用萤光抗体试验筛选能表达鸡马立克病病毒pp38基因的重组昆虫杆状病毒  

SCREENING THE RECOMBINANT BACULOVIRUS WHICH EXPRESSED MAREK'S DISEASE VIRUS pp38 GENE BY USE OF FLUORESCENCE ANTIBODY TEST

作　　者：崔治中 Lee L F 

机构地区：[1]Dept.of Anim.Sci.and Vet.Med.,Jiangsu Agric.Coll.,Yangzhou 225001 [2]美国农业部禽病及肿瘤实验室

出　　处：《江苏农学院学报》1992年第1期1-5,共5页Jiangsu Agricultural Research

基　　金：国家自然科学基金;及江苏省教委自然科学基金资助项目;亦为与美国农业部禽病及肿瘤实验室Dr LeeLF合作项目

摘　　要：用抗鸡马立克病病毒(MDV)的38Kd磷蛋白的单克隆抗体作萤光抗体试验,直接从用可转染性野生型杆状病毒AcMNPV株DNA与含MDVpp38基因的重组转基因质粒载体DNA共同转染的Sf 9单层细胞培养中筛选到能表达pp38基因产物的重组杆状病毒感染斑,并从保存于琼脂醣凝胶上的复印斑中收复并进一步克隆了该基因重组病毒.该法易于实施,即使未能熟练掌握根据杆状病毒多角体来区别野生型杆状病毒斑与基因重组病毒斑的技术,也可顺利地筛选到能表达目的基因的重组合病毒。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 recomb

关 键 词：萤光抗体 马立克氏病 病毒 鸡 

分 类 号：S858.315.3[农业科学—临床兽医学]