Abstract：Citrus tatter leaf virus (CTLV), a species of the family Capillovirus, is an important citrus (Citrus reticulate) virus and aquarantine object in obtaining virus-free citrus seedlings, which causes significant losses to citrus industry worldwide. It is very important to prepare sensitive and specific monoclonal antibodies (MAbs) against CTLV for the diagnosis and detection of CTLV in citrus groves. For this purpose, the 714 bp coat protein gene (CP) of CTLV was cloned from a CTLV-infected citrus sample collected from citrus grove in Chongqing municipality, which shared 100% nucleotide sequence identity with a Chinese isolate of CTLV deposited in GenBank. The CTLV CP then was cloned into the His-tagged prokaryotic expression vector, pET-28a. The resulting expression vector was transformed into Escherichia coli BL21 (DE3) strain. After induced by isopropylthio-β-D-galactoside (IPTG), E. coli BL21 (DE3) cells harboring the recombinant vector expressed an approximately 30 kD fusion protein. The recombinant fusion protein was purified with Ni2+-NTA agarose and used to immunize BALB/c mice (Mus musculus). Three hybridoma cell lines (6C5, 14E11 and 15F8) secreting MAbs against CTLV were prepared by fusing mouse myeloma cells (SP 2/0) with spleen cells from the immunized BALB/c mouse. The hybridomas were injected into pristine-primed BALB/c mice to prepare the ascetic fluids contained the MAbs. The titers of 3 MAbs in ascitic fluids ranged from 10-6 to 10-7 in indirect-ELISA. Isotypes and subclasses of all 3 MAbs belonged to IgG1, κ light chain. The IgG yields of 3 MAbs (6C5, 14E11 and 15F8) in ascetic fluids were 9.41, 7.83 and 9.93 mg/mL respectively. Triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) analysis indicated that the 3 MAbs could specifically react with CTLV-infected citrus leaf and CTLV-infected Chenopodium quinoa leaf crude extracts, had negative reactions with Citrus tristeza virus, Apple stem grooving virus, healthy citrus leaf and Chenopodium quinoa leaf crude extracts. Western blot demonstrated that all 3 MAbs could specifically react with an approximately 30 kD CP of CTLV in CTLV-infected citrus leaf crude extracts, but no hybridization signal was observed on the lane of healthy citrus leaf crude extracts. A dot enzyme-linked immunosorbent assay (dot-ELISA) was developed using the prepared 3 MAbs, and the established dot-ELISA could successfully detect virus in plant sap diluted at 1∶640 (W/V, g/mL). Fourteen grove samples collected from Chongqing municipality and Zhejiang Province were tested for CTLV infection using the dot-ELISA and RT-PCR. The detection results indicated that the developed dot-ELISA could accurately, reliably and sensitively detect CTLV in citrus trees in groves. The prepared anti-CTLV MAbs and the developed dot-ELISA provide technical and material support for the diagnosis, production of virus-free citrus seedlings and scientific prevention and control of CTLV in citrus groves in China.
