携带hTERT-P2A-EGFP基因真核表达质粒的构建和鉴定

doi:10.13481/j.1671-587x.20170201

Abstract

Abstract: Objective: To construct the eukaryotic expression plasmid carrying hTERT-P2A-EGFP, and to explore its expression and transfection efficiency in the HEK293FT cells. Methods: The recombinant plasmid was constructed by using pBABE-puro-hTERT and pRRLSIN-cPPT-MSCV-EGFP plasmids. The hTERT,P2A,and EGFP genes were obtained using pBABE-puro-hTERT as template by PCR. And the correct hTERT was inserted into pRRLSIN-cPPT-MSCV-EGFP vector. Then the recombinant plasmid containing hTERT-P2A-EGFP gene was obtained and identified. The HEK293FT cells were transfected by the recombinant plasmid, and the expression of green fluorescence protein(GFP) was observed by fluorescence microscope. Results: The PCR results showed that the fragments of hTERT, P2A, and EGFP were 3 400, 110 and 720 bp. And the length of gene fragment(hTERT-P2A-EGFP)was 4 300 bp by enzyme digestion. The results of sequencing showed that the 1 547 site of the target gene was mutated. Using site-directed mutagenesis, the 1 547 site was successfully mutated. And the target gene sequence was completely identical with the sequence published in GenBank. The recombinant plasmid was transfected into the HEK293FT cells, and GFP was observed in the cells.The results of flow cytometry showed that the transfection efficiency of recombinant plasmid was 44.8%. Conclusion: The recombinant plasmid carrying hTERT-P2A-EGFP gene is successfully constructed, and it can be used for cell transfection.

Key words: green fluorescence protein, plasmid construction, human telomerase reverse transcriptase

CLC Number:

Q785

CHEN Xiaona, WANG Xiaodan, SUN Liguang, FANG Fang, CUI Weiwei, YANG Yongguang, LIU Ya. Construction and identification of eukaryotic expression plasmid carrying hTERT-P2A-EGFP[J].Journal of Jilin University Medicine Edition, 2017, 43(02): 213-219.

References

[1] Wojtyla A, Gladych M, Rubis B. Human telomerase activity regulation [J].Mol Biol Rep, 2011, 38(5):3339-3349.
[2] Hayashi MT, Cesare AJ, Rivera T, et al. Cell death during crisis is mediated by mitotic telomere deprotection [J]. Nature, 2015, 522(7557):492-496.
[3] Dessauge F, Lollivier V, Ponchon B, et al. Effects of nutrient restriction on mammary cellturnover and mammary gland remodeling in lactating dairy cows [J]. J Dairy Sci, 2011, 94(9):4623-4635.
[4] Shukla S, Acharya S, Rajput D, et al. Telomere-the twilight to immortality [J]. J Assoc Physicians India, 2010, 58(31):553-560.
[5] Zvereva MI, Shcherbakova DM, Dontsova OA. Telomerase:structure, functions, and activity regulation[J]. Biochemistry (Mosc), 2010, 75(13):1563-1583.
[6] Osterhage JL, Friedman KL. Chromosome end maintenance by telomerase[J]. J Biol Chem,2009, 284(24):16061-16065.
[7] Domenech A, Raynes JG, Rodriquez EG, et al. Recombinant expression of goat milk serum amyloid A:preliminary studies of the protein and derived peptides on macrophage phagocytosis [J]. Protein Pept Lett, 2012, 19(3):299-307.
[8] Hasegawa T, Chosa N, Asakawa T, et al. Establishment of immortalized human periodontalligament cells derived from deciduous teeth [J]. Int J Mol Med, 2010, 26(5):701-705.
[9] Low KC, Tergaonkar V. Telomerase:central regulator of all of the hallmarks of cancer[J]. Trends Biochem Sci, 2013, 38(9):426-434.
[10] D'Souza Y, Chu TW, Autexier C. A translocation-defective telomerase with low levels of activity and processivity stabilizes short telomeres and confers immortalization[J]. Mol Biol Cell, 2013, 24(9):1469-1479.
[11] Miller J, Dakic A, Chen R, et al. HPV16 E7 protein and hTERT proteins defective for telomere maintenance cooperate to immortalize human keratinocytes[J]. PLoS Pathog, 2013, 9(4):e1003284.
[12] Artandi SE. Telomeres, telomerase, and human disease [J]. N Engl J Med, 2006, 355(12):1195-1197.
[13] Balducci L, Blasi A, Saldarelli M, et al. Immortalization of human adipose-derived stromal cells:production of cell lines with high growth rate, mesenchymal marker expression and capability to secrete high levels of angiogenic factors [J]. Stem Cell Res Ther, 2014, 5(3):63.
[14] Daniel M, Peek GW, Tollefsbol TO. Regulation of the human catalytic subunit of telomerase (hTERT)[J]. Gene, 2012, 498(2):135-146.
[15] Li NF, Broad S, Lu YJ, et al. Human ovarian surface epithelial cells immortalized with hTERT maintain functional pRb and p53 expression[J]. Cell Prolif, 2007, 40(5):780-794.
[16] Abdallah BM, Haack-Sørensen M, Burns JS, et al. Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite [corrected] extensive proliferation[J]. Biochem Biophys Res Commun, 2005, 326(3):527-538.
[17] Vidale P, Magnani E, Nergadze SG, et al. The catalytic and the RNA subunits of human telomerase are required to immortalize equid primary fibroblasts[J]. Chromosoma, 2012, 121(5):475-488.
[18] Yao CL, Hwang SM. Immortalization of human mesenchymal stromal cells with telomerase and red fluorescence protein expression[J]. Methods Mol Biol, 2012, 879:471-478.
[19] Ebe T. Green fluorescent protein as a marker gene expression[J]. Tanpakushitsu Kakusan Koso, 2007, 52(13 Suppl):1766-1767.
[20] 杨慧敏, 李文刚,吴高峰,等. 绿色荧光蛋白的研究进展[J]. 中国畜牧兽医, 2008, 35(8):29-32.
[21] 王晓丽, 邵卫星,单虎. 绿色荧光蛋白研究进展[J]. 动物医学进展, 2008, 29(1):56-59.
[22] 刘伟,王杰,幸永明,等.绿色荧光蛋白标记SOX9基因慢病毒载体的构建及在兔骨髓间充质干细胞中的表达[J]. 中国实验诊断学,2017,21(1):140-145.
[23] Chan HY, Sivakamasundari V, Xing X, et al. Comparison of IRES and F2A-based locus-specific multicistronic expression in stable mouse lines[J]. PLoS One, 2011, 6(12):e28885.
[24] Deng W, Yang D, Zhao B, et al. Use of the 2A peptide for generation of multi-transgenic pigs through a single round of nuclear transfer[J]. PLoS One, 2011, 6(5):e19986.

Construction and identification of eukaryotic expression plasmid carrying hTERT-P2A-EGFP

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