| Marek's disease(MD) is a contagious,lymphoproliferative disease of domestic chickens caused by highly infectious cell-asscioated oncogenic alph-herpesvirus MD virus serotype 1(MDVI). Strains of MDV are classified into three catagories based on their pathogenecity.All of them having the ability of pathopoiesis belonging to the MD virus stereotypeâ… (MDVâ… ).Previous results of investigations implicated that several candidate genes encoded by the BamHI-D,-H,I2,-I,-O2 fragments of the MDV genome were oncogenic. Among them,the meq gene is most consistently detected in MD samples and MDV1 transformed cell lines. The meq gene is only present in MDV1,and encodes a 339-amino-acid protein with a N-terminal basic-leucine zipper domain ,which resembles the Fos/Jun family of oncoprotein and a C-terminal 33-amino acid proline-rich region which is a potent transcroptinoal transactivator. meq gene is may be the most important oncoprotein in MDV genome that has been detectived. These results strongly suggest that,as a viral oncogene,meq plays a significant role in transformation and oncogenesis by MDV1. In addition,recent identification showed a 180-bp nucleotide sequence is inserted into the meq open reading frame(ORF) of a non-oncogenic MDV vaccine strain,CVI988,and this changed meq gene is termed as long-meq. Southern blot analysis also confirmed the insertion. The insertion of 180-bp sequence may explain the reason why CVI988 is not oncogenic. The present study was carried out to understand the biological function and properties of L-MEQ and its effect on telomerase activity.L-meq gene was amplified from MDVgenome by PCR and was subcloned into an expression vector,pET28a(+). The subcloned L-meq gene was sequenced and analyzed to be consistent with the one of GENEBANK. The positive recombinants were transformed into E. coli Rosseta(DE3). The expression of L-MEQ protein was induced by IPTG and the purpose protein(55kDa) was detected and analyzed by SDS-PAGE. L-MEQ protein was expressed at high level,amounting to 18.88% of the total bacterial protein confirmed by thin-layer scanning. Purified L-MEQ protein was used as antigen to immune rabbits and induce the production of polyclonal antibody against L-MEQ,of which titers is 1:12800.To study the biological function of L-MEQ,recombinant eukaryotic expression vector, pcDNA3.1(+)-1-meq,were constructed and transfected into MDCC-MSB1 cells,a chicken lymphoblastoid cell line transformed by MDV1. The positive cells transfected with pcDNA3.1(+)-1-meq and pcDNA3.1(+) was selected by G418 respectively. The positive cloned cells were picked up and enlarged culture to measure telomerase activity and to detect chTRET mRNA.In contrast to the cells transfected with pcDNA3.1(+),the cells transfected with pcDNA3.1(+)-1-meq stably expressed L-MEQ protein even until 96 hours after transfection. Updated TRAP method and Real-time PCR was used to test the telomerase activity and chTRET mRNA respectively before and after transfection. The results showed that L-MEQ can suppressed the telomerase activity,and even reduced the relative telomerase activity and decreased the amount of chTRET mRNA,but did not induce MDCC-MSB1 cells proliferation inhibition and apoptosis.In a word,we firstly investigated the partly function of L-MEQ at hom-e and abroad and the present research will promote to elucidate the role of L-MEQ on oncogenesis. It would be helpful to complete the theory foundation of prevention and cure in MD to explore the mechanism of tumorige -nesis and related tumor research.
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