Khalid Hafiz Muhammad, Zaidi Najam Us Sahar Sadaf, Rashid Naeem, Tahir Muhammad
Department of Agricultural Sciences and Technology, Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad, Pakistan.
Department of Biological and Health Sciences, Pak-Austria Fachhochschule Institute of Applied Sciences and Technology, Haripur, Pakistan.
Turk J Biol. 2024 Aug 27;48(6):390-400. doi: 10.55730/1300-0152.2714. eCollection 2024.
BACKGROUND/AIM: (SCMV; genus and family ), poses a significant threat to global sugarcane cultivars, including those in Pakistan. The aim of this study was to develop a rapid and effective diagnostic tool for detection of SCMV, enabling timely implementation of control measures to mitigate potential yield losses.
The study focused on the in silico analysis, physicochemical properties, immunogenicity, and subcellular localization of the SCMV coat protein (CP). The SCMV CP gene was synthesized, cloned, and expressed in . The recombinant fusion CP (rFCP-SCMV) was purified and used to generate polyclonal antibodies (pABs) in mice. The immunogenicity of the antibodies was evaluated through indirect ELISA and RT-PCR.
Epitope prediction using tools like the OptimumAntigen design tool from GenScript, BepiPred, and IEDB identified key B-cell epitopes on the SCMV CP, enhancing the specificity of the antibodies. Structural modeling with SWISS-MODEL and PyMOL provided insights into the 3D structures of viral proteins and their epitopes, aiding in the design of high-affinity antibodies. Molecular docking studies simulated the interaction between antibodies and viral epitopes, enabling the selection of optimal antibody candidates. The synthesized recombinant fusion CP (rFCP-SCMV) was used to produce pAbs in mice. These antibodies exhibited high sensitivity, detecting as low as 100 pg of SCMV protein in indirect ELISA. They also effectively identified SCMV in infected sugarcane field samples, confirmed by RT-PCR. The antibodies maintained high specificity and sensitivity even at a 1:10,000 dilution, proving their efficacy in recognizing both the recombinant protein and virus particles in plant sap.
The study reveals a rapid, effective immunodiagnostic technique for detecting SCMV in sugarcane cultivars, offering an accurate alternative to conventional virology methods, reducing contamination risk, and providing a valuable tool for mitigating yield losses.
背景/目的：甘蔗花叶病毒（SCMV；病毒属和病毒科）对包括巴基斯坦甘蔗品种在内的全球甘蔗品种构成重大威胁。本研究的目的是开发一种快速有效的用于检测SCMV的诊断工具，以便及时采取控制措施减轻潜在的产量损失。
本研究聚焦于甘蔗花叶病毒外壳蛋白（CP）的电子分析、理化性质、免疫原性和亚细胞定位。合成、克隆了甘蔗花叶病毒CP基因，并在……中进行表达。纯化重组融合CP（rFCP-SCMV），并用于在小鼠中产生多克隆抗体（pABs）。通过间接ELISA和RT-PCR评估抗体的免疫原性。
使用如金斯瑞的OptimumAntigen设计工具、BepiPred和IEDB等工具进行表位预测，确定了甘蔗花叶病毒CP上的关键B细胞表位，提高了抗体的特异性。利用SWISS-MODEL和PyMOL进行结构建模，深入了解了病毒蛋白及其表位的三维结构，有助于设计高亲和力抗体。分子对接研究模拟了抗体与病毒表位之间的相互作用，从而能够选择最佳的抗体候选物。合成的重组融合CP（rFCP-SCMV）用于在小鼠中产生pAb。这些抗体表现出高灵敏度，在间接ELISA中能检测低至100 pg的甘蔗花叶病毒蛋白。它们还能有效鉴定感染甘蔗田间样本中的甘蔗花叶病毒，经RT-PCR证实。这些抗体即使在1:10000稀释时仍保持高特异性和灵敏度，证明它们在识别植物汁液中的重组蛋白和病毒颗粒方面的有效性。
本研究揭示了一种用于检测甘蔗品种中甘蔗花叶病毒的快速、有效的免疫诊断技术，为传统病毒学方法提供了一种准确的替代方法，降低了污染风险，并为减轻产量损失提供了一种有价值的工具。
