SV40 LT抗原介导的蝙蝠胎儿肾细胞永生化  

Immortalization of bat fetal kidney cell by SV40 large T antigen

作　　者：张思春[1] 杨凡力[1] 余乐[1] 何彪[1] 涂长春[1] 

机构地区：[1]军事医学科学院军事兽医研究所 吉林省人兽共患病预防与控制重点实验室,吉林长春130122

出　　处：《中国预防兽医学报》2014年第5期363-366,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：NSFC-与云南联合基金(U1036601);国家“973”项目(2012CB722501);国家“863”项目(2012AA022006)

摘　　要：蝙蝠是许多新发人兽共患病病毒的自然储存宿主,由于缺乏蝙蝠细胞系,只能利用其他哺乳动物细胞系来分离蝙蝠病毒,因此成功分离的几率较低。为建立稳定的蝙蝠细胞系,本研究利用胰酶消化法培养东亚水鼠耳蝠(Myotis petax)的胎儿原代肾细胞。利用重组逆转录病毒转导法将SV40 LT抗原基因转导入蝙蝠胎儿原代肾细胞,经嘌呤霉素筛选获得LT基因整合和表达的阳性细胞,并进行连续传代培养。RT-PCR和western blot检测表明转导的细胞中有SV40 LT基因的整合和表达。转导细胞在软琼脂中培养不能够形成克隆,表明其未发生癌性转化。对第33代的转导细胞进行单细胞克隆,获得了3个衍生的细胞系,其中Mp-Ki03细胞系已培养至第60代。病毒感染试验表明蝙蝠西江病毒和蝙蝠轮状病毒均可以感染该转导细胞系。本研究通过SV40 LT抗原建立了永生化的蝙蝠胎儿肾细胞系,并且该细胞系对蝙蝠病毒易感,对分离与鉴定蝙蝠病毒以及研究病毒感染蝙蝠细胞的机制具有重要的作用。Bats are important natural reservoir hosts for a number of new and emerging zoonotic viruses. Currently the isolation of bat viruses has to be conducted mainly on cell lines derived from terrestrial mammals rather than bats and only limited bat viruses have been isolated. To increase the isolation efficiency of bat viruses an immortalized bat cell lines was established in the present study by transduction with SV40 large T antigen （LT） gene. First of all, primary fetal kidney cells of bat Myotis petax were prepared by trypsinization of the kidney tissue. Then SV40 LT gene was transduced into the primary kidney cells by retrovirus system. Positively transduced cells were selected by puromycin, followed by continuous passage. The expression of SV40 LT gene was detected by RT-PCR and western blot. Finally three immortalized cell lines were established by cloning of transduced cells at passage 33 with one of them, named Mp-Ki03, being cultured for 60 passages. This cell line is unable to form cell clumps in soft agarose indicating that it was not transformed cells. Experimental infection of Mp-Ki03 cells with previously isolated bat reovirus and rotavirus showed that Mp-Ki03 cells are susceptible to these viruses at primary passage. These results indicated that an immortalized bat fetal kidney cell line has been established and showed desirable sensitivity to bat viruses, indicating the potential use of this cellline for bat virus isolation and identification.

关 键 词：蝙蝠 肾细胞 SV40 LT抗原 永生化 

分 类 号：S852.65[农业科学—基础兽医学]