牛lncRNA H19过表达载体构建

Construction of LncRNA H19 Overexpression Vector

[目的]构建以及鉴定牛lncRNA H19过表达重组载体,为进一步探究lncRNA H19与miRNA 491和牛CART基因的互作关系奠定试验基础。[方法]NCBI获取牛lncRNA H19序列,经聚合酶链式反应(PCR)扩增,双酶切后载入pcDNA3.1-EGFP载体得到重组质粒。将pcDNA3.1-EGFP-H19、miRNA 491和CART 3种质粒共转染至HEK293T细胞,在细胞内反复扩增过表达之后,利用荧光定量技术检测pcDNA3.1-EGFP-H19的表达量。[结果]结果显示pcDNA3.1-EGFP-H19载体序列正确;293T细胞绿色荧光达50%,且强度适中,说明转染效果良好;lncRNA H19在293T细胞中显著表达。[结论]pcDNA3.1-EGFP-H19载体构建成功,试验将为后续探究lncRNA H19与miRNA 491和CART基因之间的互作关系创造试验条件。

[Objective]To construct and identify lncRNA H19 overexpression recombinant vector,to lay the experimental foundation for further exploring the interaction between lncRNA H19,CART gene and miRNA 491.[Method]LncRNA H19 sequence was obtained by NCBI,amplified by polymerase chain reaction(PCR),double digested and loaded into pcDNA3.1-EGFP vector to obtain recombinant plasmids.The three plasmids were transfected into HEK293T cells.After repeated intracellular amplification and overexpression,purification and titer detection were performed.[Result]Finally,the expression of lncRNA was detected by fluorescence quantification,and the sequence of pcDNA3.1-EGFP-H19 vector was correct.The green fluorescence of 293T cells reached 50%,and the intensity was moderate,indicating that the transfection operation was correct.lncRNA H19 was significantly expressed in 293T cells.[Conclusion]pcDNA3.1-EGFP-H19 vector was successfully constructed.This study will create experimental conditions for further exploring the interaction between lncRNA H19,miRNA 491 and CART gene.