Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (2): 352-361.doi: 10.3864/j.issn.0578-1752.2015.02.15

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Fusion Expression of Non-Structural Proteins 3a and 3b of Porcine Transmissible Gastroenteritis Virus and Influence on Cell Cycle

LIANG Ya-bing, ZHANG Qi, CHANG Rong, TONG De-wen, XU Xin-gang   

  1. College of Veterinary Medicine, Northwest A&F University, Shaanxi 712100, Yangling
  • Received:2014-03-04 Online:2015-01-16 Published:2015-01-16

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Abstract: 【Objective】 Non structural protein (nsp) 3a and 3b genes of porcine transmissible gastroenteritis virus (TGEV) were cloned and expressed in eukaryotic cell. Expression of nsp 3a and 3b proteins and their influence on cell cycle were studied.【Method】 Two pairs of primers used to amplify TGEV SX strain’s nsp 3a and 3b genes were designed according to the archived TGEV strain nucleotide by Primer 5.0. TGEV SX strain’s nsp 3a and 3b genes were cloned by RT-PCR, and ligated into eukaryotic expression vector pEGFP-N1, respectively. The recombinant plasmids were named as p3a-EGFP-N1 and p3b-EGFP-N1. The recombinant plasmids p3a-EGFP-N1 and p3b-EGFP-N1 were transfected into swine intestinal epithelial cells (IEC) using lipofectamine 2000. Expression of nsps 3a and 3b were confirmed by confocal microscopy. Transcription of TGEV 3a and 3b genes were analyzed by RT-PCR and the expression of nsp 3a and 3b was analyzed by Western blotting assay. The effects of proteins on cell cycle were investigated by flow cytometry assay. Transcription level of cyclin A and GRP78 were analyzed by qRT-PCR assay, and the expression level of GRP78, Cyclin A and Cyclin B1 were analyzed by Western blotting assay. 【Result】 The DNA fragments of 3a gene (213bp) and 3b gene (732bp) of TGEV SX strain were cloned successfully. The 3a gene of SX strain shared 97.4%—100% homology of nucleotide sequences and 98.6%—100% homology of amino acid. 3b gene shared 98.3%-99.9% homology of nucleotide sequences and 100% homology of amino acid with those of TGEV strains. Western blotting assay showed that 3a-GFP and 3b-GFP fusion proteins were in agreement with the predicted MW of 35kD and 54kD, respectively. The confocal microscopy analysis showed that 3a-GFP and 3b-GFP fusion proteins were distributed in the whole cell. Flow cytometry assay showed that the percentage of cells in G2/M phase was more than the control cells (IEC and IEC-GFP) in TGEV nsp 3b expression cells. The transcription level of Cyclin A was higher in 3b-GFP-expressing cells compared with the control cells by qRT-PCR assay. At the same time, the protein level of Cyclin A was significantly increased and Cyclin B1 level was down-regulated in TGEV 3b-GFP expressing cells by western blot assay. TGEV nsp 3a had no influence on cell cycle. The mRNA level of GRP78 was higher in 3a-GFP expressing cells than the control cells by qRT-PCR assay, and the expression level of GRP78 was up-regulated by TGEV nsp 3a compared with the control cells by western blot assay. Moreover, TGEV nsp 3b had no influence on GRP78 expression in IEC. 【Conclusion】The expressed nsp 3a and 3b of TGEV distributed in the whole transfected cells. Nsp 3a induced ER stress by up-regulated GRP78. Nsp 3b caused cell cycle arrested at G2/M phase by up-regulated Cyclin A and down-regulated Cyclin B1.

Key words: transmissible gastroenteritis virus, nsp 3a, nsp 3b, eukaryotic expression, cell cycle

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