羊痘和羊痘是小反刍动物的高度传染性和经济上重要的病毒性疾病。由于它们对动物健康构成的风险,畜牧业生产,国际贸易,卷发病毒对畜牧业经济构成相当大的威胁。在这项研究中,我们在杆状病毒表达载体系统中表达了山羊痘病毒的两个核心蛋白(A4L和A12L)和一个细胞外包膜病毒体蛋白(A33R),并评估了它们在ELISA中作为诊断抗原的用途。全长A4L,A12L,并扩增了GTPVUttarkashi菌株的A33R基因,克隆到pFastBacHTA供体载体中,并导入含有杆状病毒穿梭载体质粒的DH10Bac细胞以产生重组杆粒。通过转染在Sf-21细胞中产生重组杆状病毒,蛋白质在TN5昆虫细胞中表达。通过SDS-PAGE分析重组蛋白,并通过蛋白质印迹确认。预期大小约为30kDa,~31kDa,A4L为~32kDa,A12L,A33R,分别。纯化重组蛋白,纯化蛋白的免疫反应性通过使用抗GTPV血清的蛋白质印迹证实。表达的蛋白质作为诊断抗原的抗原特异性通过测试它们与感染的反应性来评估。已接种疫苗间接ELISA中GTPV/SPPV血清阴性,并对基于A33R的间接ELISA进行了优化。基于A33R的间接ELISA的诊断灵敏度和特异性分别为山羊的89%和94%和98%和91%,对于绵羊来说,分别。没有观察到与其他相关病毒的交叉反应性。本研究中开发的基于重组A33R的间接ELISA表明,它具有检测GTPV和SPPV感染/接种疫苗的动物中抗体的潜力。
Goatpox and sheeppox are highly contagious and economically important viral diseases of small ruminants. Due to the risk they pose to animal health, livestock production, and international trade, capripoxviruses are a considerable threat to the livestock economy. In this study, we expressed two core proteins (A4L and A12L) and one extracellular enveloped virion protein (A33R) of goatpox virus in a baculovirus expression vector system and evaluated their use as diagnostic antigens in ELISA. Full-length A4L, A12L, and A33R genes of the GTPV Uttarkashi strain were amplified, cloned into the pFastBac HT A donor vector, and introduced into DH10Bac cells containing a baculovirus shuttle vector plasmid to generate recombinant bacmids. The recombinant baculoviruses were produced in Sf-21 cells by transfection, and proteins were expressed in TN5 insect cells. The recombinant proteins were analysed by SDS-PAGE and confirmed by western blot, with expected sizes of ~30 kDa, ~31 kDa, and ~32 kDa for A4L, A12L, and A33R, respectively. The recombinant proteins were purified, and the immunoreactivity of the purified proteins was confirmed by western blot using anti-GTPV serum. The antigenic specificity of the expressed proteins as diagnostic antigens was evaluated by testing their reactivity with infected, vaccinated, and negative GTPV/SPPV serum in indirect ELISA, and the A33R-based indirect ELISA was optimized. The diagnostic sensitivity and specificity of the A33R-based indirect ELISA were found to be of 89% and 94% for goats and 98% and 91%, for sheep, respectively. No cross-reactivity was observed with other related viruses. The recombinant-A33R-based indirect ELISA developed in the present study shows that it has potential for the detection of antibodies in GTPV and SPPV infected/vaccinated animals.