Generation And Characterization Of A Monoclonal Antibody Against Bombyx Mori Nucleopolyhedrovirus Polyhedrin

Baculoviruses are a family of large DNA viruses characterized by rod-shaped virions and covalently closed double-stranded circular DNA, which replicate and assemble within the host nucleus. Baculoviruses are infectious mostly to insects within the order Lepidoptera. The virions are specifically occluded within robust crystalline particles (polyhedra) which act as protective packages allowing infectious virions to survive for long periods in harsh environments. The polyhedrin (the polyhedra protein) is one of the most conserved proteins of the virus. The structure of the polyhedrin and its organization within the polyhedra has remained obscure. In order to provide the effective detection method for the advance studies of these mechanisms, this study prepared the monoclonal antibody (mAb) against Bombyx mori Nuclear polyhedrosis virus (BmNPV) polyhedrin by the aid of hybridoma technique and identified it. Main studies and results are as follows:1. The preparation of anti-BmNPV polyhedrin mAbIn the study, the BmNPV polyhedra particles were purified by differential centrifugation and BaLb/c mice were immunized with the purified polyhedra particles by intraperitoneal injection (IP). Then, The spleen cells of immunized BaLb/c mice were fused with SP2/0 cells using PEG-4000, and hybridoma cells were screened using selective HAT medium. The hybridoma cell secreting mAb against BmNPV polyhedrin were detected by indirect ELISA and were sub-cloned by limited dilution. In the end, one positive clone were screened out and named as D7. During four months, D7 could stably secrete anti-BmNPV Polyhedrin mAb after serial passages and three times freezing and revival. It showed that the hybridoma D7 possessed a good genetic stability.2. The characterization of anti-BmNPV polyhedrin mAbThe characteristics of the mAb were identified by chromosome stability assay of hybridoma, isotype identification of mAb, ELISA titers, specificity and so on. The chromosome of D7 was counted and the number was 99～108, which demonstrated that hybridoma cell line have been obtained on the genetic point. The isotype of mAb secreted by D7 was identified as corresponding to IgG2a subclass and kappa type, and the ELISA titers of the ascites were 1:106～1:107. In addition, Western blot analysises showed that the mAb specifically reacted with the peptides of BmNPV polyhedrin and the mAb showed no cross-reactivity to AcNPV,ClbiNPV and EoNPV polyhedrin, but cross-reactivity to ApNPV and PcrNPV polyhedrin. 3. Localization of the epitope of anti-BmNPV polyhedrin mAbFirst of all, a series of recombinant AcNPV integrated different length BmNPV polyhedrin fragment(F1-6)-EGFP have been constructed successfully by using Bac-to-Bac baculovirus expression system. And then, insect cells were infected with these recombinant viruses and F1-6-EGFP proteins were expressed. Finally, the epitope of mAb was limited to the amino-acid sequence 100-180 of BmNPV polyhedrin by Western blot analysises.Taken together, a hybridoma cell strain which can secrete mAb against BmNPV polyhedrin was generated by using hybridoma technique. The mAb was developed and its characteristics were identified. The preparation of anti-BmNPV polyhedrin mAb can provide the foundation for the advance studies of baculoviruses and others.