METHOD FOR TARGETED KNOCKOUT OF NON-ESSENTIAL GENES FOR BOMBYX MORI NUCLEAR POLYHEDROSIS VIRUS REPLICATION

摘要

The present invention relates to a method for targeted knockout of non-essential genes for replication of the Bombyx mori nuclear polyhedrosis virus BmNPV comprising: using BmNPV as material to amplify a homologous sequence at both ends of a non-essential region for replication by PCR and cloning it into vector PUC19; splicing successively IE1 early promoter, marker gene (EGFP) and termination sequence SV40polyA of BmNPV by overlapping PCR and cloning them to the abovementioned vector pUC19 to obtain the recombinant transfer vector pUC19-lef7-IE1-EGFP-SV40polyA-gp64; co-transfecting BmN cells using the vector and the genome of wild BmNPV to obtain the recombinant virus RBmNPV-EGFP with a fluorescent marker via homologous recombination; co-transfecting BmN cells using genomic DNA thereof and transferring vector pUC19-lef7-gp64 without marker gene to obtain recombinant virus RBmNPV without fluorescent marker gene via homologous recombination. The present invention solves the problem of the presence of marker genes in recombinant virus genomes, improving the screening efficiency of positive recombinant viruses and thus the marker genes can be reused.