抗丝状支原体丝状亚种单克隆抗体的制备及初步应用

旨在制备抗丝状支原体丝状亚种(Mmm)的特异性单克隆抗体(MAb),为牛传染性胸膜肺炎(CBPP)病原诊断的免疫学方法提供特异性抗体。本研究利用生物信息学技术分析了Mmm国内分离株Ben-1不同传代株的全基因组序列,选取M0071蛋白作为研究对象。将原核表达的可溶性重组蛋白M0071(rM0071)作为免疫原免疫BALB/c小鼠,通过有限稀释法和间接ELISA方法筛选得到能稳定分泌抗rM0071蛋白的单克隆抗体的杂交瘤细胞株。进一步制备单抗腹水并纯化,利用Western blot方法对该单抗进行特异性鉴定,同时测定其抗体效价和抗体亚类。随后利用间接免疫荧光试验(IFA)评价该单抗对细胞感染Mmm的检测能力。结果表明成功获得1株单克隆细胞株3C4A1,将其分泌抗体命名为MAb 3C4A1。特异性结果表明,MAb 3C4A1能与Mmm的分离株和标准株发生特异性反应,而不与山羊支原体山羊肺炎亚种、丝状支原体山羊亚种、牛鼻支原体、无乳支原体、牛支原体、leachii支原体和牛A型巴氏杆菌等发生反应。抗体亚类鉴定MAb 3C4A1属于IgG1亚类、轻链为κ链。经间接ELISA测定其抗体效价为1∶256 000。IFA试验结果表明,MAb 3C4A1仅与感染EBL细胞的Mmm发生绿色荧光反应,而与牛鼻支原体、无乳支原体、牛支原体感染的细胞不发生荧光反应,特异性良好。本研究制备的MAb 3C4A1具有良好的特异性和免疫反应性,可作为CBPP病原免疫学诊断的工具,为进一步研制CBPP病原鉴别诊断试剂盒提供了基础材料。     

Experimental Pathological Study of Acute African Swine Fever

To study the clinicopathology and histopathology of African swine fever (ASF), and to explore the internal relationship between pathological changes and disease occurrence and development and its pathological mechanism, 13 Landrace pigs with bodyweight about 20 kg were intramuscular injected with African swine fever virus (ASFV), strain Pig/HLJ/18 at a dose of 10²HAD₅₀·mL^-1. During the experiment, all the dead pigs were systematically dissected and sampled, paraffin sections were produced, and haematoxylin-eosin staining was performed. Clinicopathological evaluation standards for acute ASF were established, then pathological lesions (classification variables) were expressed by counting frequency and percentage, and the lesion degree (continuous variables) was graded and scored according to different pathological changes of various tissues and organs. The results showed that all infected pigs were in line with the clinical characteristics of ASF, including acute, febrile and highly infectious, with a 100% incidence rate and 100% mortality. The dead pigs showed typical characteristics of septicemia, cadavers prone to corruption, blood clotting adverse or hemolysis, rigor mortis incomplete. The main pathological lesions were hemorrhagic necrotizing lymphadenitis, acute inflammatory splenomegaly (septic spleen), cerebral edema, pulmonary edema and lung consolidation et al. The spleen and lymphonodus are the target organs attacked by ASFV, with the most significant lesions, the earliest occurrence time, the longest duration and the highest frequency. The most prominent pathological changes are blood circulation disorders, including multiple pathological manifestations such as edema, hyperemia, congestion, hemorrhage, infarction, disseminated intravascular coagulation (DIC), and the most important characteristics are hemorrhagic lesions. The inflammatory reaction of lymphocytic exudation caused by ASFV runs through the whole process, especially in the middle and later stages of the course. The results suggest that the main pathological process of acute African swine fever is a typical immune/inflammatory cascade reaction and severe systemic blood circulation disorder, which resulted in the high incidence rate and high mortality rate of acute ASF.     

猪增生性肠炎套式PCR检测方法的建立及初步应用

本试验旨在建立检测猪增生性肠炎(porcine proliferative enteropathy,PPE)的套式PCR方法,为临床快速鉴别诊断和防控该病提供病原学依据。根据GenBank上已发表的的劳森氏胞内菌(Lawsonia intracellularis,LI)的基因组序列（登录号：AM180252）设计2对特异性引物,以疑似PPE病猪粪便中纯化的DNA为模板,建立了套式PCR方法,并对PCR产物进行序列测定,所扩增的序列与GenBank上登录的劳森氏胞内菌相应序列的同源性在99%以上。应用本试验建立的套式PCR方法对疑似PPE病猪的粪便进行检测,结果表明,本方法具有快速、方便和敏感度高等优点,可用于PPE的临床检测。     

