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）在鸡中引起恶性淋巴瘤，但其转化的分子机制尚不清楚。MDV毒株根据致病性分为3种血清型：除减毒疫苗株（即CVI988）外，只有血清型1株（MDV1）具有致癌性。虽然马立克氏病（MD）可以通过疫苗接种得到有效控制，但活疫苗的保护机制尚不清楚，并且有报道称出现了可导致疫苗断裂的高毒力MDV。为此，本研究构建了一种能够诱导肿瘤特异性凋亡的重组MDV，用于开发一种新型抗肿瘤疫苗。此外，meq基因的结构在致癌型和减毒型MDV1之间存在差异。由于meq基因是一种已知的MDV致癌基因，我们对病毒基因组的变化进行了详细的分析，以了解meq基因在MD转化中的作用。我们构建了更多的V.A基因盒，在meq启动子的控制下表达鸡传染性贫血病毒的凋亡诱导基因VP3。之所以选择meq启动子，是因为已知meq基因仅在肿瘤或MDV潜伏感染的细胞中表达。将该基因盒导入MD肿瘤来源细胞系MSB1后，MSB1的活力在24小时内下降，并且在MSB1制备的总细胞DNA中也检测到DNA断裂。然而，转染该卡带后的鸡胚成纤维细胞（CEF）的细胞活力未见明显变化。这些结果表明，该基因盒可用于肿瘤特异性诱导细胞凋亡。为了研究致癌性和非致癌性MDV1的遗传差异，采用聚合酶链反应（PCR）扩增病毒基因组中的meq基因。除了包含天然meq开放阅读框（ORF）的PCR产物外，从CVI988感染的CEF制备的DNA样本中扩增出1.2 kb的产物，但不包含致癌菌株RB1B和Md5。序列分析表明，在CVI988 （L-meq基因）的meq ORF中插入了一个178 bp的序列。该L-meq基因也在致癌MDV1菌株JM中检测到，该菌株已在体外传代超过70次。由于这种插入可能会导致meq ORF中的帧移位，因此产生的ORF可能会用不同的transactii vator域编码meq蛋白。L-meq基因也在MD肿瘤来源细胞系制备的MDV基因组中检测到。该长meq基因的生物学意义和功能尚不清楚，但它的插入也可能导致不同MDV1菌株生物学特性的变化。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