SCREENING THE RECOMBINANT BACULOVIRUS WHICH EXPRESSED MAREK'S DISEASE VIRUS pp38 GENE BY USE OF FLUORESCENCE ANTIBODY TEST
CUI Zhi-zhong Dept. of Anim. Sci. and Vet. Med., Jiangsu Agric. Coll, Yangzhou 225001
MAb H_(19)-recognized 38kd phosphorylated protein (pp38) of Marek's disease virus (MDV) is thought to be related to MDV-induced transformation. To further study its biological function in transformation, a preparation of purified pp38 would be uscful. The MDV pp38 gene including its own initiation codon, stop codon and transcriptional termination signals was integrated into baculovirus AcMNPV transfer vector pVL1392 at BamHI and KpnI cut sites, the recombinant transfer vector was des- ignated as pVLpp38Ⅱ. The insect Sf 9 cell monolayers in 6o mm plates were cotransfccted with pVLpp38 Ⅱplasmid DNA and transfectable AcMNPV DNA, and then incubated in TMN-FH media at 27℃ for 4 days. The culture superratant was collected and kept at 4℃, the Sf 9 cell monolayers were covered with 0.7% low melting point agarose in TMN-FH media and incubated for another day. The agarose gel was taken off from the plate and saved as the virus plaque replica. The plates were examined by fluorescence antibody (FA) test with anti-pp38 MAb H19 for screening the pp38 gene-expressing recombinant AcMNPV. The typical FA-positive plaques were found in one plate of five independent transfection plates, and only nontypical plaques consisting of several FA-positive infected cells were seen in other 4 transfection plates. The FA positive plaques were matched with the plaque replicas on the agarose gel, the plaque replicas were picked up from the agarose gel and resuspended into TMN-FH media for amplifica- tion and recloning. When Sf 9 cell monolayers were infected with transfected cell culture supernatant or amplified virus suspensions from the FA positive plaque replicas at a certain dilutions (less than 1 000 plaque forming units per 60 mm plate cell monolayer) and covered with 0.7% low melting point agarose in media during the incubation, the typical FA positive plaques could be easily seen in the most cases. By this way, the MDV pp38 gene-expressing recombinant AcMNPV plaques were easily screened without using differentiation of the recombinant and wild type AcMNPV plaques by determining occlusion-negative or positive plaques.