家鹑与野生日本鸣鹑群体微卫星DNA标记的遗传学分析

选择10个微卫星标记位点，合成引物对微山湖野生日本鸣鹑和家鹌鹑各40只进行PCR扩增，变性聚丙烯酰胺凝胶电泳检测分型，并对其品种内和品种间的遗传变异进行群体遗传学分析，从分子水平上揭示这两个鹌鹁群体的遗传结构关系、亲缘关系和遗传分化程度，为鹌鹑的遗传资源研究提供新资料。     

栗、白、黄3种鹌鹑羽色的基因和表型关系及应用的研究

朝鲜龙城系蛋用鹌鹑已分化出 3种毛色 :即龙城栗羽 ,北京白羽和黄羽。试验证明 ,3种羽色白均为伴性遗传并且存在如下关系 :白♂×栗♀→F1 栗♂白♀ ;黄♂×栗♀→F1 栗♂黄♀ ,后代均自别雌雄 ,而黄♂ (♀ )×白♀ (♂ )正反交 ,后代均能自别雌雄 ,黄♂×白♀→F1为栗♂黄♀ ;白♂×黄♀→F1 为栗♂白♀ ,试验进一步表明 :栗 (Y)和黄 (y) 在Z染色体上是一对等位基因 ,栗相对于黄为显性 :白羽是由位于Z染色体上的隐性白化基因b对Y或y上位作用所致。栗和黄两种鹌鹑的Z染色体上均有b的不白化等位基因B ,B相对于b为显性     

细菌CpG DNA对鸡球虫弱毒苗Immucox的免疫增强效应

采用细菌CpG DNA作鸡球虫弱毒苗Immucox佐剂以探索其应用的可行性。将制备的细菌CpG DNA和球虫疫苗Immuncox单独或联合免疫接种肉鸡,第2次免疫后7d进行攻虫,口服接种柔嫩艾美儿球虫孢子化卵囊105个/只。感染后第7天,对试验动物进行称重并屠杀,分别观察盲肠病变积分、每克粪便卵囊排出数和体增重情况。结果:在相对增重方面,细菌CpG DNA+疫苗组高于感染非免疫组、疫苗组和CpG组,且与它们均差异显著(P<0.05);在卵囊排出总数和盲肠病变平均记分方面,细菌CpG DNA+疫苗组低于感染非免疫组、疫苗组和CpG组,且与之比较差异显著(P<0.05);在抗球虫综合指数方面,疫苗+细菌CpGDNA组达到172。结果表明用细菌CpG作鸡球虫弱毒苗Immucox的佐剂可提高该疫苗免疫保护效果。     

液相色谱-质谱联用技术在检测食品中兽药残留的应用

动物性食品安全问题现已成为一个全球性的议题。动物性食品药残超标、安全性差的问题十分突出。建立一种准确、灵敏、可靠的分析动物性食品中兽药残留的方法刻不容缓。介绍了液相色谱-质谱联用技术(LC-MS)应用于动物性食品中几种常见兽药残留的检测方法。     

影响牛肉品质的宰后因素

本文从肉牛屠宰后肌肉变化,分析了几种影响屠宰后牛肉品质的相关生理过程,即肌肉pH的下降、温度,肌肉收缩以及肌肉中蛋白质的分解,合适的pH下降速度和(或)肌肉收缩的机械保护,对提高牛肉的嫩度和色泽有较大作用。     

浅析牛青贮饲料中毒的防治

青贮是一种优质的青绿多汁饲料,是草食家畜主要粗饲料之一;但是在生产中由于管理不当,饲喂不合理,极易引起中毒发生。本文从不同的方面分析了引起青贮饲料中毒的原因,指出了合理化的预防措施;同时笔者根据在生产中的治疗体会,提出了行之有效的治疗方案。     

牛瑟氏泰勒虫P33表面蛋白基因的克隆与序列分析

为分析吉林省流行的牛瑟氏泰勒虫基因序列，根据GenBank上发表的牛瑟氏泰勒虫P33表面蛋白基因序列设计合成一对引物，用PCR方法扩增出牛瑟氏泰勒虫的基因片段，并成功地将该基因纯化后克隆到pGEM-TEasy载体上，将经EcoRⅠ酶切鉴定和PCR鉴定为阳性的重组质粒进行测序。结果表明克隆的基因片段长度为868bp，编码283个氨基酸，有2个潜在的糖基化位点。核苷酸同源性分析表明，该基因片段与韩国株（AF521557）、日本株（AB016280）、俄罗斯株（AB016279）的核苷酸序列同源性分别为99．4％、88.0％、88．1％。