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| Construction of an Eukaryocyte Expression Vector of Human ANKRD49 and the Study of Function and RNA Interference Target of ANKRD49 |
| PANG Min1, WANG Hai-long2, GUO Min3, GUO Rui2 |
1. Department of Respiration, The First Hospital of Shanxi Medical University, Taiyuan 030001, China;
2. Academy of Basic Medicine, Shanxi Medical University, Taiyuan 030001, China;
3. Center of Laboratory Animal, Shanxi Medical University, Taiyuan 030001, China |
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Abstract Objective: To construct the eukaryotic expression recombinant plasmid of human ANKRD49, study its function, and identify the RNA interference targets of ANKRD49. Methods: Total RNA was extracted from A549 cells and the cDNA was synthesized. The open reading frame of human ANKRD49 gene was amplified from the cDNA by RT-PCR, and cloned into p3×Flag-CMV-14 to construct recombinant plasmid p3×Flag-CMV-14/ANKRD49. After confirming by colony-PCR, double restrict enzyme digestion and DNA sequencing, the eukaryotic expression recombinant p3×Flag-CMV-14/ANKRD49 was transfected into HEK 293T cells. The targeted protein expressed in host cells was detected by Immunoblotting and immunofluorescence staining. The distribution of ANKRD49 in host cells was detected by immunofluorescence staining. The cell proliferation of ANKRD49-expressed HEK 293T cells were measured through MTT assay. The RNA interference targets of human ANKRD49 were identified via Immunoblotting assay followed by co-transfecting both p3×Flag-CMV-14/ANKRD49 and siRNA into HEK293T cells. Results: The product of RT-PCR was 720 bp. The recombinant plasmid p3×Flag-CMV-14/ANKRD49 was confirmed successfully by colony-PCR, double restrict enzyme digestion and DNA sequencing. Immunofluorescence staining and Immunoblotting showed that human ANKRD49 was expressed successfully in the p3×Flag-CMV-14/ANKRD49 transfected-293T cells than mock transfected cells with molecular weight was approximately 27 kDa. Immunofluorescence staining also revealed that ANKRD49 was distributed in nucleus. MTT assay showed that ANKRD49 had no effect on cell proliferation. Co-transfection and Immunoblotting assays showed that the expression of human ANKRD49 was efficiently knockdown by number 1 and 4 siRNA. Conclusion: The eukaryotic expression recombinant plasmid p3×Flag-CMV-14/ANKRD49 was constructed and expressed in nucleus of HEK 293T cells. This protein had no effect on cell proliferation. Two ANKRD49 interference targets were identified.
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Received: 25 June 2014
Published: 25 October 2014
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