不同病变型J亚群禽白血病病毒LTR启动子序列分析及活性比较

Sequence and Promoter Efficacy Analysis of Avian leukosis Virus Subgroup J Strains of Different Pathotypes

从ALV-J中国地方分离株SCAU-HN06株（血管瘤病变型）、NX0101株和JS-nt株（骨髓瘤病变型）病毒的细胞培养物提取前病毒DNA,通过PCR扩增各毒株的LTR并克隆,随后进行测序分析。与国内外ALV-J参考毒株LTR序列比较发现：国内地方分离株与英国ALV-J原型株HPRS-103和美国ALV-J原型株ADOL-7501的LTR核苷酸序列相似性为88.0%~97.2%;LTR中的U5区及R区具有较高的保守性,而U3区内存在较大差异。将不同病变型ALV-J的LTR片段分别插入pCAT-basic载体CAT报告基因5＇端。用所得的重组报告基因表达质粒转染DF-1细胞,48h后通过测定转染细胞中的CAT表达量来评价LTR启动子的活性。结果表明,SCAU-HN06株与骨髓瘤病变型ALV-J（JS-nt株,NX0101株）LTR启动子活性差异不显著。

To characterize the long terminal repeat（LTR） of the ALV-J strain which can induce hemangioma,fragments of provirus LTR of the three different ALV-J strains SCAU-HN06,NX0101 and JS-nt were amplified with a pair of specific primers,then cloned and subjected to sequence analysis.In comparison with the prototype ALV-J strains HPRS-103 and ADOL-7501,the LTRs of domestic strains（SCAU-HN06,NX0101,JS-nt and SD07lk1） had an 88.0%-97.2% nucleotide sequence identity;the U5 and R regions in the LTR had a high nucleotide similarity,while the U3 region in the LTR showed significant variance.The LTR fragments from the different ALV-J strains were inserted into the upstream of bacterial CAT gene of the plasmid pCAT-Basic,respectively.The resultant recombinant plasmids were transfected into DF-1 cells.The transfected cells were harvested 48 h post-transfection,and cell lysates were prepared for CAT expression detection.The CAT assay was performed using CAT-ELISA.The results showed that the promoter activity of the LTRSCAU-HN06 was a little higher than those of LTRJS-nt and LTRNX0101,but there was no significant difference in the promoter activity among the compared LTRs.