人肝脏特异性miR-122重组慢病毒载体的构建与鉴定

Construction and identification of the human liver-specific miR-122 recombinant lentivirus

目的构建microRNA-122(miR-122)的重组过表达慢病毒,并对其进行病毒包装、鉴定与滴度测定,为进一步研究miR-122的功能和作用机制奠定基础。方法利用PCR法扩增人类基因组DNA中miR-122发夹前体结构RNA,并将其克隆至PCDH-CMV-MCS-EF1a-GFP-puro慢病毒表达载体上,经酶切及基因测序鉴定,将阳性重组PCDH-CMV-miR-122-EF1a-GFP-puro表达载体、p CMV-VSV-G和p CMVdR8.91三质粒共转染到HEK-293T细胞,收获上清液,将所得病毒悬液浓缩后梯度稀释后感染HEK-293T细胞,并利用绿色荧光蛋白进行病毒滴度测定。将重组慢病毒转染原代培养人皮肤成纤维细胞,并利用实时定量PCR检测miR-122的表达。结果重组慢病毒表达载体PCDH-CMV-miR-122-EF1a-GFP-puro酶切及测序鉴定证明载体构建成功,并得到滴度为3×108TU/m L的病毒液。病毒感染人皮肤成纤维细胞后miR-122表达明显增强。结论通过优化miRNA载体构建方法成功构建人miR-122的重组慢病毒载体并可在人类成纤维细胞内显著过表达miR-122,为miR-122的研究提供更高效稳定的基因载体。

Objective To construct, package and identify the recombinant lentivirus overexpressing microRNA - 122 （ miR - 122 ） and to lay the foundation for further study the functions and mechanism of miR - 122. Methods The small hairpin RNA of human pre - miR - 122 gene was amplified from the human genomic DNA by polymerase chain reac- tion （PCR） , which was constructed to the lentiviral expression vector PCDH - CMV - MCS - EFla - GFP - puro. After enzymatic digestion and sequencing, the positive recombinant PCDH- CMV- miR- 122 -EFla- GFP- puro expression vector, pCMV -VSV -G and pCMV -dR8.91 were co -transfected into HEK -293T cells, and the snpernatants were subsequently harvested. After the virus suspension concentrated, the gradient dilutions of infected HEK -293T cells were produced and determined by the green fluorescence protein. The expression of miR - 122 in primary cultured human skin fibroblasts HFFs, which were infected with recombinant lentivirus, was analyzed by real - time quantitative PCR. Results The recombinant lentiviral expression vector PCDH - CMV - miR - 122 - EF1 a - GFP - puro was successfully construc- ted according to enzymatic digestion and DNA sequencing. The lentivirus, with the concentration of 3 ×108 TU/mL, sig- nificantly increased the expression of miR - 122 in infected HFFs. Conclusion The recombinant lentivirus with PCDH - CMV - miR - 122 - EF1 a - GFP - puro is successfully constructed, and highly overexpresses mature miR - 122 in HFFs, providing a good carrier for miR - 122 with high efficiency and stability.