猪Ⅱ型圆环病毒ORF1基因克隆及在E.coli中的表达

作者：郗鑫; 陈焕春; 李川; 刘正飞; 曹胜波; 琚...porcinecircovirustype2orf1genecloningexpression

摘要：A pair of specific primers were designed and synthesized according to the published sequences of ORF1 gene of PCV-2. The complete DNA fragment of ORF1 gene was obtained by PCR from viral DNA of PCV-2 QD strain. Its nuclectide sequence was determined by the dideoxy-mediated chain termination method. The results showed that the complete open reading frame (ORF) of ORF1 gene encoding 314 amino acids was 945 bp in length.A comparison of the nucleotide and amino acid sequences of ORF1 gene with that of other PCV strains showed that the identity of nucleotide with PCV-1 and PCV-2 were 83% and 96.4%-99.2% respectively,and identity of the deduced amino acid with PCV-1 and PCV-2 were 84% and more than 98%respecitively. The DNA fragment of ORF1 gene was subcloned into prokaryotic expression vector pET-28a and pGEX-KG;while the specific non-fusion and fusion proteins with GST of molecular weight 38 kD and 63 kD were expressed in E.coli BL21(DE3).Western blotting assay indicated that the polyclonal antibody against PCV-2 could recognize these two proteins.

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