BVDV、BRV和BCoV TaqMan三重RT-qPCR检测方法的建立

为建立牛病毒性腹泻病毒(BVDV)、牛轮状病毒(BRV)和牛冠状病毒(BCoV)的快速检测方法,根据GenBank中登录的BVDV 5'-UTR、BRV NSP5和BCoV N基因序列设计特异性引物和探针,通过优化反应体系和条件,建立了同时检测上述3种病毒的TaqMan三重RT-qPCR方法.该方法仅对BVDV 5'-...     

基于单抗捕获牛病毒性腹泻病毒(BVDV)抗原ELISA方法的建立及应用

应用制备的牛病毒性腹泻病毒(BVDV) CC13B毒株E2蛋白的单克隆抗体与多克隆抗体,建立了基于单抗捕获牛病毒性腹泻-黏膜病病毒(Bovine viral diarrhea-mucosal disease virus,BVD-MDV)抗原的双抗体夹心ELISA方法。方阵试验确定的捕获BVDV抗原的单克隆抗体最佳抗体包被量为200 ng/孔,酶标多克隆抗体的最佳稀释浓度为1 000倍稀释。对100份BVDV阴性粪便样品进行检测与统计学处理,确定了夹心ELISA检测BVDV抗原的判定标准为待检样品D₄₉₀值≥0.156 7时,检测结果判定为阳性。特异性、敏感性和重复性试验结果显示,建立的检测BVDV抗原的方法具有特异、敏感、快速及重复性好等特点。以建立的单抗捕获BVDV抗原的夹心ELISA方法,调查了山东、河南和吉林3省部分地区牛群BVDV感染情况,发现上述地区牛群的BVDV感染率低至6%。本研究结果为BVDV感染的诊断、检疫、净化及防制提供了有效的技术手段及流行病学理论依据。     

牛病毒性腹泻病毒NS3蛋白纳米抗体的筛选及反应原性检测

本研究旨在获得高效特异性的牛病毒性腹泻病毒（BVDV）NS3（P80）非结构蛋白的纳米抗体。用BVDV灭活疫苗免疫羊驼,测得抗体效价后分离全血中的淋巴细胞。通过噬菌体展示技术构建羊驼重链抗体可变区噬菌体展示文库。经过连续3次吸附-洗脱-扩增的生物筛淘,从中挑选出与BVDV-NS3蛋白结合的噬菌体。对经菌液PCR、琼脂糖凝胶电泳鉴定到的单域抗体（VHH）克隆进行基因测序和同源性比对。用ELISA方法验证筛选出的纳米抗体的反应原性,找到与BVDV-NS3蛋白亲和力高的纳米抗体。结果表明,获得插入率为92.8%、库容为1.84×10¹⁴ CFU/mL的噬菌体展示文库。ELISA结果和氨基酸序列分析显示,成功得到1条与BVDV-NS3蛋白具有良好反应性且与VHH同源性较高的纳米抗体序列。本研究利用大肠杆菌成功表达BVDV-NS3抗原蛋白,建立BVDV纳米抗体噬菌体展示文库,筛选到针对BVDV重要抗原蛋白相应的纳米抗体且与VHH同源性较高。试验结果为牛病毒性腹泻/黏膜病的防控、诊断、治疗及纳米抗体药物的研制提供参考。     

牛病毒性腹泻病毒结构蛋白基因的克隆及其B细胞抗原表位的预测

[目的]以免疫信息学和反向疫苗学为理论根据,应用生物信息学相关软件和方法,对BVDV结构蛋白( Structural Protein,SP)的二级结构的不同方面及其B细胞表位进行了预测,综合评价了BVDV结构蛋白的抗原特异性细胞表位,并计算出一些潜在的抗原位点.[方法]利用RT- PCR扩增法获得牛病毒性腹泻病毒结构蛋白序列,通过克隆、测序分析结构蛋白基因的基因组序列和蛋白序列,采用DNAStar Protean软件对结构蛋白的二级结构、可塑性、亲水性、表面可及性及抗原指数等参数进行综合分析,并预测其B细胞的抗原表位分布.[结果]P14、gp48和gp53蛋白含有的细胞优势抗原表位较多,有的甚至有可能成为优势抗原表位或对优势抗原表位有协同作用.[结论]与已鉴定的B细胞抗原表位相比,试验所用方法预测的结果有较高的准确度,是一种较为先进的表位研究方法.     

Self-clearance from BVDV infections--a frequent finding in dairy herds in an endemically infected region in Peru

In this cross-sectional study, a stratified two-stage random sampling procedure was employed to select 221 dairy herds for bulk tank milk (BTM) sampling, and a subset of 55 dairy herds for individual blood sampling of a number of young animals (spot test), to predict presence or absence of current BVDV infection, and for data collection. The prediction was based on the high probability of seropositivity in groups of animals where PI animals are present because of the efficient spread of virus from PI animals to the surrounding group. BTM samples were collected in August 2003 (n = 192) and February 2004 (n = 195), and the 55 herds selected for spot testing and data collection were visited in December 2003. All samples were tested for presence of BVDV specific antibodies using a commercial indirect ELISA (SVANOVA Biotech AB, Uppsala, Sweden). The results demonstrated a very high level of exposure to BVDV in the region, and the proportion of herds with high antibody levels in the BTM was above 95% on both occasions. Despite this, almost two thirds of the herds had spot test results indicating absence of current infection, suggesting a high probability of self-clearance. A logistic regression model with the results from the spot tests as dependent variable was used to investigate possible herd and management factors associated with self-clearance, and suggested that this may occur regardless of herd size. Even though it is well established that the process of identification and elimination of PI animals is required within a systematic BVDV eradication programme, the present study strongly suggests that many herds may be cleared without intervention even in regions with high cattle density and high BVDV prevalence. Consequently, in any BVDV infected population (regardless of the herd-level BVDV seroprevalence), and at any given point of time, a large proportion of the herds will be free from infection due to self-clearance. Self-clearance is therefore a process that works in favour of any effort to control BVDV, which should be taken into account when planning and assessing the cost-effectiveness of a systematic control programme.