Expression Of Polyhedrin Gene By Transposon And Improve The Recombinant Baculovirus Infection Per OS TO Insect Larvae

Baculoviruses have been widely used for the expression of foreign genes in insect cells and larvae. The baculovirus expression vector system (BEVS) has a number of unique features, such as the capacity for large inserts of DNA and a high yield of recombinant protein. Proteins produced in the BEVS are very similar to naturally occurring human proteins in terms of post-translational modifications (Phosphorylation), biological activity, and protein stability. For this reason the BEVS is widely used in academia and industry. However, BEVS lack the polyhedrin (poly) gene and do not produce occlusion bodies (OBs). Thus, these kinds of recombinant baculovirus had to be inoculated in insect larvae. It made the larvae wounded (even infected by other microorganisms, such as bacteria) in the process of infection and wasted time and energy. In this study, we obtained the transgenic BmN cells that contain the poly gene by transfection, and packaged the recombinant baculovirus with the transgenic BmN cells for raise the pathogenicity of the recombinant virus in orally infected B. mori larvae. This method is a potential way to improve the recombinant baculovirus infection per os to insect larvae. We did the following work:The poly gene was amplified with a pair of primers designed according to the sequence of Bombyx mori nucleopolyhedrovirus (BmNPV) gene by PCR, and the product was cloned into the plasmid pigA3GFP-a transposon vector. Then we amplified the immediate early gene 1 (ie1) promoter of BmNPV with the special primers, and inserted it into the pigA3GFP vector with poly gene before. The resulting plasmid was named pigA3GFP -ie. At the same time, we also amplified the poly promoter of BmNPV, and inserted it into the pigA3GFP vector with poly gene before. We immediately named the recombinant plasmid as pigA3GFP -poly.DNA of the resulting vector plasmid pigA3GFP- ie1 (or pigA3GFP -poly) and helper plasmid pHA3PIG were transfected into BmN cells by the Lipofectamin. We got the BmN cells that emit fluoresce. After several rounds of selecting, the number of GFP-positive cells increased significantly. These results indicated that the stable transgenic BmN cells were obtained. And we examined the inserted poly gene in transgenic BmN cells by RT-PCR., and demonstrated mRNA of the poly gene was transcripted in the transgenic BmN cells.In order to examine whether the expression product of the poly gene could package the recombinant virus, the transgenic BmN cells were infected with the recombinant baculoviruses without the poly gene, BmPAK6 and BmGFP, respectively. The results showed that the transgenic BmN cells package the recombinant virus, and the OBs were observed in the nuclei of the transgenic BmN cells at 72 h post infection.The B. mori larvae (50 larvae in each experiment), in the first day of fifth instar were orally inoculated with the recombinant virus (vBmGFP and vBmPAK6). The dead larvae were collected daily, and confirmed to be infected by the two kinds of the OBs by fluorescence observation or X-Gal staining test. The results indicated that the OBs of the recombinant baculoviruses that were packaged by the transgenic BmN cells (vBmPAK6 and vBmGFP) all exhibited a high infectivity phenotype relative to the origin recombinant baculoviruses. However, the B. mori larvae mortality by vBmPAK6 (or vBmGFP) compare to that by BmNPV was about 40%, suggesting that the infectivity of vBmPAK6 (or vBmGFP) was lower than that of wild type BmNPV in orally infected B. mori larvae.These results indicate that the products of poly gene expressed in the transgenic BmN cells could package the recombinant baculoviruses when the viruses infected the cells and raise the pathogenicity of the recombinant virus in orally infected B. mori larvae. This is a potential way to improve the recombinant baculovirus infection per os to insect larvae...