Development Of Qualitative And Quantitative For Detection Of The Ectropis Obliqua Nucleopolyhedrovirus

Tea looper nuclear polyhedrosis virus (Ectropis oblique nucleopolyhedrovirus,EoNPV) belongs to the genera Nucleopolyhedrovirus (family Baculoviridae). EoNPV is an important pathogen of the tea looper. As an efficient biological insecticide, EoNPV has been widely used in tea looper control in Zhejiang Province and neighboring provinces. However, the applications of EoNPV rely mainly on biometrics at present, the process of using virus and preparation of the virus could not be effective monitored. In this study, we developed a more reliable method for qualitative and quantitative detection of EoNPV, including fluorescent quantitative PCR and the preparation of monoclonal antibodies.1.Prokaryotic expression of p10 gene and vp39 gene and preparation of monoclonal antibodyUsing the EoNPV genome DNA as a template, The DNA fragments of p10 and vp39 were amplified by PCR, respectively. The segments of p10 and vp39 were cloned into pGEX-4T-2 vector, prokaryotic expression vector pGEX-4T-2-p10 and pGEX-4T-2-vp39 were constructed, respectively. Then pGEX-4T-2-p10 and pGEX-4T-2-vp39 were transferred into Escherichia.coli BL21(DE3) for expression. BALB/c mice were immunized conventionally with the expressed proteins of P10 and VP39, respectively. Mice spleen cells were then fused with myeloma cells (line Sp2/0). By indirect ELISA screening and cloning, a stable hybridoma cell lines 3E12 was cloned. The cell line can secrete stablely monoclonal antibody which can react with P10 protein and EoNPV, but can't react with VP39 protein. However, the titer of ascite was low.2. Production of monoclonal antibodies against polyhedrin of EoNPVBALB/c mice were immunized conventionally with the purified EoNPV, and the mice spleen cell was then fused with myeloma cell line Sp2/0. By indirect ELISA screening and cloning, a stable hybridoma cell line that can secrete monoclonal antibody was obtained, and was called 7D3. The titer of ascites as determined by ELISA was 1:105. Meanwhile, we cloned the polyhedrin gene and expressed it. Western blotting result showed that the monoclonal antibody produced by the hybridoma cell line 7D3 could specifically react with the expressed polyhedrin of EoNPV. Thus, a McAb against EoNPV was successfully prepared. It provides a basis for developing methods for detection of EoNPV in Ectropis oblique and EoNPV biopesticides.3. Development of the Fluorescent Quantitative PCR for Detection of EoNPVA pair of primers was designed according to the EoNPV genome sequence. The genome DNA of EoNPV was extracted from the larvae of Ectropis oblique which were infected with the EoNPV. A DNA fragment was amplified by PCR, and the fragment was cloned in to pMD18-T vector and transferred into E. coli TG1. A single clone was selected and sequencedwed. Then the recombinant plasmid was used as a quantitative template to establish a standard curve. The standard curve showed an excellent linear relationship when EoNPV concentration is between 103～108 copies/μL, correlation coefficient is 0.989, and the repeatability and specificity of quantitative PCR were higher than conventional PCR. The fluorescent quantitative PCR method for detecting EoNPV was developed, providing a basis for the research of EoNPV replicating in the host body and detection of the EoNPV in biopesticide products.