Preparation of the recombinant Marek's disease virus which can induce apoptosis in tumor cells and its application for an anti-tumor vaccine.

马立克氏病病毒(MDV)可引起鸡恶性淋巴瘤，但其转化的分子机制尚不清楚。根据MDV毒株的致病力将其分为3个血清型：除弱毒疫苗株(即CVI988)外，只有MDV 1型毒株是致癌的。虽然马立克氏病(MD)通过疫苗接种得到了有效控制，但活疫苗的保护机制尚不清楚，已有报道出现可导致疫苗破损的高毒力MDV。为此，本研究构建了能诱导肿瘤特异性细胞凋亡的重组MDV，为新型抗肿瘤疫苗的研制奠定了基础。此外，致癌MDV1和减毒MDV1在MEQ基因结构上的差异也已被鉴定。由于meq基因被称为mdv癌基因，因此对病毒基因组的变化进行了详细的分析，以深入了解meq基因在MD…转化中的作用。构建了表达鸡传染性贫血病毒VP3基因的更多V.A基因盒，该基因是由MEQ启动子控制的。之所以选择MEQ启动子，是因为已知MEQ基因只在肿瘤或MDV潜伏感染细胞中表达。将该基因盒导入MD肿瘤细胞系MSB1后，MSB1细胞活力在24小时内下降，并在MSB1细胞总DNA中检测到DNA片断。但在鸡胚成纤维细胞(CEF)中，未观察到该盒对细胞活力的影响。为了研究致癌MDV1和非致癌MDV1之间的遗传差异，采用聚合酶链式反应(PCR)扩增病毒基因组中的meq基因。除了含有天然MEQ开放阅读框(ORF)的PCR产物外，从感染CVI988的CEF制备的DNA样品中还扩增出一条1.2kb的产物，但与致癌菌株RB1B和MD5没有扩增产物。序列分析表明，在CVI988(L-meq基因)的meq ORF上插入了一个178bp的序列。在体外传代70多次的致癌新城疫病毒株JM中也检测到该L-meq基因。由于这种插入可能会导致MEQ ORF的帧移位，因此所得到的ORF可能编码具有不同转录因子结构域的MEQ蛋白。从MD肿瘤细胞系制备的MDV基因组中也检测到L-meq基因。这个长的MEQ基因的生物学意义和功能尚不清楚，但这种插入也可能导致不同MDV1毒株生物学特性的变化。较少

Marek's disease virus (MDV) causes malignant lymphomas in chickens, but the molecular mechanism of the transformation remains unknown. Strains of MDV are classified into 3 serotypes based on their pathogenicity : only serotype 1 strains of MDV (MDV1), except for attenuated vaccine strains (i.e. CVI988), are oncogenic. Although Marek's disease (MD) is effecivecontrolled by vaccination, the protection mechanism by live vaccines is not known, and emergence of highly virulent MDV which can cause vaccine breaks has been reported. For this reason, a recombinant MDV which can induce tumor-specific apoptosis was constructed for the development of a newly invented anti-tumor vaccine in this study. In addition, differencein the structure of the meq gene has been identified between oncogenic and attenuated MDV1. Since the meq gene is known as an MDV oncogene, detailedanalysis on the change in the viral genome was done to obtain the insight into the role of the meq gene on the transformation by MD … More V.A gene cassette to express an apoptosis-inducing gene, the VP3 gene cloned from chicken infectious anemia virus, under the control of the meq promoter was constructed. The meq promoter was chosen since the meq gene has been known to be expressed only in tumor or MDV latently-infectedcells. When this gene cassette was introduced into an MD tumor-derived cell line, MSB1, viability of MSB1 was decreased within 24 hours, and DNA fragmentation was also detected in total cellular DNA prepared from MSB1. However, no change in the cell viability was observed in chick embryo fibroblast (CEF) transfected with this cassette. These results suggest that this gene cassette could be used for the tumor-specific induction of apoptosis.To study genetic differences between oncogenic and nononcogenic MDV1, polymerase chain reaction (PCR) was performed to amplify the meq gene in the viral genome. In addition to a PCR product including the native meq open reading frame (ORF), a 1.2-kb product was amplified from the DNA sample prepared from CEF infected with CVI988, but not with oncogenic strains, RB1B and Md5. Sequence analysis showed that a 178-bp sequence was inserted to the meq ORF of CVI988 (L-meq gene). This L-meq gene was also detected in an oncogenic MDV1, strain JM, which had been passaged for more than 70 times in vitro. Since this insertion could potentially cause a frame shift in the meq ORF, the resultant ORF could encode for the Meq protein with a different transacti vator domain. The L-meq gene was also detected in the MDV genome prepared from MD tumor-derived cell lines. The biological significance and function of this long meq gene is not yet elucidated, but this insertion may also contribute to the changes in the biological properties of different MDV1 strains. Less