A novel way to purify recombinant baculoviruses by using bacmid.
In the present study, we studied the feasibility of deleting essential genes in insect cells by using bacmid and purifying recombinant bacmid in Escherichia coli DH10B cells. To disrupt the orf4 (open reading frame 4) gene of BmNPV [Bm (Bombyx mori) nuclear polyhedrosis virus], a transfer vector was constructed and co-transfected with BmNPV bacmid into Bm cells. Three passages of viruses were carried out in Bm cells, followed by one round of purification. Subsequently, bacmid DNA was extracted and transformed into competent DH10B cells. A colony harbouring only orf4-disrupted bacmid DNA was identified by PCR. A mixture of recombinant (white colonies) and non-recombinant (blue colonies) bacmids were also transformed into DH10B cells. PCR with M13 primers showed that the recombinant and non-recombinant bacmids were separated after transformation. The result confirmed that purification of recombinant viruses could be carried out simply by transformation and indicated that this method could be used to delete essential genes. Orf4-disrupted bacmid DNA was extracted and transfected into Bm cells. Viable viruses were produced, showing that orf4 was not an essential gene.
Su
Wu-Jie
WJ
Institute of Life Sciences, Jiangsu University, Xuefu Road 301, Zhenjiang 212013, China.
Shen
Wei-De
WD
Li
Bing
B
Wu
Yan
Y
Gao
Guang
G
Wang
Wen-Bing
WB
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D012831:Bombyx / Q000166:cytology / Q000235:genetics / Q000821:virology*; D004274:DNA, Recombinant / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D005240:Feasibility Studies; D055786:Gene Knockout Techniques; D017344:Genes, Insect; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D004279:DNA, Viral; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins