| Based on site-specific transporation of an expression cassette into a baculovirus shuttle vector (Bacmid) which propagated in Escherichia coli, the Bac to Bac System provides a rapid and efficient method to generate recombinant baculoviruses and is widely used for high level expression of heterologous proteins from viruses, fungi, plant, and animals. In this study, we describe the development of an egfp-harboring Bacmid which would allow the tracing of the recombinant Bacmid transfection or recombinant baculoviral infection under fluorescence microscopes, and be used as a vector to generate recombinant virus the same way as the parent bacmid.In brief, plasmid pGEM-egfpΔAc74, which contains a 1.9 kb AcMNPV p74 gene ORF with a Ppolh-egfp cassette insertion, was co-transfected with Bacmid DNA into Sf9 cells, where a new vector, Bacmid-egfp, was generated through homologous recombination between the p74 sequences in pGEM-egfpΔAc74 and Bacmid DNA. The recombinant was isolated through five rounds of plaque purification by selecting single fluorescent plaque under fluorescence microscope, and the recombinant Bacmid-egfp DNA was extracted and subjected to PCR verification. Then, the verified Bacmid-egfp DNA and the helper plasmid pMON7124 DNA, which was isolated from E. coli DH10Bac, were transformed together into E. coli DH10B competent cells. The resulting bacterium, designated as E. coli DH10Bac-egfp, was capable of maintaining and propagating the recombinant Bacmid-egfp.To evaluate if the insertion of the EGFP expression easselte and the resulting silencing of p74 gene in the Bacmid-egfp have any adverse impact on the modified Bac to Bac system, a recombinant pFastBac plasm id that contains a polyhedrin gene (of AcMNPV) expression cassette flanked by Tn7L and Tn7R was transformed into E. coli DHlOBac-egfp competent cells. The polyhedrin expression cassette was transposed into Bacmid-egfp at the attTn7 locus, through the Tn7L and Tn7R sequences flanking it, to generate recombinant Bac-egfp-polh. Bac-egfp-polh DNA was isolated and transfected into Sf9 cells to check EGFP expression under a fluorescence microscope and polyhedron formation under a phase-contrast microscope. The result revealed that polyhedron was formed in each EGFP expressing cell, indicating that the modified Bac to Bac system remained working properly. This modified system was also used to express SLAM, the receptor of measles virus, by Dr. Xin Liu in our laboratory. With the egfp as marker, she found that SLAM was expressed on the surface of Sf-9 cells and it could mediate measles virus infection into the nonpermissive insect cells, indicating that this eg#-harboring system facilitate baculovirus-based local gene function investigation using confocal microscopic technology.The polyhedrin-egfp fusion expression cassette was transposed into bacmid genome at the attTn7 locus to generate the recombinant vector Bac-gfp/pol, which expressed the Polh/EGFP fusion protein of approximately 59kD in insect Sf-9 cells. 4 days after Bac-gfp/pol DNA transfected into Sf-9 cells, occlusion bodies formation and EGFP expression was observed under phase-contrast or fluorescence microscope, and fluorescence was only located on the surface of occlusion bodiesjio fluorescence appearing in cytoplasm and other part of nucloplasm. It indicated that EGFP was displayed on the surface of occlusion bodies through fusion with polyhedrin. But the recombinant virus vAc-pol/gfp yielded less occlusion bodies than wilt AcMNPV in Sf-9 cells, and the recombinant occlusion bodies were irregular in morphogenesis observed under electron microscope.In order to study the function of envelope protein P74 of Baculovirus, threerecombinant bacmids, Ac-H74-poK AcG-H74-pol and Ha-A74-pol,were constructed by transposing heterologous p74 gene driven by AcMNPV polyhedrin promoter and intact AcMNPV polyhedrin gene into the polyhedrin locus of AcBacmid, AcBacmid-egfp and HaBacmid through three Bac to Bac system respectively. As Bacmid-egfp was modified by inserting egfp gene driven by AcMNPV polyhedrin promoter into p74 locus of Bacmid, the resulting AcG-H74-pol harbored only heterologous Ha-p74 gene, while the Ac-H74-pol and Ha-A74-pol harbored both the native and heterologous p74 gene.Three recombinant viruses, vAc-H74-pol > vAcG-H74-pol and vHa-A74-pol were obtained by transfecting three recombinant bacmids into insect cells respectively. 4-5 days later, a large quantity of occlusion bodies yield in infected cells, and fluorescence was observed in cells infected by vAcG-H74-pol. SDS-PAGE assay revealed that P74 protein was expressed in cells infected with three recombinant viruses, and RT-PCR assay confirmed that the native and heterologous p74 gene were transcripted. To determine whether the heterologous P74 protein was assembled onto the surface of recombinant ODVs, the membrane proteins of ODVs fractionated by NP-40 were resolved by SDS-PAGE and probed by Western-Blot with anti-His-tag mAb. The results showed that heterologous His-tagged Ha-P74 protein was assembled onto the envelope of recombinant ODVs of vAc-H74-pol and vAcG-H74-polOral-infection assay with Noctuid larvae showed that vAc-H74-pol,harboring both native and heterologous p74 gene, could only infect its own host Spodoptera exigna, and could not infect Helicoverpa armigera.the host of HaSNPV, indicating that the extra heterologous p74 gene could not expand the host range of the recombinant virus. A similar result was obtained for vHa-A74-pol,who harbors both native and heterologous p74 gene. It could only infect its own host H. armigera,and could not infect S.exigua.Xht host of AcMNPV. The bioassay demonstrated that native p74-deficient vAcG-H74-pol lost infectivity to its host,^. exigua.and the heterologous p74 gene could not rescue its infectivity to host.lt elucidates that p74 is a species-specific gene which could not be substituted by heterologous p74 gene.The ecology and biosafely of recombinant bacoluvirus rAcII was evaluated in a field release trial of about 70 ha crops, which indicated that rAcII could greatly reduce the populations of target pest, while did not damage the structure of insect community in the field. The pest mortality was up to 86.8% 9 days after virus-spraying,while in the chemicals-spraying field,the mortality was high immediately after application, but it declined several days later. The insect community diversity and evenness went up gradually, showing its application stabilized insect community. And by the analysis of the insect community similarity and the populations of pest enemies in the fields, it demonstrated that the recombinant virus rAcII did little harm to beneficial insects and eco-system. and it could balance insect community, without resulting in pest recurrence. The yields of vegetables in the fields sprayed with rAcII increased 5-8% than that of farmer practice, while the nutrient ingredients and quality of vegetables were not damaged. Detecting the viruses in the environments by PCR, the recombinant virus rAcII proved to be less persistence than that of wtAcMNPV. All these field tests demonstrated that the recombinant virus rAcII is efficient and safe in crop protection, and it worth to be widely application in the future. |
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