去除信号肽有利鸡白细胞介素-10在哺乳动物细胞胞浆的表达  

Removing Signal Peptide Accumulate Chicken Interleukin-10 in the Cytoplasm of Mammal Animal Cell Line

作　　者：彭蔚宇[1] 王丽廷 王兴童 杨柳[1] 孟兴[1] 陈洪岩[1] 韩凌霞[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所实验动物与比较医学团队/兽医生物技术国家重点实验室,哈尔滨150001 [2]山西农业大学动物科技学院,太谷030801

出　　处：《实验动物科学》2017年第2期16-20,25,共6页Laboratory Animal Science

基　　金：国家科技支撑计划项目(No.2015BAI07B02-02)

摘　　要：目的分析鸡白细胞介素10(chicken interleukin-10,chIL-10)的信号肽对体外表达的影响,以获得稳定表达chIL-10的细胞系。方法采用RT-PCR方法从经脂多糖(LPS)体外刺激的鸡外周血淋巴细胞中分别扩增含信号肽(euchIL-10F)和去信号肽(euchIL-10M)的chIL-10编码基因,克隆入含有报告基因e GFP和新霉素抗性基因(neo)的真核表达载体pEGFP-N1中,获得重组质粒N1-euchIL-10F和N1-euchIL-10M。分别转染中国仓鼠卵巢细胞系(CHO-K1),经G418加压筛选、纯化,获得稳定表达chIL-10蛋白的CHO-K1细胞系,利用激光共聚焦试验观察外源蛋白的细胞定位。结果通过RT-PCR、间接免疫荧光试验和免疫印迹试验,表明euchIL-10F和euchIL-10M均整合入细胞基因组中,且N1-euchIL-10M在胞浆大量表达较强的绿色荧光,而N1-euchIL-10F表达的荧光较弱,且胞浆内含量很低。结论去除信号肽利于chIL-10基因在体外真核细胞胞浆的表达。Objective To analyze the effect of signal peptide of chicken interleukin-10 （chIL-10） on expressing in CHO-K1 cell line, and to establish a stable chIL-10 expressing cell line. Method Whole chIL-10F and signal peptide-deleting genetic fragment of chIL-10 were amplified with two pairs of primers and inserted into pEGFP-N1 vector. The obtained recombinant plasmids of NI-chIL-10F and NI-chIL-10M were transfected into CHO-K1 cells under G418 screening. Result Two stable cell lines of CHO-chIL-10F and CHO-chIL-10M were obtained, respectively, confirmed by RT-PCR and western blot assays. Confocal examination showed that the reporter protein GFP was expressed in the cytoplasm of CHO-chIL-IOM and stronger than in the CHO-chIL-10F. Conclusion Removing signal peptide of chIL-10 improves expression of chIL-10 in the cytoplasm in vitro.

关 键 词：鸡白细胞介素-10 信号肽 真核表达 CHO-K1细胞 

分 类 号：Q789[生物学—分子生物学]