中文摘要：

为了制备牛病毒性腹泻病毒（BVDV）p80蛋白单克隆抗体,试验采用BVDV接种牛肾细胞（MDBK）,提取病毒RNA,通过RT-PCR扩增出BVDV的p80基因,将其定向克隆到原核表达载体pET30a,得到重组表达质粒pET30a-p80。将重组质粒转化大肠杆菌BL21（DE3）表达菌,挑选单菌落并扩大培养,经IPTG诱导和SDS-PAGE以及Western-blot分析表明重组蛋白p80获得了表达,分子质量约为44ku。同时用浓缩的BVDV培养液免疫6-8周龄的Balb/c小鼠,取免疫鼠脾细胞与SP2/0细胞进行融合。利用纯化的重组蛋白p80作为检测抗原的间接ELISA筛选阳性杂交瘤细胞,经过3次亚克隆后对获得的杂交瘤细胞进行间接免疫荧光（IFA）和抗体亚类鉴定及腹水的制备和效价的测定。结果表明：重组p80蛋白具有很好的反应性;获得8株稳定分泌针对BVDVp80蛋白的杂交瘤细胞株,分别为3E3、2A4、8H9、3G3、2B3、3G2、2G2和4A11;8株杂交瘤细胞株接种Balb/c小鼠制备腹水,采用BVDV作为包被原的间接ELISA方法测得其效价为1×10^5-1×10^7;8株杂交瘤细胞所分泌的MAb具有良好的反应性和特异性;8株单克隆抗体的抗体亚类均为IgM/κ。说明制备的重组p80蛋白单克隆抗体可试用于建立检测BVDV抗原的诊断方法。

英文摘要：

To prepare monoclonal antibodies against the p80 protein of bovine viral diarrhea virus （BVDV） ,Madin - Darby bovine kidney （MDBK） cells were inoculated with BVDV to extract viral RNA. The pS0 gene was amplified by RT -PCR and directionally cloned into the prokaryotic expression vector pET30a, and then a recombinant expression plasmid pET30a- 1）80 was obtained. The recombinant plasmid was transformed into E. coli BL21 （ DE3 ） , a single colony was picked for expanded cultivation. SDS - PAGE and Western - blot analysis showed that recombinant protein pS0 was expressed after induction with IPTG, the expressed protein had a molecular weight of approximately 44 ku. At the same time, six - and eight - week - old female Balb/e mice were immunized with concentrated BVDV culture solution. The immunized spleen cells of the mice were extracted and fused with SP2/0 cells. Positive hybridoma cells were screened by indirect ELISA using purified re- combinant pS0 protein as a detection antigen. The hybridoma ceils were detected using indirect immunofluorescence assay （IFA） after 3 times subcloning, and the antibody subclasses were also determined. The ascites were prepared, and then their titers were determined. The results showed the recombinant p80 protein had good reactivity. Eight secreting hybridoma cell lines against BVDV of p80 protein were obtained, including 3E3, 2A4, 8H9, 3G3 ,2B3, 3G2, 2G2 and 4Al 1. The Balb/c mice were inoculated with the eight hybridoma cell lines to prepare ascites, and their ascite titers ranged from 1 × 10^5 to 1 × 10^7 , which were detected using ELISA coated with BVDV. The monoclonal antibodies （MAbs） secreted by the eight hybridoma cell lines had good reactivity and specificity. All of the eight mAbs subtypes were tgM/K. The results indicate that the prepared mAbs against the recombinant p80 protein can be applicable the establishment of a diagnostic method to test the BVDV antigen