牛lncRNA H19过表达载体构建

Construction of LncRNA H19 overexpression vector

中文关键词: pcDNA3.1-EGFP-H19  荧光定量技术  CART  细胞转染

英文关键词: lncRNA H19  overexpression vector  the CART  cell transfection

中文摘要:

      目的 构建以及鉴定lncRNA H19过表达重组载体，为进一步探究lncRNA H19与miRNA 491和CART基因的互作关系奠定试验基础。方法 NCBI获取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三种质粒共转染至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 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，本试验将为后续探究lncRNA H19与miRNA 491和CART基因之间的互作关系创造试验条件。

英文摘要:

      Objective To construct and identify lncRNA H19 overexpression recombinant vector, so as to lay the experimental foundation for further exploring the interaction between lncRNA H19, CART gene and miRNA 491. Methods 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. 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.