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| Fusion Expression of ORF5 gene of Porcine reproductive and respiratory syndrome virus |
| CHEN Yong-jun;SU Xin-ming;GAO Xiao-fei;ZHENG Qi-sheng;YU Chun-mei;CAO Rui-bing;ZHOU Bin;CHEN Pu-yan |
| Key Laboratory of Animal Disease Diagnosis and Immunology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China |
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Abstract Three pairs of specific primers were designed according to the sequence of Porcine reproductive and respiratory syndrome virus ORF5 gene published on GenBank(accession No.AY737282). Two modified ORF5 gene fragments ORF5-1 and ORF5-2 which deleted the N-terminal transmembrane region (28 residues, including signal peptides) sequence and the middle tansmembrane region (60 residues) sequence of ORF5 gene respectively were obtained from recombinant plasmid pMD-ORF5 by PCR amplification using these primers. ORF5-1 and ORF5-2 gene fragments were inserted into prokaryotic expression vector pET-32 a(+), resulting in the recombinant plasmids pET-ORF5-1 and pET-ORF5-2. After transformed into Escherichia coli BL21(DE3) cells and induced with IPTG, SDS-PAGE analysis revealed that ORF5-1 and ORF5-2 recombinant proteins with a expression level amounted to 12.2% and 39% of the total bacterial proteins respectively . Expression of ORF5 gene has been improved by removing the dual transmembrane regions. Western blotting analysis showed that the recombinant proteins had immunoreactivity and proved that the deleting of transmembrane regions had no significant effect on the antigenicity of the recombinant proteins.
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Received: 17 June 2005
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Corresponding Authors:
CHEN Pu-yan
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