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| Gene Cloning,Site-directed Mutagenesis and Prokaryotic Expression of Porcine Sep15 |
| ZHOU Ji-chang1,2, LI Dai-lin2, TANG Jia-yong2, ZHAO Hua2, LIU Xiao-li1, ZHU Yu-mei1, XU Jian1 |
1. Molecular Biology Lab,Shenzhen Center of Chronic Disease Control,Shenzhen 518020,China;
2. Institute of Animal Science,Sichuan Agricultural University,Chengdu 611134,China |
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Abstract To clone and identify the gene of porcine selenoprotein 15kDa(Sep15), Sep15 ,conduct the site-directed mutagenesis and prokaryotic expression for the further functional study of Sep15 using pig models. The total RNA was extracted from pig spleen for RT-PCR,and then the Sep15 cDNA sequence of 1230 bp containing the open reading frame(ORF)till to poly(A)tail was cloned. The 489 bp of ORF sequence had 85.1% identity to that of human,while their amino acid sequences had 92.7% identity. Like those in various mammals,the porcine Sep15 cDNA bore the common characteristics of an in-frame TGA for selenocysteine(Sec)and a Sec insertion sequence element of Form 2 located in the 3'-untranslated region. By inverse PCR,the TGA codon for Sec was mutated into TGC for cysteine(Cys). The mutant was recombined to pET30 vector and expressed in E. coli BL21(DE3)under the induction of 0.4 mmol/L IPTG for 3h. The expressed recombinant protein with His-tag detected by SDS-PAGE was about 23kDa. In Western blot assays,both the rabbit anti-sera generated by the purified recombinant protein and the commercial murine antibody against the peptide downstream the Sec residue of human Sep15 were able to detect the recombinant protein. In conclusion,the porcine Sep15 was cloned and identified for the first time,and its molecular characteristics shared high similarity with those of human SEP15 . And the Cys-mutant of porcine Sep15 cross reacted with the antibody against the C-terminal of human Sep15.
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Received: 12 November 2010
Published: 25 August 2011
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[1] Behne D,Kyriakopoulos A. Mammalian selenium-containing proteins. Annu Rev Nutr,2001,21:453-473.
[2] Johansson L,Gafvelin G,Arnér E S. Selenocysteine in proteins—properties and biotechnological use. Biochim Biophys Acta,2005,1726(1):1-13.
[3] Schook L,Beattie C,Beever J,et al. Swine in biomedical research:creating the building blocks of animal models. Anim Biotech,2005,16(2):183-190.
[4] Kryukov G V,Castellano S,Novoselov S V,et al. Characterization of mammalian selenoproteomes. Science,2003,300(5624):1439-1443.
[5] Merk-Novagen. pET System Manual. 10th Edition, 2002.
[6] Ferguson A D,Labunskyy V M,Fomenko D E,et al. NMR structures of the selenoproteins Sep15 and SelM reveal redox activity of a new thioredoxin-like family. J Biol Chem,2006,281(6):3536-3543.
[7] Gill R W,Sanseau P. Rapid in silico cloning of genes using expressed sequence tags(ESTs). Biotechnol Annu Rev,2000,5:25-44.
[8] Buettner C,Harney J W,Larsen P R. The 3'-untranslated region of human Type 2 iodothyronine deiodinase mRNA contains a functional selenocysteine insertion sequence element. J Biol Chem,1998,273(50):33374-33378.
[9] Mix H,Lobanov A V,Gladyshev V N. SECIS elements in the coding regions of selenoprotein transcripts are functional in higher eukaryotes. Nucleic Acids Res,2007,35(2):414-423.
[10] Grundner-Culemann E,Martin G W,Harney J W,et al. Two distinct SECIS structures capable of directing selenocysteine incorporation in eukaryotes. RNA,1999,5(5):625-635.
[11] Driscoll D M,Copeland P R. Mechanism and regulation of selenoprotein synthesis. Annu Rev Nutr,2003,23:17-40.
[12] Zhou J C,Zhao H,Li J G,et al. Selenoprotein gene expression in thyroid and pituitary of young pigs is not affected by dietary selenium deficiency or excess. J Nutr,2009,139(6):1061-1066.
[13] Korotkov K V,Kumaraswamy E,Zhou Y,et al. Association between the 15-kDa selenoprotein and UDP-glucose:glycoprotein glucosyltransferase in the endoplasmic reticulum of mammalian cells. J Biol Chem,2001,276(18):15330-15336.
[14] Easwari K,Malykh A,Korotkov K V,et al. Structure-expression relationships of the 15 kDa selenoprotein:possible role of the protein in cancer etiology? J Biol Chem,2000,275(45):35540-35547.
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