The Transgenic BmN Cells with Polyhedrin Gene: A Potential Way to Improve the Recombinant Baculovirus Infection Per Os to Insect Larvae

Abstract

The principle of baculovirus expression system is that substitute exogenous gene for polyhedrin (polh) gene, and the recombinant baculovirus lacks the ability to infect insect larvae by oral inoculation. In this study, we cloned the polh gene with immediate early gene 1 (ie1) promoter of Bombyx mori nucleopolyhedrovirus (BmNPV) into transposon pigA3GFP vector, transported it into BmN cells by lipofectamine and obtained the transgenic BmN cell line. The mRNA transcription of the polyhedrin gene was demonstrated by reverse transcription-polymerase chain reaction. Then the polh gene negative viruses (BmPAK6 and BmGFP), infected the transgenic BmN cells and Polyhedrin-like structures were observed in the infected cells. Subsequently, the viruses (vBmPAK6 and vBmGFP) from infected cells were used to orally inoculated the fifth instar larvae of B. mori, respectively. The results showed that B. mori larvae could be infected per os with the recombinant baculoviruses vBmPAK6 and vBmGFP, respectively. These results suggest that the products of polh 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.

Trans-complementation of polyhedrin by a stably transformed Sf9 insect cell line allows occ− baculovirus occlusion and larval per os infectivity

“…On the other hand, when using recombinant baculoviruses as biological pesticides, an occ+ virus could be not safe, as the subsequent generations would be able to propagate in nature. Chen et al (2009) were able to occlude Bombyx mori nucleopolyhedrovirus (BmNPV) by infection of a BmN cell line stably transformed by using a transposon-based methodology with the polyhedrin gene under the regulation of a constitutive promoter. However, the generation of a Sf9 cell line expressing polyhedrin only after AcNPV infection could be advantageous over polyhedrin constitutive expression due to the possible side-effects in cellular growth and viability.…”

Establishment of a Novel Baculovirus–Silkworm Expression System

“…This novel strategy of packaging a recombinant baculovirus is convenient and safer for the inoculation of B. mori larvae. Chen et al have tried to inoculate the virus via oral inoculation [ 11 ]. However, in our study, we found that the growth of our constructed polh -expressing BmN cells seems abnormal, which may contribute to the constitutive expression of polh (unpublished data).…”

“…For example, foreign proteins are usually expressed at a later stage of infection, leading to insufficient protein modification, while the invasion ability of the virus drops soon after being introduced into host cell populations [ 8 , 9 , 10 ]. More importantly, the virus vector needs to be injected into the silkworm to initiate infection and protein production because the polyhedrin gene ( polh ) has been disrupted so that no occlusion bodies could be formed to infect silkworms via oral inoculation [ 11 , 12 ]. Thus, it is necessary to construct a novel recombinant virus expression system for better performance.…”

“…In fact, Chen et al showed the assembly strategy is feasible. They cloned the polh gene under the control of the IE1 promoter of BmNPV into a transposon pigA3GFP vector and then transported it into BmN cells to successfully construct a transgenic BmN cell line [ 11 ].…”