人巨细胞病毒miR-UL22A的重组载体构建以及表达分析  

Construction of Recombinant Vectors and Expressions of Human Cytomegalovirus miR-UL22A

作　　者：王鸿雁[1] 王博[1] 齐莹[1] 马艳萍[1] 黄郁晶[1] 柳中洋 阮强[1] 

机构地区：[1]中国医科大学附属盛京医院病毒研究室,辽宁沈阳110004

出　　处：《微生物学杂志》2017年第4期34-39,共6页Journal of Microbiology

基　　金：辽宁省自然科学基金项目(2013021008)

摘　　要：优化构建人巨细胞病毒(human cytomegalovirus,HCMV)miR-UL22A的真核表达载体;明确HCMV临床株及实验室株感染过程中miR-UL22A-5p及miR-UL22A-3p的表达情况。扩增HCMV UL22A编码区不同长度(975、494、209及140 bp)的序列,构建表达miR-UL22A的重组p Silencer(p S)载体,转染人胚肾293细胞后提取总RNA;收集HCMV临床株Han株以及实验室株Towne株感染人胚肺成纤维细胞后6、12、24、48和72h以及不同感染时相(即刻早期、早期、晚期)的总RNA标本;应用Taq Man颈环-实时荧光定量PCR技术检测上述标本miR-UL22A-5p及miR-UL22A-3p的表达量。成功构建了包含上述不同长度miR-UL22A编码序列的重组载体;侧翼序列长度各为40 bp左右的pre-miRNA(即插入140 bp的编码序列)表达成熟miRNA的效果最佳;在病毒的一个复制周期内,HCMV临床株和实验室株miR-UL22A的表达趋势无明显差异,均在感染后6h即检测到表达,72 h达到峰值,且在即刻早期即有miRNA的明确表达;无论是在重组载体表达状态下还是自然感染状态下,miR-UL22A-5p始终为miR-UL22A前体优势表达的miRNA。结果表明,有140 bp片段miRUL22A编码序列的重组载体能够高效地异源表达miR-UL22A-5p及miR-UL22A-3p;miR-UL22A-5p始终为miR-UL22A前体优势表达的miRNA。为进一步研究miRNA转录后调控作用提供了参考。Eukaryotic expression vectors of human cytomegalovirus （HCMV） miR-UL22A were optimized and con- structed, to clarify the expression situation of the infection process of HCMV clinical strain and the lab strain. Se- quences of different lengths of HCMV miR-UL22A encoding fragments （975 bp, 494 bp, 209 bp, and 140 bp） were amplified and constructed a recombinant the expression vector pSilencers （pS） , and extracted total RNA after trans- fected into human embryo kidney 293 cell, and collected total RNA samples at 6, 12, 24, 48, and 72 hours after transfection of embryo lung fibroblast with HCMV clinical strain, Han strain and lab Towne strain, as well as different infection time phase （immediate-early stage, early stage, and late stage）. The expressions of miR-UL22A-Sp and miR-UL22A-3p were determined and tested by Taqman stem-loop RT-PCR. The results showed that the recombinant vectors containing different lengths of miRNA encoding sequences were constructed successfully. The flank sequences each about 40 bp of pre-miRNA （ i. e. tbe inserted 140 bp coding sequence） achieved the best expression effect of the mature miRNA. No obvious difference of miR-UL22A expression trend was observed during one viral replication cycle of HCMV clinical strain and lab strain. All the expressions were determined and tested 6 hours after the infection, and reached the peak value at 72 hour, moreover, the definite expression of miRNA was immediately observed at immedi- ate-early stage; and whatever expressed by recombinant vector or infected during natural condition, miR-UL22A-5p has always been prosomatically dominant expression of miR-UL22A. Therefore, recombinant vector containing 140 bp of miR-UL22A encoding sequence of miR-UL22A could high-efficiently heterogenetic express miR-UL22A-5p and miR-UL22A-3p; And miR-UL22A-5p has always been prosomatically dominant expression of miRNA; This study pro- vides a foundation for further study on the regulations after transcription of miRNA.

关 键 词：人巨细胞病毒 miR-UL22A-5p miR-UL22A-3p 

分 类 号：Q939.93[生物学—微生物学] R37[医药卫生—病原生物学]