牛病毒性腹泻病毒河南分离株部分特性研究及卵黄抗体制备

发布时间：2018-06-10 05:26

  本文选题：牛病毒性腹泻病毒 + 分子鉴定　； 参考：《河南农业大学》2015年硕士论文

【摘要】：牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV),又称牛病毒性腹泻-粘膜病病毒(Bovine viral diarrhea-mucosal disease virus,BVD/MDV),是一种世界范围内广泛分布的危害动物健康的重要病原体。它能感染牛、羊、猪等多种动物,造成机体的呼吸系统或生殖系统等发生疾病,感染牛免疫力降低,易继发其他病原感染,导致疾病的发病率和死亡率增加。特别是形成持续性感染的牛,没有任何临床症状但可以排毒形成传播源,给疫病的防控造成极大困难。为了解河南肉牛BVDV分离株的培养和遗传特性,制备免疫诊断和防治试剂,本研究比较了致细胞病变型(cytopathic,CP)BVDV标准毒和BVDV分离株的细胞培养特性和分子遗传特性,并通过制备免疫原免疫产蛋鸡,获得了高效的抗BVDV IgY,为BVD免疫学检测和临床治疗该病提供物质和技术参考。首先,利用细胞培养技术对RT-PCR检测为阳性的血清样品进行病毒分离培养,获得一个稳定的分离株,命名为BVDV-MQ03。经光学显微镜观察,该分离株为致细胞病变型病毒,细胞培养48h左右即开始出现部分单层细胞呈拉网状、细胞死亡、脱落等病变。根据分离株MQ03与瘟病毒属参考株进行5'-UTR序列分析构建进化树显示,其属于BVDV-I型,其与NY-1和Osloss两个BVDV-Ib亚型毒株处于同一进化分支上,进化关系较为密切。通过遗传进化分析和序列相似性分析,MQ03与Osloss毒株的同源性最高,为97.1%,与NY-1的同源性达到96.2%,而与其它毒株的同源性均低于92.0%,证实MQ03属于BVDV-Ib亚型。通过MDBK细胞增殖MQ01、MQ03和Oregon C24V共三个毒株,收集病毒细胞培养物。利用PEG、蔗糖密度梯度离心纯化以上三个毒株。提纯病毒经磷钨酸负染,电镜观察到略呈圆形,直径30~80 nm的有囊膜病毒颗粒。将纯化的标准毒作为抗原,建立了间接ELISA方法。其中抗原包被浓度为1μg/mL,被检样品的稀释倍数为1:100,包被液为0.05mol/L,PH 9.6的碳酸盐缓冲液,酶标抗体工作浓度为1:8000稀释。最佳封闭条件、血清和酶标抗体的作用最佳条件均为37℃,1 h。上述条件确定后,特异性和重复性检验发现,除BVDV阳性卵黄抗体外,该纯化抗原不与抗鸡新城疫病毒、鸡减蛋综合征病毒、H5亚型禽流感病毒、H9亚型禽流感病毒的阳性血清反应,随机抽取的五个卵黄样品检测的批内变异系数和批间变异系数在3.54%和7.28%之间,均小于10%。以浓缩的标准毒和分离毒制作抗原免疫健康产蛋鸡,收集鸡蛋,用水稀释硫酸铵盐析法提取卵黄抗体。间接ELISA法测定IgY效价,发现蛋鸡首次免疫后第14 d,在卵黄中可以检测到特异性抗体,经过3次免疫后,抗MQ01、MQ03和Oregon C24V IgY的效价分别达到1:25600、1:51200和1:51200。获得的高免卵黄抗体可进一步开发为抗体制剂并用于临床该病的预防和治疗。
[Abstract]:Bovine viral diarrhea virus (Bovine viral diarrhea virus), also known as bovine virus viral diarrhea-mucosal disease virus (BVD / MDV), is an important pathogen which is widely distributed in the world and endangers animal health. It can infect many animals, such as cattle, sheep, pigs and so on, and cause diseases such as respiratory system or reproductive system. In particular, cattle with persistent infection, without any clinical symptoms, but can detoxify to form a source of transmission, causing great difficulties in the prevention and control of epidemic disease. In order to understand the culture and genetic characteristics of BVDV isolates from Henan beef cattle, and to prepare immunodiagnostic and preventive agents, the cell culture characteristics and molecular genetic characteristics of cytopathicus CPVDV standard virus and BVDV isolates were compared. The immunogen immunized laying hens were prepared and the highly effective anti-BVDV IgYs were obtained, which provided material and technical reference for the detection of BVD immunology and the clinical treatment of the disease. Firstly, a stable virus isolate was obtained from the serum samples detected by RT-PCR and named BVDV-MQ03. It was observed by optical microscope that the isolated strain was cytopathic virus. After 48 hours of cell culture, some monolayer cells began to appear in the form of pulling net, cell death, exfoliation and so on. The phylogenetic tree analysis of MQ03 and reference strain showed that MQ03 belongs to BVDV-I type, and it is closely related to NY-1 and Osloss BVDV-Ib subtype in the same evolutionary branch. The genetic evolution analysis and sequence similarity analysis showed that MQ03 had the highest homology with Osloss strain (97.1), 96.2% homology with NY-1, and lower homology with other strains than 92.0%. It was confirmed that MQ03 belonged to BVDV-Ib subtype. The virus cell cultures were collected by proliferation of MDBK cell lines MQ01mq03 and Oregon C24V. The above three strains were purified by PEG and sucrose density gradient centrifugation. The purified virus was negatively stained with phosphotungstic acid and was observed to be a circular virus particle with a diameter of 3080 nm by electron microscope. Using the purified standard virus as antigen, an indirect Elisa method was established. The concentration of antigen coating is 1 渭 g / mL, the dilution multiple of the tested sample is 1: 100, the coating solution is 0.05 mol / L PH9.6 carbonate buffer, and the working concentration of enzyme labeled antibody is 1: 8000 dilution. The best conditions for blocking the serum and the enzyme labeled antibody were 37 鈩，

本文编号：2002124