A Study On Knockout Of The Orf129 Of Autographa Californica Multicapsid Nucleopolyhedrovirus

Baculoviruses are double stranded DNA viruses that are rod shaped with genomes ranging in size from 80kb to 180kb. Among the members of baculoviruses the most widely studied is Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). The sequencing of the AcMNPV genome has lead to a breakthrough in the research of the virus because it now puts into perspective research for both the present and the future. Ac orf 129 codes for the protein p24. Homolog's of the Ac 129 are present in the genomes of all Group I/II and GV genomes.P24 is associated with both BV and ODV of AcMNPV and OpMNPV.P24 was previously shown to be interrupted by a transposable element in the E strain of AcMNPV. This data forms the hypothesis that Ac 129 is likely to be non essential in the life cycle of the baculovirus.This study was done to knockout orf 129 from the genome of AcMNPV.Homologous recombination was used to carry out the knockout. Ac orf 129 is from nt (109900-110496) and is flanked by gp64 (108179-109717) and gp16 (110524-110844). The segment of Ac orf 129 that was targeted for deletion in this study was from nt 109899-110192. Knowing the flanking genes helped in the designing of p24 homologous arms, designated H1 and H2.Each contained a 40 nucleotide (nt) long homologous arm at the 5'end with H1 (nt 109899-109939) and the 3'end H2 (nt110331-110370) of the AcMNPV genome (GENEBANK accession number NC₀01623).H2 was later changed to(nt110192-110231). Homologous recombination utilizes the lambda red recombinase strategy. It involves two main steps. The first step was the amplification of a gentamicin acetyl transferase resistance gene cassette (CAT) flanked by H1 and H2.The CAT gene cassette was used to replace Ac 129. This amplification was achieved by using a PCR cycle that had double annealing temperatures of 55℃and 50℃. The second step to theλ-Red recombinase method involves the transformation of E.coli DH10BAC which was made competent for electroporation. This bacterium contains an AcMNPV bacmid and pKD46 plasmid that contains the genes that encode theλ-Red recombinase that catalyzes recombination between bacmid with the PCR product. This was not successful as the verification with PCR primes K01 and K02 showed bands of less than 2000bp and the control of Bacmid wild type confirmed the result.