、膜整体组件通路和质膜通路共7条信号通路;共有130个QTLs与3个共同CNVRs重叠,其中与背膘厚、肉质和乳头数相关的QTLs分别有11、9和6个。[结论] 隆林猪CNV可能与嗅觉功能、繁殖性能、背膘厚、肉质和乳头数性状相关。     

猪流行性腹泻病毒S2基因B细胞表位的筛选及其单克隆抗体的制备与鉴定

目的 应用生物学软件与单克隆抗体技术相结合的方法鉴定PEDV S2基因B细胞表位肽。方法 利用CLC Sequence viewer 6.8软件及在线数据库IEDB筛选出PEDV S2基因B细胞表位,并人工合成表位肽。将表位肽与钥孔血蓝蛋白（KLH）耦联后作为抗原,免疫雌性BALB/c小鼠,通过ELISA法筛选出抗体效价较高的小鼠进行1次加强免疫,3 d后取脾脏制备脾细胞悬液进行细胞融合。经HAT选择培养基培养筛选有效杂交瘤细胞,采用ELISA法筛选阳性克隆继续进行扩大培养。将部分阳性杂交瘤细胞进行小鼠腹腔注射,并收集腹水。通过ELISA方法分别检测小鼠腹水和单克隆细胞株培养上清液抗体效价,确定最高效价作为后备细胞株。结果 筛选出B细胞表位肽序列为:MQYVYTPTYYML;免疫多肽抗原后融合前血清抗体效价达到1:2 000;BALB/c小鼠腹水及单克隆细胞株培养物上清液ELISA检测结果显示抗体效价达到1:4 000。结论 筛选出了PEDV S2基因B细胞表位,为PEDV表位肽疫苗载体构建研究提供参考。     

福建莆田地区猪圆环病毒病的血清学调查

为了解莆田地区猪圆环病毒病的流行状况,2013—2014年间在莆田笏石、埭头、灵川、仙游地区等部分养猪场(户)共采集了322份未经PCV2疫苗免疫的猪血清样本,采用ELISA(酶联免疫吸附试验)方法对送检血清样品进行PCV2抗体水平检测。结果表明,322份被检血清中检出117份阳性血清,总阳性率为36.33%,其中以6~12周龄的小猪感染率最高,阳性率为55.56%。说明猪圆环病毒病在莆田市多数猪场中存在。     

猪繁殖与呼吸综合征疫苗的研究进展

猪繁殖与呼吸综合征(PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起的严重危害世界养猪业的一种传染病,该病可造成猪的繁殖障碍、呼吸系统病症与生长受阻。本文着眼于PRRSV疫苗的免疫原性、保护效力和安全性,涉及市场上现有的商品化疫苗与已报道的处于试验阶段的相关疫苗研究成果,以期为PRRSV的防控与疫苗研发提供有益的参考。     

膜分离法纯化浓缩的猪细小病毒灭活疫苗效果研究

为了研究纯化浓缩的猪细小病毒灭活疫苗的免疫效果,应用微滤/超滤管式陶瓷膜分离系统对猪细小病毒细胞收获液进行纯化浓缩,使浓缩液病毒平均含量由10~(4.7)TCID_(50)/mL提高到10~(6.5)TCID_(50)/mL;将纯化浓缩的病毒液经检验合格后,进行甲醛灭活,与注射用矿物白油佐剂制成油包水灭活疫苗;按照猪细小病毒灭活疫苗的质量标准,对制备的灭活疫苗进行检验与免疫效果试验。结果显示:纯化浓缩的猪细小病毒液杂蛋白去除率平均达到71.6%左右;制备的三组灭活疫苗检验均合格;免疫至63 d时,免疫猪体内抗猪细小病毒抗体(HI)水平分别为:纯化浓缩的灭活疫苗平均高达9.62 log_2稀释倍数,常规疫苗平均为8.78log_2稀释倍数,差异显著(P0.05)。试验表明:疫苗病毒细胞收获液进行膜纯化技术处理后,免疫效果显著优于未经纯化的常规灭活疫苗。     

硒蛋白X基因克隆、定点突变、原核表达及多克隆抗体制备

本试验旨在克隆鉴定猪硒蛋白X(Sel X)基因,将其定点突变后进行原核表达,获得重组蛋白,并制备猪Sel X多克隆抗体,为以猪为模型Sel X功能研究奠定基础。利用c DNA末端快速克隆(3'-RACE)技术从猪肝脏总RNA扩增出含开放阅读框(ORF)至poly(A)共1 215 bp的Sel X基因c DNA片段,其348 bp的ORF编码区和对应的氨基酸残基与人相应序列分别有88%和91%序列同源性,猪Sel X基因提交NCBI Gen Bank数据库,序列号为EF113597。ORF编码氨基酸残基中硒代半胱氨酸(Sec)位于C-端第95个残基,其编码密码子TGA经定点突变为半胱氨酸(Cys)的TGT后,将其插入载体p ET30,转入大肠杆菌BL21(DE3)中,在0.5 mmol/L异丙基硫代-β-D-半乳糖苷(IPTG)诱导2 h获得融合表达产物,经His-tag亲和层析后,获得分子质量为18.0 ku的纯化蛋白。将纯化获得的Sel X基因突变体融合蛋白(Sel Xm)免疫兔子,得到效价高达1∶20 000的多克隆抗体,免疫印迹(Western-blot)结果显示该抗体能特异性识别纯化的Sel Xm和猪肝脏中相应Sel X。本试验成功克隆并鉴定了猪Sel X基因,并制备了其多克隆抗体。     

PRV、PCV2与PPV三重PCR检测方法的建立与初步应用

为建立可同时检测猪伪狂犬病毒(PRV)、猪圆环病毒2型(PCV2)与猪细小病毒(PPV)的三重PCR方法,根据Gen Bank登录的病毒相关基因序列,选择保守区域设计引物,经过反应条件的优化,建立了可同时检测以上3种病毒的PCR诊断方法,扩增的目的片段长度分别为192 bp(PRV)、255 bp(PCV2)和759 bp(PPV)。对PRV、PCV2和PPV的核酸检测最低浓度分别为:2.5×10-2,2.2×10-3和3.7×10-2ng,证明该方法具有良好的特异性和较高的敏感性。应用该方法对56份临床样品进行检测发现,PRV、PCV2和PPV的阳性率分别为16.1%、50.0%和19.6%,二重感染率为12.5%,三重感染阳性率为3.6%。该方法的成功建立为快速高效地检测以上3种病毒提供了有效手段。     

The effects analysis of two neonicotinoid insecticides on in vitro maturation of porcine oocytes using hanging drop monoculture method

Acetamiprid (ACE) and imidacroprid (IMI) are known neonicotinoid insecticides with strong affinities for the insect-selective nicotinic acetylcholine receptor. These provide insect control by hyperstimulating insect nerves and are used for agricultural pest management. However, it has also been reported that ACE and IMI affect mammalian reproductive function. We determined the effects of ACE and IMI on the in vitro maturation of porcine oocytes. Significant decreases in nuclear maturation rates were observed in the ACE or IMI-exposed groups. Also, in matured oocytes from the ACE or IMI-exposed groups, irregular chromosomes were observed. Our results suggest that ACE and IMI exposure was detrimental to porcine oocytes and the extent of the effects depends on the concentration of exposure.     

Pathogenicity of emerging Japanese type 1 porcine reproductive and respiratory syndrome virus in experimentally infected pigs

To clarify the pathogenicity of Japanese type 1 porcine reproductive and respiratory syndrome virus (PRRSV) isolate in experimentally infected pigs, we evaluated clinical signs and monitored viremia for 21 days post-inoculation (dpi). Lungs were mottled, tanned and reddish in appearance; had lesions predominantly in the cranial, middle and accessory lobes; and failed to collapse at 10 dpi. Although microscopic lesions of lungs were reproduced using the Japanese emerging type 1 PRRSV isolate under experimental conditions, no significant differences were noted between the challenge and control groups regarding mean rectal temperature and daily weight gain. These results provide useful insights into the limited pathogenicity of single infection with the Japanese type 1 PRRSV isolate in piglets, which differ from findings in reported field cases.     

Downregulation of the expression of inhibin α subunit and betaglycan in porcine cystic follicles

Inhibins, as members of the transforming growth factor beta (TGF-β) superfamily, downregulate the synthesis and secretion of follicle-stimulating hormone (FSH) in an endocrine manner. The role of inhibin/betaglycan in the ovary regulation recently gained attention. To date, no data exist on the function of inhibin α subunit and betaglycan in cystic follicles. In this study, the expressions of inhibin α subunit and betaglycan in cystic follicles were investigated using immunohistochemistry, real-time PCR and Western blot analysis. Both inhibin α subunit and betaglycan immunoreactivities were mainly localized in the granulosa cells of follicles. Expression of inhibin α subunit and betaglycan was inferior in cystic follicles compared with that in normal large follicles. However, the result of enzyme-linked immunosorbent assay showed no significant difference in the decreasing in concentration of inhibin α subunit in cystic follicular fluid compared with the control (P>0.05). In this study, we explored the effects of FSH on betaglycan expression in granulosa cells in vitro. As expected, a significant increase in the expressions of betaglycan mRNA and protein in granulosa cells was observed in response to exogenous FSH (30 ng/ml) (P<0.05) compared with the control. Consequently, this study provides evidence that the expressions of inhibin α subunit and betaglycan are inferior in cystic follicles, and this may be caused by the decrease in FSH in the presence of a cystic follicle.