Subcellular Localization of Orf virus 129 Anchor Protein in Goat Testis Cells

doi:10.7668/hbnxb.20191063

Abstract

Abstract: This experiment was conducted to study the localization of ORFV129 ankyrin repeat proteins from Orf virus on transfected cells. According to the sequence of ORFV SY 17 complete gene (GenBank accession number:MG 712417.1), primers were designed to amplify the 129 gene of ORFV,and ORFV129 gene was cloned into pEGFP-N1 eukaryotic expression vector directionally. The recombinant expression plasmid was identified by restriction enzyme digestion analysis,sequencing and named as pEGFP-ORFV129. Preliminary bioinformatic analysis of ORFV129 gene was predicted and analyzed,including the amino acid sequence,protein transmembrane region and subcellular localization. The recombinant plasmid pEGFP-ORFV129 was transfected into the primary testis cells of newborn goat,and identification of ORFV129 protein expression by Western Blot. Subcellular localization of ORFV129 was examined with laser confocal microscopy after DAPI staining. The sequence of ORFV129 gene was amplified and cloned successfully,with a total length of 1 551 bp. Amino acid sequence analysis showed that the gene encoded a protein containing 516 amino acids,composing of 8 multiple copies of the ankyrin repeat motifs(ANKs). The transmembrane region analysis showed that ORFV129 had no transmembrane regions,and subcellular localization predicted that the protein was mainly located in the cytoplasm. The result of Western Blot showed that recombinant plasmid pEGFP-ORFV129 transfected into the primary testis cells of newborn goat was successfully expressed. Subcellular localization experiment results showed that the ORFV129 protein was located in the cytoplasm,which was identical with the predicted ones.The results might lay a foundation for further research the function of ORFV129.

Key words: Goat, Orf virus, ORFV129 ankyrin repeat proteins, Bioinformatic analysis, Eukaryotic expression, Subcellular localization

CLC Number:

S855.3
Q78

XIANG Hua, HUANG Ren, ZHU Jiangjiang, YUE Hua, TANG Cheng, ZHANG Huanrong. Subcellular Localization of Orf virus 129 Anchor Protein in Goat Testis Cells[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(4): 222-229. doi: 10.7668/hbnxb.20191063.

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