摘要
Citrus tristeza virus （CTV） is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid protein （CP） gene of CTV was expressed in Escherichia coli BL21 （DE3） using the expression vector pET-28a and purified through Ni＊-NTA affinity chromatography. The recombinant protein was used to immunize BALB/c mice. Four hybridoma cell lines （14B10, 14Hll, 20D5, and 20G12） secreting monoclonal antibodies （MAbs） against CTV were obtained through conventional hybridoma technology. The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10-6 to 10.7 in indirect enzyme-linked immunosorbent assay （ELISA）. Western blots showed that all four MAbs could specifically react with CTV CP. Using the prepared MAbs, dot-ELISA, Tissue print-ELISA, and triple antibody sandwich （TAS）-ELISA were developed to detect CTV in tree nurseries and epidemiological studies. The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1：2560 and 1：10, 240 （w/v, g/mL）, respectively. Tissue print-ELISA was particularly useful for large-scale field sample detection, mainly owing to its simplicity and lack of sample preparation requirements. The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality, Jiangxi Province, and Zhejiang Province of China. The coincidence rate of serological and RT-PCR test results reached more than 99.5%. The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.
Citrus tristeza virus(CTV) is one of the most economically important citrus viruses and harms the citrus industry worldwide.To develop reliable and effective serological detection assays of CTV,the major capsid protein(CP) gene of CTV was expressed in Escherichia coli BL21(DE3) using the expression vector p ET-28 a and purified through Ni^+-NTA affinity chromatography.The recombinant protein was used to immunize BALB/c mice.Four hybridoma cell lines(14B10,14H11,20D5,and20G12) secreting monoclonal antibodies(MAbs) against CTV were obtained through conventional hybridoma technology.The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10-6 to 10-7 in indirect enzyme-linked immunosorbent assay(ELISA).Western blots showed that all four MAbs could specifically react with CTV CP.Using the prepared MAbs,dot-ELISA,Tissue print-ELISA,and triple antibody sandwich(TAS)-ELISA were developed to detect CTV in tree nurseries and epidemiological studies.The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1:2560 and1:10,240(w/v,g/m L),respectively.Tissue print-ELISA was particularly useful for large-scale field sample detection,mainly owing to its simplicity and lack of sample preparation requirements.The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality,Jiangxi Province,and Zhejiang Province of China.The coincidence rate of serological and RT-PCR test results reached more than 99.5%.The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.
基金
supported by Public Science and Technology Research Funds Projects of Agriculture (20120307605)
