马TRIM5α基因的扩增及抗EIAV作用评价  

The cloning of equine TRIM5α gene and the anti-equine infectious anemia virus activity of the protein

作　　者：蔡伟刚[1] 那雷[1] 刘荻萩[1] 马建 尹鑫[1,2] 张泽力[1,3] 艾有为[1,3] 王晓钧[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001 [2]东北农业大学动物医学院,黑龙江哈尔滨150030 [3]东北林业大学野生动物资源学院,黑龙江哈尔滨150040

出　　处：《中国预防兽医学报》2014年第8期620-623,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金项目(31072113;31222054);兽医生物技术国家重点实验室自主课题(SKLVBP201205;201304)

摘　　要：TRIM5α是一种重要的天然免疫因子,能够限制多种逆转录病毒的跨种感染。为研究马的TRIM5α是否具有抗病毒作用,本实验采用RT-PCR技术从马外周血液细胞的总RNA中扩增TRIM5α基因。测序结果表明,与GenBank中唯一的马TRIM5α(eqTRIM5α)基因序列相比马基因组预测的TRIM5α(eqTRIM5α-predicted)基因在其C末端部位有208 bp片段的插入,该插入造成TRIM5α的截短突变(eqTRIM5α-S)。通过删除插入片段后得到与eqTRIM5α-predicted基因相同的全长马的TRIM5α(eqTRIM5α-F)基因。同时将eqTRIM5α-S基因和eqTRIM5α-F基因分别插入pLPCX-HA真核表达载体中构建重组质粒,并分别转染293T细胞,转染24 h后感染马传染性贫血(EIAV)假病毒。通过检测荧光素酶活性,实验结果表明相对于阴性对照组eqTRIM5α-S可以有效地降低EIAV假病毒的复制,而eqTRIM5α-F则无该生物学功能。但通过将两种eqTRIM5α重组质粒与EIAV-gag表达重组质粒共转染293T细胞,结果表明eqTRIM5α-S和eqTRIM5α-F均不能降解Gag蛋白,这表明eqTRIM5α-S可能存在另一种机制限制EIAV的复制。TRIM5α is an indispensable innate immune factor which is capable to block interspecies transmission of retrovirus. To investigate whether equine TRIM5α （eqTRIM5α） harbors antivirus activity, the eqTRIM5α gene was amplified from horse blood cells by RT-PCR, and the sequencing result showed that an only eqTRIM5α gene with additional insertion of 208 bp in C terminal of the gene was amplified which was different from the predicted sequence of eqTRIM5α. While, the 208 bp insertion of the gene resulted in the expression of truncated eqTRIM5a （eqTRIM5α-S）. By removing the additional inserted sequence, the full length of eqTRIM5α gene was created as the predicted eqTRIM5a （eqTRIM5α-F）. In addition, the eqTRIM5α-F and eqTRIM5α-S encoding sequences were inserted into the eukaryotic expression vector of pLPCX-HA and transfected into 293T cells, respectively, and infected with pseudotyped equine infectious anemia virus. （EIAV） at 24 hours post the transfections. The resultsshowed that the replication of pseudotyped EIAV was dramatically declined in eqTRIM5α-S expressed （transfected） cells compared with mock transfected cells, but not in eqTRIM5α-F expressed （transfected） cells detected by the activity of luciferase expressed from pseudotyped EIAV. However, both eqTRiM5α-S and eqTRIM5α-F were unable to degrade the Gag protein, indicating that eqTRIM5α-S might have an alternative approach to inhibit the virus replication.

关 键 词：马TRIM5α 马传染性贫血病毒 抗病毒作用 衣壳蛋白 降解作用 

分 类 号：S852.65[农业科学—基础兽医学]