不同亚群禽白血病病毒5′LTR序列及启动子活性分析

Sequence and Promoter Activity Analysis of 5′LTR from Different Subgroups of Avian Leukosis Viruses

对不同亚群禽白血病病毒(avian leukosis virus,ALV)5′LTR序列及其启动子活性进行了比较分析,以探讨LTR对ALV复制和致病力的影响。通过PCR分别扩增克隆了中国分离株GD08(ALV-A)、CD08(ALV-B)、HN06(血管瘤病变型ALV-J)和NX0101(骨髓瘤病变型ALV-J)毒株基因组5′LTR片段。与国内外不同亚群ALV分离株5′LTR核苷酸序列比较发现,NX0101株和HN06株与ALV-J国内外分离株的同源性最高,达90.8%～97.5%;GD08株与ALV-A国内分离株SDAU09C1的同源性最高,为94.6%;CD08株与GD08株和ALV-J各株的同源性高达90%以上。LTR中的R区具有较高的保守性,但CD08株U3区缺失11bp,GD08株U5区与其它毒株的U5区差异较大。将LTR片段插入到pCAT-Basic载体的CAT报告基因前,通过转染DF-1细胞和测定CAT表达量来评价LTR的启动子活性。HN06株和NX0101株之间,以及GD08株和CD08株之间LTR启动子活性有差异,但差异不显著;而ALV-J毒株与GD08株和CD08株之间的LTR启动子活性差异显著。

To characterize the long terminal repeat（LTR） of different subgroups of ALVs,fragments of 5′ LTR of subgroup J strains HN06 and NX0101,subgroup A isolate GD08 and subgroup B isolate CD08 were cloned and subjected to sequence analysis.The 5′ LTRs of HN06 and NX0101 had an 90.8% to 97.5% nucleotide sequence identity in comparison with other ALV-J strains.The 5′LTR of GD08 had the highest identity of 94.6% with that of the domestic strain SDAU09C1 of ALV-A and had a lower nucleotide sequence identity of 91.7% and 92.4% with HN06 and NX0101,respectively.The nucleotide sequence of CD08 5′LTR was highly identified with that of ALV-J 5′LTRs.The R region had a high nucleotide similarity,while the U3 and U5 regions showed variance.To compare the promoter activities,the 5′LTR fragments of different ALV isolates were inserted into the pCAT-Basic vector and transfected into DF-1 cells,respectively.Kinetic analysis for CAT activity showed that the promoter activities of 5′LTR from HN06 and NX0101 were similar,as well as that form GD08 and CD08.While there were significant differences in activity between ALV-J and GD08 or CD08.