摘要
为初步研究猪TRIM11的免疫学功能,以猪颌下淋巴结cDNA为模板扩增TRIM11基因cDNA并对该基因进行了蛋白质结构预测和组织表达谱分析,将该基因与PiggyBac载体相连获得真核表达载体PB-TRIM11,之后转染PK15细胞获得稳定表达细胞系。用猪伪狂犬病毒(PRV)刺激过表达TRIM11PK15细胞和对照组细胞后,于不同时间收获细胞上清,比较两组细胞上清中病毒滴度变化。结果表明,猪TRIM11的ORF长度为1 407bp,编码468个氨基酸,该氨基酸序列含有3个TRIM家族保守的结构域。组织表达谱分析显示,猪TRIM11在脂肪组织、肺的表达量较高,而在心、回肠、十二指肠、直肠中的转录量较低。成功克隆了猪TRIM11cDNA序列并构建了真核转录载体PB-TRIM11,qRT-PCR检测结果表明TRIM11在PK15细胞系中成功表达。检测感染PRV后过表达TRIM11PK15细胞和对照组细胞上清液病毒的TCID50发现过表达TRIM11组的病毒滴度始终高于对照组。过表达TRIM11有一定的促进PRV增殖的作用,为进一步研究猪TRIM11在天然免疫中的作用奠定了基础。
In order to preliminary study the immunologic functions of porcine TRIM11,the cDNA of the target gene was amplified from the cDNA of porcine submandibular lymph node,the protein structure of porcine TRIM11 was predicted and its tissue expression profile was analysed,the cloned porcine TRIM11 cDNA was connected into PiggyBac（PB）transposon vector,then the recombinant vector（PB-TRIM11）was transfected to PK15 cells to establish stably transfected PK15 cell line.The results showed that the coding length of the cloned porcine TRIM11 was1 407 bp and the sequence code 468 amino acids.Porcine TRIM11 protein was characterized by the presence of the tripartite motif.Tissue transcription profile analysis showed that,the transcription of TRIM11 was higher in fat,lung,whereas the transcription of porcine TRIM11 was lower in heart,ileum,recutum,duodenum.DNA sequence analysis of TRIM11 showed that the cloned TRIM11 cDNA was consistent with sequence from GenBank and the recombinant vector（PBTRIM11）was constructed.The transfected PK15 cell line stably expressing TRIM11 was estab-lished successfully and the transcription of TRIM11 was identified by qRT-PCR.PK15 cell line that stably express porcine TRIM11 and control cells were infected with pseudorabies virus（PRV）,respectively,then the cell supernatants were harvested at various time points after infection and the virus titers were detected.The results showed that the viral TCID50 of the TRIM11over-expression group was higher than the control all the time.It indicates that the TRIM11over-expression cells might be more susceptible to PRV.The study provides the experimental basis for the role of porcine TRIM11 in the innate immunity system in the future.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2016年第6期1239-1246,共8页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(3149600030)
农业部转基因专项(2014ZX0801015B)
河南省基础与前沿技术研究计划项目(142300413209)
关键词
三基序结合蛋白
猪伪狂犬病毒
病毒增殖
稳定细胞系
tripartite motif protein
pseudorabies virus
virus multiplication
stable cell line
