Reddy J R, Kwang J, Okwumabua O, Kapil S, Loughin T M, Lechtenberg K F, Chengappa M M, Minocha H C
Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Manhattan, KS 66506, USA.
Vet Microbiol. 1997 Sep;57(2-3):119-33. doi: 10.1016/s0378-1135(97)00128-4.
The National Animal Disease Laboratory (NADL) vaccine strain of bovine viral diarrhea virus (BVDV) genes for gp48 and p80 were expressed in Escherichia coli. The BVDV-NADL gene for gp62 was integrated into a baculovirus genome for expression in Spodoptera frugiperda (Sf-9) insect ovarian cells. The antigenicity of baculovirus expressed BVDV protein was detected by anti-BVDV specific antibodies in an enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent assay (IFA) and radio-immunoprecipitation (RIP). The recombinant proteins isolated from bacteria showed antigenic properties when analyzed by ELISA and immunoblotting using BVDV antibodies. The recombinant proteins were then used in ELISA or IFA to detect BVDV infection by testing 54 independent bovine serum samples. The baculovirus-expressed BVDV protein was used as an ELISA and IFA antigen, and the bacteria-expressed proteins were used as ELISA antigens. BVDV-NADL-infected Madin-Darby bovine kidney (MDBK) cell monolayers served as a control antigen. Statistical analysis showed a high degree of correlation between the reactivity of recombinants and natural antigens in ELISA using bovine sera. The results of ELISA or IFA proved there is a high degree of correlation with the virus neutralization. In the comparative ELISA assays, the insect-cell-mediated expression revealed greater specificity and sensitivity than the bacterial expression or the natural BVDV antigens produced by cell cultures.
牛病毒性腹泻病毒（BVDV）的gp48和p80基因的国家动物疾病实验室（NADL）疫苗株在大肠杆菌中表达。BVDV-NADL的gp62基因被整合到杆状病毒基因组中，以便在草地贪夜蛾（Sf-9）昆虫卵巢细胞中表达。在酶联免疫吸附测定（ELISA）、间接免疫荧光测定（IFA）和放射免疫沉淀（RIP）中，用抗BVDV特异性抗体检测杆状病毒表达的BVDV蛋白的抗原性。当使用BVDV抗体通过ELISA和免疫印迹分析时，从细菌中分离的重组蛋白显示出抗原特性。然后，将重组蛋白用于ELISA或IFA，通过检测54份独立的牛血清样本检测BVDV感染。杆状病毒表达的BVDV蛋白用作ELISA和IFA抗原，细菌表达的蛋白用作ELISA抗原。BVDV-NADL感染的Madin-Darby牛肾（MDBK）细胞单层用作对照抗原。统计分析表明，在使用牛血清的ELISA中，重组体与天然抗原的反应性之间存在高度相关性。ELISA或IFA的结果证明与病毒中和存在高度相关性。在比较ELISA试验中，昆虫细胞介导的表达比细菌表达或细胞培养产生的天然BVDV抗原显示出更高的特异性和敏感性。
